Polyoligonucleotide capable of triggering gene silencing

Abstract

Disclosed are a class of polyoligonucleotides capable of triggering gene silencing and a use thereof, and specifically disclosed are a compound as shown in formula (I) and a use thereof.

Description

Polyoligonucleotides that can trigger gene silencing

[0001] This application claims priority to Chinese Patent Application No. 2024118500941, filed on December 14, 2024; Chinese Patent Application No. 2024118556025, filed on December 16, 2024; Chinese Patent Application No. 2025112848109, filed on September 9, 2025; and PCT Patent Application No. PCT / CN2025 / 120444, filed on September 10, 2025. The full text of the aforementioned patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to a class of oligonucleotides that can trigger gene silencing and their applications. Background Technology

[0003] RNA interference (RNAi) technology refers to the highly conserved evolutionary phenomenon of 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 metabolic diseases, infectious diseases, and malignant tumors.

[0004] However, inhibition of a single target sometimes fails to achieve the desired therapeutic effect, necessitating simultaneous regulation of two or more pathways within the same cell. In such cases, administering siRNA drugs targeting two different pathways to the same patient typically fails to achieve the desired therapeutic effect. This may be due to the influence of pharmacokinetic (PK) mechanisms, leading to different drug concentrations of the two siRNA drugs within the same cell, thus preventing effective simultaneous inhibition of both targets within the same cell and achieving synergistic effects.

[0005] One solution to the above problems is to develop chemically coupled dual siRNA trigger siRNA drug molecules. A common method is to link two siRNA triggers together using a linker. Once inside the cell, changes in the cellular environment (pH) or the capabilities of enzymes (hydrolysis, redox reactions, etc.) cause the two triggers to separate, thus activating the drug.

[0006] Dual-target and multi-target RNAi technologies, which can target multiple genes simultaneously, will have a wider range of applications and are expected to show great potential in clinical treatment. Summary of the Invention

[0007] This invention provides a compound capable of inhibiting the expression of a first target gene and a second target gene, the structure of which is shown in formula (I):

[0008] in:

[0009] DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, the first sense strand and the first antisense strand forming a first double-stranded portion of 15-27 nucleotide pairs in length, and a first 5' extension in the first antisense strand located upstream of the 5' of the first double-stranded portion, the first 5' extension being at least 3 nucleotides in length.

[0010] DS2 is a double-stranded oligonucleotide containing a second sense strand and a second antisense strand, which together form a second double-stranded portion of 15-27 nucleotide pairs in length.

[0011] L is a structural unit that serves as a connector. One end of L is connected to the first justice chain, and the other end of L is connected to the second justice chain.

[0012] The first target gene may be the same as or different from the second target gene.

[0013] In some schemes, DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, the first sense strand and the first antisense strand forming a first double-stranded portion of 15-27 nucleotide pairs in length, and a first 5' extension in the first antisense strand located upstream of the 5' of the first double-stranded portion. The first 5' extension is at least 3 nucleotides in length and can be cleaved at the 3' end nucleotide of the first 5' extension to obtain a cleaved DS1 product containing the first double-stranded portion. The cleaved DS1 product can silence a first target RNA or inhibit the expression of a first target gene through RNA interference.

[0014] In some embodiments, the compound represented by formula (I),

[0015] in:

[0016] DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, the first sense strand and the first antisense strand forming a first double-stranded portion of 15-27 nucleotide pairs in length, and a first 5' extension in the first antisense strand located upstream of the 5' of the first double-stranded portion. The first 5' extension is at least 3 nucleotides in length and can be cleaved at the 3' end nucleotide of the first 5' extension to obtain a cleaved DS1 product containing the first double-stranded portion. The cleaved DS1 product can silence a first target RNA or inhibit the expression of a first target gene through RNA interference.

[0017] DS2 is a double-stranded oligonucleotide comprising a second sense strand and a second antisense strand, the second sense strand and the second antisense strand forming a second double-stranded portion of 15-27 nucleotide pairs in length, and a second 5' extension in the second antisense strand located upstream of the 5' of the second double-stranded portion. The second 5' extension is at least 3 nucleotides in length and can be cleaved at the 3' end nucleotide of the second 5' extension to obtain a cleaved DS2 product containing the second double-stranded portion. The cleaved DS2 product can silence a second target RNA or inhibit the expression of a second target gene through RNA interference.

[0018] L is a structural unit that serves as a connector. One end of L is connected to the first justice chain, and the other end of L is connected to the second justice chain.

[0019] The first target RNA or the first target gene may be the same as or different from the second target RNA or the second target gene.

[0020] In some designs, L is a connecting structural unit, with one end of L connected to the 5' or 3' end of the first justice chain, and the other end of L connected to the 5' or 3' end of the second justice chain. In some designs, L is a connecting structural unit, with one end of L connected to the 5' end of the first justice chain, and the other end of L connected to the 5' end of the second justice chain. In some designs, L is a connecting structural unit, with one end of L connected to the 3' end of the first justice chain, and the other end of L connected to the 3' end of the second justice chain. In some designs, L is a connecting structural unit, with one end of L connected to the 3' end of the first justice chain, and the other end of L connected to the 5' end of the second justice chain. In some designs, L is a connecting structural unit, with one end of L connected to the 5' end of the first justice chain, and the other end of L connected to the 3' end of the second justice chain.

[0021] In some embodiments, one end of L is connected to a nucleotide at position 6 from the 5' end of the first sense strand, the other end of L is connected to the 5' end of the second sense strand, and the second sense strand includes the second 5' extension.

[0022] In some embodiments, the aforementioned first antisense chain further includes a first 3' extension located downstream of the 3' of the first double-stranded portion.

[0023] In some embodiments, the aforementioned second antisense chain further includes a second 3' extension located downstream of the 3' portion of the second double chain.

[0024] In some embodiments, the first antisense chain further includes a first 3' extension downstream of the first double-stranded portion, and the second antisense chain further includes a second 3' extension downstream of the second double-stranded portion.

[0025] In some embodiments, the first double-stranded portion is formed by base pairing of a first segment of the first antisense strand and a second segment of the first sense strand, wherein the first and second segments are of the same length; and / or the second double-stranded portion is formed by base pairing of a fourth segment of the second antisense strand and a fifth segment of the second sense strand, wherein the fourth and fifth segments are of the same length.

[0026] In some schemes, the lengths of the first double-stranded portion and / or the second double-stranded portion are independently:

[0027] 15-25, 15-24, 15-23, 16-24, 16-23, 16-22, 16-21, 16-20, 17-23, 17-22, 17-21, 17-20, 18-23, 18-22, 18-21, 18-20, 19-23, 19-22, 19-21, or 19-20 nucleotide pairs.

[0028] In some schemes, the lengths of the first double-stranded portion and / or the second double-stranded portion are independently 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotide pairs.

[0029] In some schemes, the lengths of the first and / or second sense strands are independently: 15-35, 16-35, 16-30, 16-27, 16-26, 16-25, 16-21, 17-35, 17-30, 17-25, 17-21, 18-35, 18-30, 18-25, 18-23, 18-21, 19-35, 19-30, 19-25, 19-21, 20-35, 20-30, 20-25, 20-23, 21-35, 21-30, 21-25, or 21-23 nucleotides.

[0030] In some schemes, the lengths of the first and / or second sense strands are independently 25, 24, 24, 23, 22, or 21 nucleotides, respectively.

[0031] In some schemes, the lengths of the first 5' extension and / or the second 5' extension are independently at least 3, 4, 5, 6, 7 or more nucleotides.

[0032] In some schemes, the lengths of the first 3' extension and / or the second 3' extension are each independently at least 1, 2 or more nucleotides.

[0033] In some embodiments, the lengths of the first 5' extension and / or the second 5' extension are independently at least 3, 4, 5, 6, 7 or more nucleotides, and the lengths of the first 3' extension and / or the second 3' extension are independently at least 1, 2 or more nucleotides.

[0034] In some schemes, the first fragment includes a first target region that is fully complementary to the first target RNA or the mRNA encoding the first target gene, and the fourth fragment includes a second target region that is fully complementary to the second target RNA or the mRNA encoding the second target gene. The first target region and the second target region may be the same as or different from each other.

[0035] In some schemes, the above cleavage is enzyme-catalyzed cleavage, optionally by specific cleavage of endonucleases, optionally by specific cleavage of ribonucleases (RNases).

[0036] In some schemes, the above-mentioned compounds, wherein:

[0037] a) The first antisense strand described above contains a first cleavage region, which contains the nucleotide sequence shown in Formula A-1.

[0038] Equation A-1: ​​(3'-5')X2-YZ

[0039] The above-described cleavage occurs between X2 and Y, where X2 is the 5' end nucleotide of the first fragment, and Y and Z are the two nucleotides at the 3' end of the first 5' extension; and / or

[0040] b) The second antisense strand described above contains a second cleavage region, which contains the nucleotide sequence shown in Formula A-2.

[0041] Equation A-2: (3'-5')X2'-Y'-Z',

[0042] The above cleavage occurs between X2' and Y', where X2' is the nucleotide at the 5' end of the fifth fragment, and Y' and Z' are the two nucleotides at the 3' end of the second 5' extension.

[0043] In some schemes, the above-mentioned compounds, wherein:

[0044] a) The first cleavage region further comprises a nucleotide (N1), where N1 is the third nucleotide from the 3' end of the first 5' extension, and the first cleavage region comprises the nucleotide sequence shown in Formula B-1.

[0045] Formula B-1:(3'-5')X2-YZ-N1,

[0046] The aforementioned splitting occurs between X2 and Y; and / or

[0047] b) The second cleavage region further comprises a nucleotide (N1'), where N1' is the third nucleotide from the 3' end of the second 5' extension, and the second cleavage region comprises the nucleotide sequence shown in Formula B-2.

[0048] Formula B-2: (3'-5')X2'-Y'-Z'-N1',

[0049] The above-mentioned splitting occurs between X2' and Y'.

[0050] In some schemes, the above-mentioned compounds, wherein:

[0051] a) The first cleavage region further comprises a third fragment (N), the third fragment comprising at least one nucleotide, wherein the nucleotide at the 3' end of the third fragment is N1, and the first cleavage region comprises the nucleotide sequence shown in Formula B'-1.

[0052] Equation B'-1:(3'-5')X2-YZN,

[0053] The above-described cleavage occurs between X2 and Y, where N is 1-10 nucleotides in length, preferably 1-5 nucleotides, more preferably 1 nucleotide; and / or

[0054] b) The second cleavage region further comprises a sixth fragment (N'), which contains at least one nucleotide, wherein the nucleotide at the 3' end of the sixth fragment is N1', and the second cleavage region comprises the nucleotide sequence shown in Formula B'-2.

[0055] Formula B'-2:(3'-5')X2'-Y'-Z'-N',

[0056] The above-mentioned cleavage occurs between X2' and Y', and the length of N' is 1-10 nucleotides, preferably 1-5 nucleotides, more preferably 1 nucleotide.

[0057] In some schemes, the above-mentioned compounds, wherein:

[0058] a) The above DS1 includes the double-stranded oligonucleotide represented by formula C-1.

[0059] The first fragment and the second fragment form the first double-stranded portion through base pairing, and the first fragment and the second fragment are of the same length. The first 5' extension contains at least 3 nucleotides, the first antisense strand contains the first cleavage region, the first cleavage region contains the nucleotide at the 5' end of the first fragment (X2) and the two nucleotides at the 3' end of the first 5' extension (YZ), and the first cleavage region contains the nucleotide sequence shown in Formula A-1.

[0060] Equation A-1: ​​(3'-5')X2-YZ

[0061] The aforementioned cleavage occurs between X2 and Y, and Formula A-1 is as defined in any of the preceding claims, wherein the length of the first sense strand is 15–35, 15–23, 15–22, or 15–21 nucleotides, and the length of the first antisense strand is 25–35, 26–35, 26–30, 25–27, or 26–27 nucleotides, and the 3' or 5' end of the first sense strand is connected to one end of L; and / or

[0062] b) The above DS2 includes the double-stranded oligonucleotide represented by formula C-2.

[0063] The fourth and fifth fragments mentioned above form the second double-stranded portion through base pairing, and the fourth and fifth fragments are of the same length. The second 5' extension contains at least 3 nucleotides, and the second antisense strand contains the second cleavage region. The second cleavage region contains the nucleotide at the 5' end of the fourth fragment (X2') and the two nucleotides at the 3' end of the second 5' extension (Y'-Z'). The second cleavage region contains the nucleotide sequence shown in Formula A-2.

[0064] Equation A-2: (3'-5')X2'-Y'-Z',

[0065] The aforementioned cleavage occurs between X2' and Y', and Formula A-2 is as defined in any of the preceding claims, wherein the length of the second sense strand is 15–35, 15–23, 15–22, or 15–21 nucleotides, the length of the second antisense strand is 25–35, 26–35, 26–30, 25–27, or 26–27 nucleotides, and the 3' or 5' end of the second sense strand is connected to one end of L.

[0066] In some schemes, the above-mentioned compounds, wherein:

[0067] a) The above-mentioned DS1 includes the double-stranded oligonucleotide represented by formula D-1.

[0068] The first fragment and the second fragment form the first double-stranded portion through base pairing, and the first fragment and the second fragment are of the same length. The first 5' extension contains at least 3 nucleotides, the first antisense strand contains a first cleavage region, the first cleavage region contains the 5' end nucleotide (X2) of the first fragment and the two nucleotides (YZ) at the 3' end of the first 5' extension, and the first cleavage region contains the nucleotide sequence shown in Formula A-1.

[0069] Equation A-1: ​​(3'-5')X2-YZ

[0070] The aforementioned cleavage occurs between X2 and Y, and Formula A-1 is as defined in any of the preceding claims, wherein the length of the first sense strand is 15–35, 15–23, 15–22, 15–21, 16–25, 17–23, 18–23, 19–23, 19–21, 20–23, 20–21, 21–23, 17, 18, 19, 20, 21, 22, or 23 nucleotides, and the length of the first antisense strand is 25–35, 25–30, 26–35, 26–30, 25–27, 26–27, 25, 26, 27, 28, 29, or 30 nucleotides, and the 3' or 5' end of the first sense strand is connected to one end of L; and

[0071] b) The DS2 comprises the double-stranded oligonucleotide represented by formula D-2:

[0072] The fourth and fifth segments form the second double-stranded portion through base pairing, and the fourth and fifth segments are of the same length. The second 3' protrusion and the second 5' protrusion each independently contain 0, 1, 2, or 3 nucleotides. The length of the second sense strand is 15–35, 15–23, 15–22, 15–21, 16–25, 17–23, 18–23, 19–23, 19–21, 20–23, 20–21, or 21–23, 17, 18, 19, 20, 21, 22, or 23 nucleotides. The length of the second antisense strand is 15–35, 19–30, 21–35, 21–30, 19–23, 21–23, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. The 3' or 5' end of the second sense strand is connected to one end of L.

[0073] In some schemes, the above-mentioned compounds, wherein:

[0074] a) The above-mentioned DS1 includes the double-stranded oligonucleotide represented by formula D-1.

[0075] The first fragment and the second fragment form the first double-stranded portion through base pairing, and the first fragment and the second fragment are of the same length. The first 5' extension contains at least 3 nucleotides, the first antisense strand contains a first cleavage region, the first cleavage region contains the 5' end nucleotide (X2) of the first fragment and the two nucleotides (YZ) at the 3' end of the first 5' extension, and the first cleavage region contains the nucleotide sequence shown in Formula A-1.

[0076] Equation A-1: ​​(3'-5')X2-YZ

[0077] The aforementioned cleavage occurs between X2 and Y, and Formula A-1 is as defined in any of the preceding claims, wherein the length of the first sense strand is 15–35, 15–23, 15–22, 15–21, 16–25, 17–23, 18–23, 19–23, 19–21, 20–23, 20–21, 21–23, 17, 18, 19, 20, 21, 22, or 23 nucleotides, and the length of the first antisense strand is 25–35, 25–30, 26–35, 26–30, 25–27, 26–27, 25, 26, 27, 28, 29, or 30 nucleotides, and the 3' or 5' end of the first sense strand is connected to one end of L; and

[0078] b) The above DS2 includes the double-stranded oligonucleotide shown in formula D-2:

[0079] The fourth and fifth fragments mentioned above form the second double-stranded portion through base pairing, and the fourth and fifth fragments are of the same length. The second 5' extension contains at least 3 nucleotides, and the second antisense strand contains the second cleavage region. The second cleavage region contains the nucleotide at the 5' end of the fourth fragment (X2') and the two nucleotides at the 3' end of the second 5' extension (Y'-Z'). The second cleavage region contains the nucleotide sequence shown in Formula A-2.

[0080] Equation A-2: (3'-5')X2'-Y'-Z',

[0081] The aforementioned cleavage occurs between X2' and Y', and Formula A-2 is as defined in any of the preceding claims, wherein the length of the second sense strand is 15–35, 15–23, 15–22, 15–21, 16–25, 17–23, 18–23, 19–23, 19–21, 20–23, 20–21 or 21–23, 17, 18, 19, 20, 21, 22 or 23 nucleotides, and the length of the second antisense strand is 25–35, 25–30, 26–35, 26–30, 25–27, 26–27, 25, 26, 27, 28, 29 or 30 nucleotides, and the 3' or 5' end of the second sense strand is connected to one end of L.

[0082] In some schemes, the first justice chain and the second justice chain are not connected to the same end of L.

[0083] In some schemes, Z and Z' are independently G, a natural analog of G, a non-natural analog of G, A, a natural analog of A, or a non-natural analog of A.

[0084] In some schemes, Z and Z' are independently natural analogs of G and G or non-natural analogs of G.

[0085] In some schemes, X2 and X2' are respectively independently A, a natural analog of A, a non-natural analog of A, U, a natural analog of U, or a non-natural analog of U.

[0086] In some schemes, the above formulas A-1 and A-2 each independently have 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 their natural or non-natural analogues.

[0087] In some schemes, Y and Y' are respectively independently A, a natural analog of A, a non-natural analog of A, U, a natural analog of U, or a non-natural analog of U.

[0088] In some schemes, Equations A-1 and A-2 above each independently have a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IUG, IAG, IUA and IAA.

[0089] In some schemes, formulas B-1 and B-2 above each independently have a sequence (3'-5') selected from the following: UUGC, UAGC, AUGC, AAGC, AAGU, UUAC, UAAC, AUAC, AAAC, UCGC, UGGC, ACGC, AGGC, UCAC, UGAC, ACAC, AGAC, AAGG, AAGCC, AAUC, AUUU, AAGAG, AAGAGC, AAGACCA, or their natural or non-natural analogs.

[0090] In some schemes, Equations B-1 and B-2 above each independently have a sequence (3'-5') selected from the following: UUGC, UAGC, AUGC, AAGC, AAGG, UUAC, UAAC, AUAC, and AAAC.

[0091] In some schemes, Equations B-1 and B-2 above each independently have 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.

[0092] In some embodiments, the first cleavage region and the second cleavage region each independently contain at least one modified nucleotide. Preferably, all nucleotides in the first cleavage region and / or the second cleavage region are modified nucleotides. In some embodiments, formulas A-1, A-2, B-1, and B-2 each independently contain at least one modified nucleotide. Preferably, all nucleotides in formulas A-1, A-2, B-1, and B-2 are modified nucleotides.

[0093] In some schemes, the modified nucleotides described above contain modified bases, modified glycosides, and / or modified nucleoside bonds.

[0094] In some schemes, the first cleavage region is not sufficiently complementary to the first target RNA or the mRNA encoding the first target gene, and / or the second cleavage region is not sufficiently complementary to the second target RNA or the mRNA encoding the second target gene.

[0095] In some schemes, the above-mentioned compounds, wherein:

[0096] (a) The first cleavage region described above contains at least one nucleoside internucleotide bond that is not a thiophosphate bond;

[0097] (b) The internucleotide bond between X2 and Y is not a thiophosphate bond;

[0098] (c) The internucleotide bond between Y and Z is not a thiophosphate bond;

[0099] (d) All internucleotide bonds in the first cleavage region described above are not phosphate thioester bonds;

[0100] (e) The first cleavage region described above contains at least one phosphate diester bond between nucleosides;

[0101] (f) The internucleotide bond between X2 and Y is a phosphodiester bond;

[0102] (g)The nucleoside bond between Y and Z is a phosphodiester bond;

[0103] (h) All internucleotide bonds in the first cleavage region described above are phosphodiester bonds;

[0104] (i) The nucleoside bond between the first cleavage region and the first fragment is a phosphodiester bond;

[0105] (a') The second cleavage region described above contains at least one nucleoside internucleotide bond that is not a thiophosphate bond;

[0106] (b') The internucleotide bond between X2' and Y' is not a thiophosphate bond;

[0107] (c')The internucleotide bond between Y' and Z' is not a thiophosphate bond;

[0108] (d') All internucleotide bonds in the second cleavage region described above are not thiophosphate bonds;

[0109] (e') The second cleavage region described above contains at least one phosphate diester bond between nucleosides;

[0110] The internucleotide bond between (f')X2' and Y' is a phosphodiester bond;

[0111] The internucleotide bond between (g')Y' and Z' is a phosphodiester bond;

[0112] (h') All internucleotide bonds in the second cleavage region described above are phosphodiester bonds; and / or

[0113] (I') The nucleoside bond between the second cleavage region and the fourth fragment is a phosphodiester bond.

[0114] In some schemes, the above-mentioned compounds, wherein:

[0115] (a) The first cleavage region described above contains at least one nucleotide selected from nucleotides modified with 2'-OMe or nucleotides modified with 2'-F;

[0116] (b) Each nucleotide in the first cleavage region described above is a nucleotide modified with 2'-OMe or a nucleotide modified with 2'-F;

[0117] (c) The first cleavage region described above contains at least one nucleotide modified with 2'-F;

[0118] (d) The first cleavage region described above contains no more than two 2'-F modified nucleotides;

[0119] (e) Z in formula A-1, formula B-1 or formula B'-1 is a nucleotide modified by 2'-F, and optionally X2 in formula A-1, formula B-1 or formula B'-1 is a nucleotide modified by 2'-F;

[0120] (f) N1 in formula B-1 or formula B-1 is a nucleotide modified with 2'-F;

[0121] (g) X2 and Y in formula A-1, formula B-1 or formula B'-1 are nucleotides modified with 2'-OMe, and Z is a nucleotide modified with 2'-F, and further, N1 in formula B-1 or formula B'-1 is a nucleotide modified with 2'-F.

[0122] (h) X2, Y and Z in formula A-1, formula B-1 or formula B'-1 are all nucleotides modified with 2'-OMe, and further N1 in formula B-1 or formula B'-1 is a nucleotide modified with 2'-F;

[0123] (a') The second cleavage region described above contains at least one nucleotide selected from nucleotides modified with 2'-OMe or nucleotides modified with 2'-F;

[0124] (b') Each nucleotide in the second cleavage region described above is a nucleotide modified with 2'-OMe or a nucleotide modified with 2'-F;

[0125] (c') The second cleavage region described above contains at least one nucleotide modified with 2'-F;

[0126] (d') The second cleavage region described above contains no more than two 2'-F modified nucleotides;

[0127] (e') Z' in formula A-2, formula B-2 or formula B'-2 is a nucleotide modified with 2'-F, and optionally X2' in formula A-2, formula B-2 or formula B'-2 is a nucleotide modified with 2'-F;

[0128] (f') N1' in formula B-2 or formula B-2 is a nucleotide modified with 2'-F;

[0129] (g') In formula A-2, B-2, or B'-12, X2' and Y' are both nucleotides modified with 2'-OMe, and Z' is a nucleotide modified with 2'-F, and further, N1' in formula B-2 or B'-2 is a nucleotide modified with 2'-F; and / or

[0130] (h') X2', Y' and Z' in formula A-2, formula B-2 or formula B'-2 are all nucleotides modified with 2'-OMe, and further, N1' in formula B-2 or formula B'-2 is a nucleotide modified with 2'-F.

[0131] In some embodiments, the first double-stranded portion of DS1 or DS1 further comprises at least one nucleoside interchain selected from thiophosphate bonds or methylphosphonate bonds, and / or the second double-stranded portion of DS2 or DS2 further comprises at least one nucleoside interchain selected from thiophosphate bonds or methylphosphonate bonds.

[0132] In some schemes, the above-mentioned compounds, wherein:

[0133] (a) The first and / or second nucleotide internucleotide bond from the 5' end of the first and / or fourth segments mentioned above is a thiophosphate bond or a methylphosphonate bond; and / or

[0134] (b) The first and / or second internucleotide bond from the 3' end of the first and / or fourth segments above are thiophosphate bonds or methylphosphonate bonds.

[0135] In some embodiments, the above-mentioned compound, wherein: the lengths of the first sense strand and / or the second sense strand are independently 17–23, 17–22, or 17–21 nucleotides, respectively; and the lengths of the first antisense strand and / or the second antisense strand are independently 22–28, 22–27, or 22–26 nucleotides, respectively.

[0136] In some schemes, the above-mentioned compounds, wherein:

[0137] a) The lengths of the first sense strand and the first antisense strand described above are (a) 19 and 25 nucleotides, (b) 20 and 25 nucleotides, (c) 21 and 25 nucleotides, (d) 19 and 26 nucleotides, (e) 20 and 26 nucleotides, (f) 21 and 26 nucleotides, or (g) 21 and 27 nucleotides, respectively; and / or

[0138] b) The lengths of the second sense strand and the second antisense strand are (a) 19 and 25 nucleotides, (b) 20 and 25 nucleotides, (c) 21 and 25 nucleotides, (d) 19 and 26 nucleotides, (e) 20 and 26 nucleotides, (f) 21 and 26 nucleotides, or (g) 21 and 27 nucleotides, respectively.

[0139] In some embodiments, the above-mentioned compounds, wherein L is selected from bonds, degradable linkers or non-degradable linkers.

[0140] In some schemes, the above-mentioned compounds, wherein L is selected from DNA, RNA, functionalized monosaccharides, or oligosaccharides.

[0141] In some schemes, the above-mentioned compounds are used, where L is a non-degradable linker.

[0142] In some embodiments, the compounds described above are capable of cleavage within cells but possess sufficient stability outside cells.

[0143] In some schemes, the above-mentioned compounds, wherein L has sufficient stability in vivo or in vitro environments.

[0144] In some of the formulations, the aforementioned compound, wherein L remains intact in the appropriate environment for a period of time before the compound comes into contact with the mRNA, for example, in plasma for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48 or 72 hours, with a breakage of no more than 5%, 10%, 20%, 30%, 40% or 50%.

[0145] In some embodiments, the compound can maintain the DS1 and DS2 linkage state outside the cell. In some embodiments, the compound is resistant to degradation factors (e.g., enzymes, pH, etc.) in the in vitro or in vivo environment, maintaining the DS1 and DS2 linkage state for a sufficiently long time. In some embodiments, the compound, after mixing with human plasma, has a half-life of not less than 55 hours (e.g., not less than 54 hours, not less than 53 hours, not less than 52 hours). In some embodiments, the compound, after mixing with human plasma, degrades by no more than 15% within 24 hours (e.g., not more than 14%, not more than 13%, not more than 12%, not more than 11%, not more than 10%, not more than 9%, or not more than 8%).

[0146] In some embodiments, the compounds provided herein possess sufficient stability in vivo or in vitro environments, and more preferably remain unbroken for a period of time in the appropriate environment (e.g., extracellular or in blood circulation) before the compound contacts mRNA (e.g., before entering cells), for example, remaining unbroken in plasma for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, or 72 hours, with breakage not exceeding 5%, 10%, 20%, 30%, 40%, or 50%, respectively. In some embodiments, the compounds provided herein do not contain deoxyribonucleosides, disulfide bonds, pH-sensitive cleavage groups, polypeptide hydrolase substrates, or sugars. In some embodiments, the compounds provided herein do not contain deoxyribonucleotides, abasic ribonucleic acid, abasic deoxyribonucleic acid, inverted abasic ribonucleic acid, or inverted abasic deoxyribonucleic acid. In some embodiments, the compounds provided herein do not contain deoxyribonucleosyl (e.g., dT), disulfide bonds, pH-sensitive cleavage groups, polypeptide hydrolase substrates, or sugars in or near the adjacent region (e.g., the first 5' extension, L, the second 3' extension, formula A-1, formula B-1, formula B'-1, and one or two nucleotides linked thereto). In some embodiments, the compounds provided herein do not contain deoxyribonucleotides, abasic ribonucleic acid, abasic deoxyribonucleic acid, inverted abasic ribonucleic acid, or inverted abasic deoxyribonucleic acid in or near the adjacent region. In some embodiments, L is a bond or chemical linker.

[0147] In some embodiments, the molar ratio of the cleaved DS1 product that interacts with the mRNA encoding the first gene and the DS2 product that interacts with the mRNA encoding the second gene is approximately 1 (e.g., approximately 0.8, 0.9, 1, 1.1, or 1.2).

[0148] In some embodiments, the molar ratio of the cleaved DS1 product that interacts with the first target RNA or the mRNA encoding the first target gene and the cleaved DS2 product that interacts with the second target RNA or the mRNA encoding the second target gene is about 1 (e.g., about 0.8, 0.9, 1, 1.1 or 1.2).

[0149] In some embodiments, the above-mentioned compound, wherein L is as shown in formula (I-1') or formula (I-1),

[0150] in:

[0151] X is either O or S;

[0152] Y represents a single bond, -O-, or -S-;

[0153] L1 is selected from single bonds, -O-, -S-, -SS-, -(C=O)-, -NH-, -NH-(C=O)-, -CH2-, -CH2CH2-, -CH2CH2CH2-,

[0154] L2, L3, L4, L6, L7, L8, and L9 are independently selected from single bonds, -O-, -S-, -SS-, -(C=O)-, -NH-, -NH-(C=O)-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -O-CH2-, -S-CH2CH2-, -O-CH2CH2-, -S-CH2CH2-, -CH2-O-CH2CH2-, - CH2CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-O-, -CH2-O-CH2-O-, -CH2CH2CH2-O-, -CH2CH2CH2-S-, -CH2-(C=O)-, -CH2-NH-(C =O)-, -O-(C=O)-NH-, -C(=O)O-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)O-, -OC(=O)NH-, -S(O)2NH-, -NHS(O)2-, and -CH2-NH-;

[0155] L5 is selected from single bonds, -O-, -S-, -SS-, -(C=O)-, -NH-, -NH-(C=O)-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -O-CH2-, -S-CH2-, -O-CH2CH2-, -S-CH2 CH2-, -CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-O-, -CH2-O-CH2-O-, -CH2CH2 CH2-O-, -CH2CH2CH2-S-, -CH2-(C=O)-, -CH2-NH-(C=O)-, -O-(C=O)-NH-, -CH2-NH-, -C(=O)O-, The 3' or 5' end of the second justice chain mentioned above is connected.

[0156] In some embodiments, the above-mentioned compound, wherein the above-mentioned L is as shown in formula (I-2') or formula (I-2),

[0157] in:

[0158] Ring A is absent and L6 is directly connected to triazole;

[0159] Alternatively, ring A can be selected from 5-16 membered heterocycles.

[0160] In some embodiments, the above-mentioned compound, wherein the L contains 1, 2 or 3 dT, and optionally, the L also contains the structure shown in the aforementioned formula (I-1') or formula (I-1) or formula (I-2') or formula (I-2).

[0161] In some embodiments, the compounds described above, with L as shown in (I-3), (I-4), or (I-5):

[0162] In some embodiments, the above-described compounds, wherein the L is as shown in formula (I-6), (I-7), or (I-8):

[0163] In some embodiments, the above-mentioned compounds, wherein: structural unit Selected from the arbitrarily replaced locations

[0164] In some embodiments, the above-mentioned compounds, wherein the above-mentioned L is as shown in formulas (I-9), (I-10), (I-11), (I-12), (I-13), (I-14), (I-15), (I-16), (I-17) or (I-18),

[0165] In some embodiments, the above-mentioned compound, wherein L is selected from...

[0166] In some embodiments, in the above-described compound, the 5' or 3' nucleotide of the first sense strand is linked to the 5' or 3' nucleotide of the second sense strand via an L-link. Preferably, the 5' nucleotide of the first sense strand is linked to the 5' nucleotide of the second sense strand via an L-link.

[0167] In some embodiments, the compound described above, wherein one end of the L is connected to a nucleotide at position 6 from the 5' end of the first sense strand, and the other end of the L is connected to the 5' end of the second sense strand.

[0168] In some embodiments, the compounds described above, wherein at least one nucleotide is a modified nucleotide, preferably all nucleotides are modified nucleotides, and the modifications include one, two or more 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 modifications that enhance the affinity of double-stranded ribonucleic acid for ARGO protein.

[0169] In some embodiments, the above-described compound, wherein the first antisense chain and / or the second antisense chain further comprises a capping group at the 5' end, optionally said capping group being connected to the 5' end of the first 5' extension and / or the second 5' extension.

[0170] In some embodiments, the above-described compound, wherein the capping group comprises an inverted non-base deoxynucleotide or MO6.

[0171] In some embodiments, the above-described compounds, wherein the capping group is connected to the 5' end of the first 5' extension and / or the second 5' extension via a nucleoside interbond, the nucleoside interbond optionally being modified or unmodified.

[0172] In some embodiments, the above-described compound, wherein the capping group is connected to the 5' end of the first 5' extension and / or the second 5' extension via a thiophosphate bond.

[0173] In some embodiments, the aforementioned compound further comprises one or more (e.g., one, two, three, four or more) delivery systems, optionally each of which is independently connected to DS1, DS2 or L.

[0174] In some embodiments, the compounds described above, wherein each of the delivery systems described above is an independent ligand, preferably the ligand alters the distribution, targeting, or lifetime of the compounds described above, more preferably the ligand provides enhanced affinity to targets, such as molecules, cells or cell types, compartments, receptors, such as cell or organ compartments, tissues, organs, or body regions, and even more preferably the ligand enables the compounds to be delivered to the target tissue and produce an RNA interference effect.

[0175] In some schemes, the compounds described above, wherein the ligands are each independently selected from GalNAc ligands, lipophilic ligands, or other ligands that target receptors to promote the endocytosis of the compounds, such as TfR-targeting ligands, LDL-R-targeting ligands, or integrin-targeting ligands.

[0176] In some embodiments, the compound is a GalNAc ligand, and the number of ligands is at least one; further, the number of GalNAc ligands is one or two. In some embodiments, the number of GalNAc ligands is one. In some embodiments, the number of GalNAc ligands is two.

[0177] In some embodiments, the above-mentioned compound, wherein: a) there are two GalNAc ligands, each linked to the 3' end nucleotide of the first and second sense strands respectively; or b) there is one GalNAc ligand linked to the 3' end nucleotide of either the first or second sense strand; or c) there are two GalNAc ligands, one linked to the 3' end nucleotide of the first sense strand and the other linked to the 5' end nucleotide of the second sense strand.

[0178] In some embodiments, the above-mentioned compounds, wherein the GalNAc ligands are independently L96 or NAG37; further, a) the number of the above-mentioned GalNAc ligands is two, one of which is L96 and is linked to the 3' end nucleotide of the first sense strand, and the other is NAG37 and is linked to the 5' end nucleotide of the second sense strand, or b) the number of the above-mentioned GalNAc ligands is two and both are L96, or c) the above-mentioned GalNAc ligand is one L96 and is linked to the 3' end nucleotide of the first sense strand or the second sense strand.

[0179] In some embodiments, the compounds described above are wherein the first target RNA or the first target gene is the same as the second target RNA or the second target gene; or the first target RNA or the first target gene and the second target RNA or the second target gene are different segments of the same RNA or the same gene; or the first target RNA or the first target gene and the second target RNA or the second target gene are different RNAs or different genes.

[0180] In some embodiments, the above-mentioned compounds or their pharmaceutically acceptable salts are any of the compounds or their pharmaceutically acceptable salts shown in Tables 1-85. SEQ ID NO in this text refers to the nucleobase sequence.

[0181] Table 1. Structure of polyoligonucleotide S1-1

[0182] Table 2. Structure of polyoligonucleotide S1-2

[0183] Table 3. Structure of polyoligonucleotide S2-1

[0184] Table 4. Structure of polyoligonucleotide S2-2

[0185] Table 5. Structure of polyoligonucleotide S3-1

[0186] Table 6. Structure of polyoligonucleotide S3-2

[0187] Table 7 Structure of polyoligonucleotide S4-1

[0188] Table 8. Structure of polyoligonucleotide S4-2

[0189] Table 9. Structure of polyoligonucleotide S5-1

[0190] Table 10 Structure of polyoligonucleotide S5-2

[0191] Table 11 Structure of polyoligonucleotide S6-1

[0192] Table 12 Structure of S6-2 Polyoligonucleotide

[0193] Table 13 Structure of polyoligonucleotide S7-1

[0194] Table 14 Structure of polyoligonucleotide S7-2

[0195] Table 15 Structure of polyoligonucleotide S8-1

[0196] Table 16 Structure of Polyoligonucleotide S8-2

[0197] Table 17 Structure of polyoligonucleotide S9-1

[0198] Table 18 Structure of Polyoligonucleotide S9-2

[0199] Table 19 Structure of polyoligonucleotide S10-1

[0200] Table 20 Structure of polyoligonucleotide S10-2

[0201] Table 21 Structure of polyoligonucleotide conjugate S11-1

[0202] Table 22 Structure of polyoligonucleotide conjugate S11-2

[0203] Table 23 Structure of S12-1, a polyoligonucleotide conjugate

[0204] Table 24 Structure of the polyoligonucleotide conjugate S12-2

[0205] Table 25 Structure of the polyoligonucleotide conjugate Z1-1

[0206] Table 26 Structure of the polyoligonucleotide conjugate Z1-2

[0207] Table 27 Structure of the polyoligonucleotide conjugate Z2-1

[0208] Table 28 Structure of the polyoligonucleotide conjugate Z2-2

[0209] Table 29 Structure of the polyoligonucleotide conjugate Z3-1

[0210] Table 30 Structure of the polyoligonucleotide conjugate Z3-2

[0211] Table 31 Structure of the polyoligonucleotide conjugate Z4-1

[0212] Table 32 Structure of the polyoligonucleotide conjugate Z4-2

[0213] Table 33 Structure of the polyoligonucleotide conjugate Z5-1

[0214] Table 34 Structure of the polyoligonucleotide conjugate Z5-2

[0215] Table 35 Structure of the polyoligonucleotide conjugate Z6-1

[0216] Table 36 Structure of the polyoligonucleotide conjugate Z6-2

[0217] Table 37 Structure of the polyoligonucleotide conjugate Z7-1

[0218] Table 38 Structure of the polyoligonucleotide conjugate Z7-2

[0219] Table 39 Structure of the polyoligonucleotide conjugate Z8-1

[0220] Table 40 Structure of the polyoligonucleotide conjugate Z8-2

[0221] Table 41 Structure of the polyoligonucleotide conjugate Z9-1

[0222] Table 42 Structure of the polyoligonucleotide conjugate Z9-2

[0223] Table 43 Structure of the polyoligonucleotide conjugate Z10-1

[0224] Table 44 Structure of the polyoligonucleotide conjugate Z10-2

[0225] Table 45 Structure of the polyoligonucleotide conjugate Z11-1

[0226] Table 46 Structure of the polyoligonucleotide conjugate Z11-2

[0227] Table 47 Structure of the polyoligonucleotide conjugate Z12-1

[0228] Table 48 Structure of the polyoligonucleotide conjugate Z12-2

[0229] Table 49 Structure of the polyoligonucleotide conjugate Z13

[0230] Table 50 Structure of the polyoligonucleotide conjugate Z14

[0231] Table 51 Structure of the polyoligonucleotide conjugate Z15

[0232] Table 52 Structure of the polyoligonucleotide conjugate Z16

[0233] Table 53 Structure of the polyoligonucleotide conjugate Z17

[0234] Table 54 Structure of the polyoligonucleotide conjugate Z18

[0235] Table 55 Structure of the polyoligonucleotide conjugate Z19

[0236] Table 56 Structure of the polyoligonucleotide conjugate Z20

[0237] Table 57 Structure of the polyoligonucleotide conjugate Z21

[0238] Table 58 Structure of the polyoligonucleotide conjugate Z22

[0239] Table 59 Structure of the polyoligonucleotide conjugate Z23

[0240] Table 60 Structure of the polyoligonucleotide conjugate Z24

[0241] Table 61 Structure of the polyoligonucleotide conjugate Z25

[0242] Table 62 Structure of the polyoligonucleotide conjugate Z26

[0243] Table 63 Structure of the polyoligonucleotide conjugate Z27

[0244] Table 64 Structure of the polyoligonucleotide conjugate Z28

[0245] Table 65 Structure of the polyoligonucleotide conjugate Z29

[0246] Table 66 Structure of the polyoligonucleotide conjugate Z30

[0247] Table 67 Structure of the polyoligonucleotide conjugate Z31

[0248] Table 68 Structure of the polyoligonucleotide conjugate Z32

[0249] Table 69 Structure of the polyoligonucleotide conjugate Z33

[0250] Table 70 Structure of the polyoligonucleotide conjugate Z34

[0251] Table 71 Structure of the polyoligonucleotide conjugate Z35

[0252] Table 72 Structure of the polyoligonucleotide conjugate Z36

[0253] Table 73 Structure of the polyoligonucleotide conjugate Z37

[0254] Table 74 Structure of the polyoligonucleotide conjugate Z38

[0255] Table 75 Structure of the polyoligonucleotide conjugate Z39

[0256] Table 76 Structure of the polyoligonucleotide conjugate Z40

[0257] Table 77 Structure of the polyoligonucleotide conjugate Z41

[0258] Table 78 Structure of the polyoligonucleotide conjugate Z42

[0259] Table 79 Structure of the polyoligonucleotide conjugate Z43

[0260] Table 80 Structure of the polyoligonucleotide conjugate Z52

[0261] Table 81 Structure of the polyoligonucleotide conjugate Z55

[0262] Table 82 Structure of the polyoligonucleotide conjugate Z56

[0263] Table 83 Structure of the polyoligonucleotide conjugate Z57

[0264] Table 84 Structure of the polyoligonucleotide conjugate Z58

[0265] Table 85 Molecular structures targeting multiple genes

[0266] Table 86 Structures of multi-gene oligonucleotide conjugates

[0267] The present invention also provides a pharmaceutical composition comprising the above-described compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0268] The present invention also provides a method for inhibiting the expression of a target gene in a subject in need, comprising administering to the subject a pharmaceutically effective amount of the above-described compound or a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition.

[0269] The present invention also provides a method for treating a disease or condition in a subject in need, comprising administering to the subject a pharmaceutically effective amount of the above-described compound or a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition, optionally wherein the disease or condition is related to a first gene and / or a second gene.

[0270] The present invention also provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition, in the preparation of an agent for inhibiting the expression of a target gene, or in the preparation of a medicament for treating a disease or condition; optionally, the disease or condition is related to a first gene and / or a second gene.

[0271] The present invention also provides the above-described compound or a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition, for inhibiting target gene expression or for treating diseases or conditions; optionally, the diseases or conditions are related to a first gene and / or a second gene. The present invention also provides the following non-limiting embodiments.

[0272] Implementation Method 1. The compound shown in formula (I),

[0273] in:

[0274] DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, the first sense strand and the first antisense strand forming a first double-stranded portion of 15 to 27 nucleotide pairs in length, and a first 5' extension in the first antisense strand located upstream of the 5' of the first double-stranded portion. The first 5' extension is at least 3 nucleotides in length and can be cleaved at the 3' end nucleotide of the first 5' extension to obtain a cleaved DS1 product containing the first double-stranded portion. The cleaved DS1 product can silence a first target RNA or inhibit the expression of a first target gene through RNA interference.

[0275] DS2 is a double-stranded oligonucleotide comprising a second sense strand and a second antisense strand, the second sense strand and the second antisense strand forming a second double-stranded portion of 15 to 27 nucleotide pairs in length, and a second 5' extension in the second antisense strand located upstream of the 5' of the second double-stranded portion. The second 5' extension is at least 3 nucleotides in length and can be cleaved at the 3' end nucleotide of the second 5' extension to obtain a cleaved DS2 product containing the second double-stranded portion. The cleaved DS2 product can silence a second target RNA or inhibit the expression of a second target gene through RNA interference.

[0276] L is a structural unit that serves as a connector. One end of L is connected to the 5' end of the first justice chain, and the other end of L is connected to the 5' end of the second justice chain.

[0277] The first target RNA or the first target gene is PCSK9, and the second target RNA or the second target gene is LPA;

[0278] The first antisense chain contains the following sequence (5'-3'): a*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a (SEQ ID NO:2);

[0279] The second antisense strand contains the following sequence (5'-3'): a*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g (SEQ ID NO:4) (a: 2'-OMe adenine nucleoside; u: 2'-OMe uracil nucleoside; c: 2'-OMe cytosine nucleoside; g: 2'-OMe guanine nucleoside; Af: 2'-F adenine nucleoside; Uf: 2'-F uracil nucleoside; Cf: 2'-F cytosine nucleoside; Gf: 2'-F guanine nucleoside; (dT): thymine deoxynucleoside; (dC): cytosine deoxynucleoside; *: thiophosphate bond).

[0280] Implementation 2. The compound according to Implementation 1, wherein the first antisense chain comprises the following sequence (5'-3'): CfGfaa*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a (SEQ ID NO:2).

[0281] Implementation 3. The compound according to Implementation 1 or 2, wherein the second antisense chain comprises the following sequence (5'-3'): CfGfaa*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g (SEQ ID NO:4).

[0282] Embodiment 4. The compound according to any one of Embodiments 1-3, wherein the first antisense chain and / or the second antisense chain further includes a capping group at the 5' end, optionally said capping group being connected to the 5' end of the first 5' extension and / or the second 5' extension.

[0283] Implementation 5. The compound according to Implementation 4, wherein the first antisense chain comprises or has the following sequence (5'-3'): (invAB)*CfGfaa*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a (SEQ ID NO:2).

[0284] Implementation 6. The compound according to Implementation 4 or 5, wherein the second antisense chain comprises or has the following sequence (5'-3'): (invAB)*CfGfaa*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g (SEQ ID NO:4).

[0285] Embodiment 7. The compound according to any one of Embodiments 1-6, wherein the first sense chain comprises or has the following sequence (5'-3'): c*uagacCfuGfu(dT)uugcuuuu*g*u (SEQ ID NO:1).

[0286] Embodiment 8. The compound according to any one of Embodiments 1-7, wherein the second sense chain comprises or has the following sequence (5'-3'): a*augguaaUfgGfaCfagaguuau (SEQ ID NO:3).

[0287] Embodiment 9. The compound according to any one of Embodiments 1-8, wherein L is

[0288] Embodiment 10. The compound according to any one of Embodiments 1-9, further comprising one or more delivery systems, optionally each of the delivery systems being independently connected to DS1 or DS2; preferably, the compound comprises one delivery system. Embodiment 11. The compound according to Embodiment 10, wherein each of the delivery systems is independently connected to the 5' end of the first positive chain, the 3' end of the first positive chain, the 5' end of the second positive chain, or the 3' end of the second positive chain; preferably, the delivery system is connected to the 3' end of the second positive chain.

[0289] Embodiment 12. The compound according to Embodiment 10 or 11, wherein the delivery system is each independently a ligand, preferably the ligand alters the distribution, targeting or lifetime, more preferably the ligand provides enhanced affinity to a target, such as a molecule, cell or cell type, compartment, receptor, such as a cell or organ compartment, tissue, organ or body region, and more preferably the ligand causes the compound to be delivered to the target tissue and produce an RNA interference effect.

[0290] Embodiment 13. The compound according to any one of Embodiments 10-12, wherein each of the ligands is independently selected from GalNAc ligands, lipophilic ligands, or other ligands that target receptors to promote the endocytosis of the compound, such as TfR-targeting ligands, LDL-R-targeting ligands, or integrin-targeting ligands; optionally, each of the ligands is independently NAG37 or L96; preferably, the ligand is L96.

[0291] Embodiment 14. The compound according to any one of Embodiments 10-13, wherein the second positive chain comprises or has the following sequence (5'-3'): a*augguaaUfgGfaCfagaguuau[L96](SEQ ID NO:3).

[0292] Implementation Method 15. The compound according to any of the foregoing embodiments is selected from Z1-1 or a pharmaceutically acceptable salt thereof.

[0293] Embodiment 16. A pharmaceutical composition comprising a compound according to any of the foregoing embodiments, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0294] Implementation Method 17. A method for inhibiting the expression of a target gene in a subject in need, comprising administering to the subject a pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of Implementation Methods 1-15, or a pharmaceutical composition according to Implementation Method 16.

[0295] Implementation 18. A method of treating a disease or condition in a subject in need, comprising administering to the subject a pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of Implementations 1-15, or a pharmaceutical composition according to Implementation 16, wherein the disease or condition is optionally related to a first target RNA or a first target gene and / or a second target RNA or a second target gene.

[0296] Implementation Method 19. According to the method of Implementation Method 18, the disease or condition includes, but is not limited to, atherosclerotic cardiovascular diseases (e.g., coronary artery disease (CAD), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD)), calcific aortic stenosis (CAVS), familial hypercholesterolemia (FH), diabetes mellitus with CVD, chronic kidney disease (CKD) with cardiovascular disease, etc.

[0297] 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 according to the embodiments of the present invention without inventive effort are considered technical solutions of the present invention and fall within the protection scope of the present invention.

[0298] definition

[0299] Unless otherwise specified, the following terms and phrases used in this invention 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 trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0300] 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.

[0301] 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.

[0302] 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, or steps may be included.

[0303] 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.

[0304] Natural nucleotides are composed of natural bases, natural ribose, and phosphate. The natural nucleotides used in this article refer to adenine ribonucleotides, adenine deoxyribonucleotides, guanine ribonucleotides, guanine deoxyribonucleotides, cytosine ribonucleotides, cytosine deoxyribonucleotides, uracil 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.

[0305] The natural bases of RNA include A (adenine), G (guanine), C (cytosine), U (uracil), and T (thymine).

[0306] 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-arabinoseurerylcytosine), and decitabine (5-aza-2'-deoxycytidine). Examples of uracil analogues include, but are not limited to, 5-fluorouracil, pseudouracil, 5-bromouracil, 4-thiouracil, and 5-azidouracil.

[0307] 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 nucleoside bonds, including single-stranded RNA and double-stranded RNA. The natural nucleoside bonds are phosphodiester bonds. In some embodiments, the ribose, bases, and nucleoside bonds in RNA may be modified independently and optionally.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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, ligands (e.g., GalNAc ligands, such as L96 or NAG37), natural nucleotides, non-natural nucleotides (e.g., modified nucleotides, nucleotide analogs, inverted non-base deoxynucleotides, GNA, LNA, etc.), and end-capping groups (e.g., M06).

[0312] As used herein, the term "nucleoside bond" refers to a bond (e.g., a linking group) between two parts of an oligonucleotide disclosed herein (e.g., between two monomers), including bonds between nucleosides, between a nucleoside and a ligand, between a nucleoside and a capping group, and between a nucleoside and a baseless nucleoside disclosed herein.

[0313] In this application, the term "capping group" refers to a group that can selectively bind to the compound described in this application, said capping group being located on the sense strand, antisense strand, or both strands. The capping group may be attached to the 3' end, 5' end, or both ends of the sense strand, antisense strand, or both strands. In some embodiments, the capping group binds to the antisense strand, particularly the 5' end of the antisense strand. In some embodiments, the capping group binds to an oligonucleotide (e.g., the 5' end of the antisense strand) via an internucleotide link, and this internucleotide link may optionally be modified as described above. In some embodiments, the capping group is linked to a double-stranded oligonucleotide via a phosphosulfate ester. In some embodiments, the capping group is linked to a double-stranded oligonucleotide via a phosphodiester. In this application, to reduce or inhibit exonuclease degradation, the "capping group" may bind to the 5' end of the first and / or second antisense strand of the compound described in this application. In some embodiments, the capping group may reduce or inhibit the RNA interference effect of the oligonucleotide. In some embodiments, the capping group is cleaved from the oligonucleotide before providing an RNA interference effect. Examples of end capping groups include, but are not limited to, non-base nucleotides, inverted non-base nucleotides, and (M06).

[0314] 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, 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.

[0315] 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.

[0316] 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 the invention, 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 the invention, the two nucleic acid chains may have multiple regions forming complementary double helixes. In some embodiments of the invention, in DNA, adenine (A) always pairs with thymine (T), and in RNA, adenine (A) pairs with uracil (U); guanine (G) always pairs with cytosine (C). In some embodiments of the invention, 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 the invention, nucleotides containing hypoxanthine as their base may pair with nucleotide bases containing adenine, cytosine, or uracil. In some embodiments of the invention, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequence of the invention by nucleotides containing, for example, inosine (in this application, the capital "I" can represent a hypoxanthine base, inosine, or an inosine-containing nucleotide, depending on the context) (this replacement is abbreviated as I modification). In some embodiments of the present invention, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a GU swing base pair with the mRNA.

[0317] 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.

[0318] The term "mismatch" as used in this article includes, but is not limited to:

[0319] 1) Two opposing (independent natural or non-natural) nucleotides (other than AT, AU or GC) pairing;

[0320] 2) No hydrogen bonds are formed between two opposing (independent natural or non-natural) nucleotides;

[0321] 3) A base is missing between two opposing (independent natural or non-natural) nucleotides.

[0322] In some embodiments, mismatches include wobbly base pairing and Hoogstein base pairing.

[0323] 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. "Sufficiently 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 first targeting region contained in the first fragment provided herein is sufficiently complementary to a portion of the mRNA encoding the first target gene. In some embodiments, the first targeting region has at least 80%, 85%, 90%, or 95% complementarity to a portion of the mRNA encoding the first target gene. In some embodiments, the first targeting region has 100% complementarity (fully complementary) to a portion of the mRNA encoding the first target gene. In some embodiments, the second targeting region contained in the third fragment provided herein is sufficiently complementary to a portion of the mRNA encoding the second target gene. In some embodiments, the second targeting region has at least 80%, 85%, 90%, or 95% complementarity to a portion of the mRNA encoding the second target gene. In some embodiments, the second targeting region has 100% complementarity (fully complementary) to a portion of the mRNA encoding the second target gene.

[0324] As used herein, the term "modified nucleotide" refers to a nucleotide having modified nucleotide 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.

[0325] 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, unlocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, tricyclic DNA (tcDNA) modification, (S)-restricted ethyl bicyclic nucleic acid ((S)-cEt-BNA) modification, thiophosphate (PS) modification, dithiophosphate (PS2) modification, methylphosphonate (MP) modification, methoxypropylmethylphosphonate (MOP) modification, phosphoselenate modification, phosphodiselenate modification, phosphorylaminosulfate modification, phosphoryl amide salt modification, phosphoramidate modification, peptide nucleic acid (PNA) modification, 5'-(E)-vinyl phosphate ( Modifications include: VP modification, N6-methyladenosine (m6A) modification, 5-methylcytidine (m5C) modification, 3-methyluridine (m3U) modification, 5-methylureaside (m5U) modification, pseudoureaside modification, 2-thioureaside (s2U) modification, propynouracil (5-pU) modification, linking the 5' or 3' end of the nucleotide to an inverted abase-free nucleotide (invAB) modification, replacing the nucleotide with an inverted abase-free nucleotide (invAb) modification, replacing the nucleotide with 2,4-difluorotolyl ribonucleotide (rF) modification, replacing the nucleotide with (S)-glycerol nucleic acid modification, replacing the nucleotide with hypoxanthine nucleotide (I) modification, replacing the nucleotide base with xanthine, replacing the nucleotide base with 7-methylguanine, replacing the nucleotide base with 5,6-dihydrouracil, and linking the 5' or 3' end of the nucleotide to M06 (M06) modification, etc. In some embodiments, at least one nucleotide comprises one, two, three, or more modifications. In some embodiments, at least one nucleotide is unmodified. In some embodiments, at least one nucleotide comprises one modification. In some embodiments, at least one nucleotide comprises two modifications. In some embodiments, at least one nucleotide comprises three modifications. In some embodiments, all nucleotides are modified, and each nucleotide independently comprises one, two, or three modifications.

[0326] As used herein, in some embodiments, "G", "A", "C", "U", and "T" refer to guanine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, and thymine ribonucleotide, respectively. Exemplary structures are as follows:

[0327] 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" and "T" are connected at the 5' end 1 position of the chain.

[0328] When “G”, “A”, “C”, “U” and “T” are connected at the 3' end of the chain, the structure is as follows:

[0329] As used herein, the prefix "d" before a monomer (such as nucleotides A, U, C, G, and T) indicates that the monomer is 2'-deoxy modified. An example nucleotide structure with 2'-deoxy modification is shown below:

[0330] As used herein, the "f" label following a monomer (such as nucleotides A, U, C, G, and T) indicates that the monomer is 2'-deoxy-2'-fluorine modified (2'-F modified). An example nucleotide structure with 2'-F modification is shown below:

[0331] As used herein, the prefix "GNA-" before monomers (such as nucleotides A, U, C, G, and T) indicates that the monomer has been modified with ethylene glycol-based nucleic acids (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:

[0332] As used herein, lowercase letters (a, u, c, g, t, etc.) indicate that the nucleotide represented by the corresponding uppercase letters (A, U, C, G, and T, etc.) is modified with 2'-O-methyl (2'-OMe). An example nucleotide structure modified with 2'-OMe is shown below:

[0333] 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:

[0334] 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:

[0335] 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:

[0336] 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 a precursor It is bonded to a nucleotide.

[0337] As used in this article, Uhd represents 2'-O-C16 alkyl-modified uracil ribonucleotide:

[0338] As used in this article, marking "*" between monomers (such as A, U, C, G, and T) indicates that the two monomers are linked by a thiophosphate bond (i.e., a thiophosphate diester bond), meaning they are modified by thiophosphate (PS).

[0339] As used in this article, the absence of an asterisk (*) between monomers (such as A, U, C, G, and T) indicates that the two nucleotides are linked by a phosphate ester bond (i.e., a phosphodiester bond).

[0340] As used in this article, This indicates the chemical bond (such as a single bond) connecting the site to other groups.

[0341] For example, structural unit and When combined, they represent a structure as For example, structural unit When combined, the structure is represented as "ug*cucaac*u*(dT)".

[0342] Unless otherwise specified, the sequence of characters in this application is written in 5'-3' order, for example... The 5' end of a 2'-F modified cytosine ribonucleotide is connected to another structural unit (e.g., L or another nucleotide) marked with Connected at the point; The 3' end of a 2'-OMe modified uracil ribonucleotide is connected to another structural unit (e.g., L or another nucleotide) marked with The two sides are connected.

[0343] For example, The structure is The structure is The structure is The structure is The structure is The structure is The structure is The structure is

[0344] For example, “5'-A(dU)gCf*(dT)-3'” means that the sequence starts from the 5' end, with adenine ribonucleotide at position 1, uracil deoxyribonucleotide at position 2, guanine ribonucleotide modified with 2'-methoxy at position 3, cytosine ribonucleotide modified with 2'-fluorine at position 4, and thymine deoxyribonucleotide linked to position 4 by a phosphate thioester bond at position 5.

[0345] For example, the structure shown as “5'-(M06)*A(dT)gCf*(invAB)-3'” is as follows:

[0346] For example, the structure shown as “5'-u(PEG6)a*u-3'” is as follows:

[0347] For example, the structure shown in “5'-u(C12)a-3'” is as follows:

[0348] As used herein, “small interfering RNA,” “siRNA,” “RNAi agent,” or “iRNA agent” are used interchangeably to refer to a double-stranded RNA molecule that is long enough to trigger an interferon response and enter the RISC (RNAi-induced silencing complex) and short enough that it does not induce a harmful interferon response in human cells (e.g., siRNA agents or their cleavage products can downregulate target genes by, for example, inducing RNAi with respect to target RNA, wherein the target may include endogenous or pathogenic target RNA). In some embodiments of the invention, the siRNA is at least partially complementary to the coding sequence in the target gene expressed in the cell. In some embodiments of the invention, after delivery of the siRNA to cells expressing the gene, the siRNA is able to inhibit or block gene expression in vitro or in vivo. Typically, the 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 of the present invention, 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 of the present invention, the sense and antisense strands of the siRNA are completely complementary, with a length of 15-30 base pairs. In some embodiments of the present invention, the sense and antisense strands of the siRNA are completely complementary, with a length of 17, 18, 19, 20, 21, or 22 base pairs.

[0349] In some embodiments of the present invention, 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.

[0350] In some embodiments, the compounds disclosed herein (e.g., the first 5' extension and / or the second 5' extension) undergo enzyme-mediated cleavage (e.g., endonuclease) prior to mediating RNAi. In some embodiments, the cleavage is mediated by ribonuclease (RNase). In some embodiments, the cleavage is mediated by Dicer endonuclease. In some embodiments, the cleavage is specific cleavage.

[0351] As used herein, with respect to the cleavage of a compound (e.g., enzyme-mediated cleavage), the term "specific" or "specific" refers to controlled or selective cleavage at a specific location (i.e., between two desired nucleotides of the oligonucleotide chain undergoing specific cleavage). The products of specific cleavage may include a variety of cleavage products, wherein the desired cleavage product (i.e., the product obtained from cleavage at a specific or desired location) is the most predominant (i.e., the product with the highest yield). In some embodiments, the desired cleavage product constitutes at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% (molar or weight) of all cleavage products. In some embodiments, the amount of the desired cleavage product (molar or weight) is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of any other cleavage product. While not wishing to be bound by theory, specific cleavage can be based on the recognition by the cleavage-mediating enzyme of a specific sequence, characteristic, motif, or combination thereof of the compound undergoing cleavage (e.g., an oligonucleotide chain, specifically the first 5' extension and / or the second 5' extension). Without being bound by any theory, there is reason to expect that specific types and modifications of certain nucleotides at certain positions in the cleavage region (e.g., the first and / or second cleavage region) will provide the desired superior effect.

[0352] In some embodiments, the specific cleavage occurs at the 3'th nucleotide of the first 5' extension. In some embodiments, the specific cleavage occurs at the 3'th nucleotide of the second 5' extension. In some embodiments, the specific cleavage occurs at both the 3'th nucleotide of the first 5' extension and the 3'th nucleotide of the second 5' extension.

[0353] In some embodiments, the specific cleavage occurs between X2 and Y. In some embodiments, the specific cleavage occurs between X2' and Y'. In some embodiments, the specific cleavage occurs between X2 and Y and between X2' and Y'.

[0354] Unbound by any theoretical constraints, the compounds disclosed herein can be specifically cleaved between X2 and Y, and between X2' and Y', resulting in the departure of the first and second 5' extensions, thereby producing cleaved DS1 and cleaved DS2 products. The cleaved DS1 and / or cleaved DS2 products can mediate RNAi.

[0355] In some embodiments, the cleaved DS1 product has a blunt end at the 5' end of the first antisense strand, wherein the first base pair at the 5' end of the first antisense strand consists of X2 and a complementary nucleotide in the first sense strand. In some embodiments, the first base pair at the 5' end of the first antisense strand is an AU base pair.

[0356] In some embodiments, the cleaved DS2 product has a blunt end at the 5' end of the second antisense strand, wherein the first base pair at the 5' end of the second antisense strand consists of an X2' nucleotide and a complementary nucleotide in the second sense strand. In some embodiments, the first base pair at the 5' end of the second antisense strand is an AU base pair.

[0357] As used in this article, the structures of NAG37 monomer and L96 monomer are respectively

[0358] [NAG37] and [L96] represent its residues, respectively. For example, the sequence 5'-[NAG37]AfGfu*[L96]-3' structure is...

[0359] This paper also provides a delivery system D02, where (D02) represents its residues and (D02)* represents residues linked to nucleotides via thiophosphate bonds, and their structures are shown below.

[0360] As used herein, the term "carbocyclic group" or "carbocyclic" refers to a non-aromatic cyclic hydrocarbon group having 3 to 20 cyclic carbon atoms ("C3-20 carbocyclic group") and zero heteroatoms in a non-aromatic ring system. In some embodiments of the invention, the carbocyclic group has 3 to 18 cyclic carbon atoms ("C3-18 carbocyclic group"). In some embodiments of the invention, the carbocyclic group has 3 to 16 cyclic carbon atoms ("C3-16 carbocyclic group"). In some embodiments of the invention, the carbocyclic group has 3 to 12 cyclic carbon atoms ("C3-12 carbocyclic group"). In some embodiments of the invention, the carbocyclic group has 3 to 10 cyclic carbon atoms ("C3-10 carbocyclic group"). In some embodiments of the invention, the carbocyclic group has 3 to 8 cyclic carbon atoms ("C3-8 carbocyclic group"). In some embodiments of the invention, the carbocyclic group has 3 to 7 cyclic carbon atoms ("C3-7 carbocyclic group"). In some embodiments of the invention, the carbocyclic group has 3 to 6 cyclic carbon atoms (“C3-6 carbocyclic group”). In some embodiments of the invention, the carbocyclic group has 4 to 6 cyclic carbon atoms (“C4-6 carbocyclic group”). In some embodiments of the invention, the carbocyclic group has 5 to 6 cyclic carbon atoms (“C5-6 carbocyclic group”). In some embodiments of the invention, the carbocyclic group has 5 to 10 cyclic carbon atoms (“C5-10 carbocyclic group”). Some exemplary C3-6 carbocyclic groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Some exemplary C3-8 carbon cyclogroups include, but are not limited to, the aforementioned C3-6 carbon cyclogroups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Some exemplary C3-10 carbon cyclogroups include, but are not limited to, the aforementioned C3-8 carbon cyclogroups, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), bicyclo[6.1.0]non-4-enyl (C9), bicyclo[6.1.0]nonyl (C9), bicyclo[6.1.0]non-4-ynyl (C9), decahydronaphthyl (C10), spiro[4.5]decyl (C10), etc. As previously mentioned, in some embodiments of the present invention, the carbocyclic group is a monocyclic (“monocyclic carbocyclic”) or polycyclic (e.g., comprising fused rings, bridged rings or spirocyclic systems such as bicyclic systems (“bicyclic carbocyclic”) or tricyclic systems (“tricyclic carbocyclic”)) and may be saturated or may contain one or more carbon-carbon double or triple bonds."Carbocyclic group" also includes ring systems in which the carbocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the bonding point is on the carbocyclic ring, and in such cases, the number of carbons continues to indicate the number of carbons in the carbocyclic ring system. Unless otherwise stated, each instance of a carbocyclic group is independently unsubstituted ("unsubstituted carbocyclic group") or substituted with one or more substituents ("substituted carbocyclic group"). In some embodiments of the invention, the carbocyclic group is an unsubstituted C3-14 carbocyclic group. In some embodiments of the invention, the carbocyclic group is a substituted C3-14 carbocyclic group. In some embodiments of the invention, the carbocyclic group is an unsubstituted C5-16 carbocyclic group. In some embodiments of the invention, the carbocyclic group is a substituted C5-16 carbocyclic group. In some embodiments of the invention, "carbocyclic group" is a monocyclic saturated carbocyclic group ("C3-14 cycloalkyl group") having 3 to 14 ring carbon atoms. In some embodiments of the invention, the cycloalkyl group has 3 to 10 cyclic carbon atoms (“C3-10 cycloalkyl”). In some embodiments of the invention, the cycloalkyl group has 3 to 8 cyclic carbon atoms (“C3-8 cycloalkyl”). In some embodiments of the invention, the cycloalkyl group has 3 to 6 cyclic carbon atoms (“C3-6 cycloalkyl”). In some embodiments of the invention, the cycloalkyl group has 4 to 6 cyclic carbon atoms (“C4-6 cycloalkyl”). In some embodiments of the invention, the cycloalkyl group has 5 to 6 cyclic carbon atoms (“C5-6 cycloalkyl”). In some embodiments of the invention, the cycloalkyl group has 5 to 10 cyclic carbon atoms (“C5-10 cycloalkyl”). Some examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Some examples of C3-6 cycloalkyl groups include the above-described C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise stated, each example of a cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”). In some embodiments of the invention, the cycloalkyl group is an unsubstituted C3-14 cycloalkyl group. In some embodiments of the invention, the cycloalkyl group is a substituted C3-14 cycloalkyl group.

[0361] As used herein, the term "heterocyclic group" or "heterocycle" refers to a group comprising a 3- to 20-membered non-aromatic ring system having a ring carbon atom and 1 to 8 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("3- to 20-membered heterocyclic group"). When valence permits, in a heterocyclic group containing one or more nitrogen atoms, the linking point can be either a carbon or a nitrogen atom. Heterocyclic groups can be monocyclic ("monocyclic heterocyclic group") or polycyclic (e.g., fused ring, bridged ring, or spirocyclic systems, such as bicyclic ("bicyclic heterocyclic group"), tricyclic ("tricyclic heterocyclic group"), or tetracyclic ("tetracyclic heterocyclic group")), and can be saturated or may contain one or more carbon-carbon double or triple bonds. Polycyclic heterocyclic systems may contain one or more heteroatoms in one, two, or three rings. "Heterocyclic group" also includes ring systems in which the heterocyclic ring as defined above is fused with one or more carbocyclic groups, wherein the connection point is on the carbocyclic ring or the heterocyclic ring; or includes ring systems in which the heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the connection point is on the heterocyclic ring, and in such cases, the number of ring members continues to indicate the number of ring members in the heterocyclic ring system. Unless otherwise stated, each instance of a heterocyclic group is independently unsubstituted ("unsubstituted heterocyclic group") or substituted with one or more substituents ("substituted heterocyclic group"). In some embodiments of the invention, the heterocyclic group is an unsubstituted 3- to 20-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is a substituted 3- to 20-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is an unsubstituted 5- to 18-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is a substituted 5- to 18-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is an unsubstituted 5- to 16-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is a substituted 5- to 16-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is an unsubstituted 5- to 12-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is a substituted 5- to 12-membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is a 5- to 10-membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 10-membered heterocyclic group”). In some embodiments of the invention, the heterocyclic group is a 5- to 8-membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 8-membered heterocyclic group”). In some embodiments of the invention, the heterocyclic group is a 5- to 6-membered non-aromatic ring system having a cyclic carbon atom and 1 to 4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 6-membered heterocyclic group”). In some embodiments of the invention, the 5- to 6-membered heterocyclic group has 1 to 3 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments of the invention, the 5- to 6-membered heterocyclic group has 1 to 2 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur.In some embodiments of the present invention, the 5- to 6-membered heterocyclic group has one cyclic heteroatom selected from nitrogen, oxygen, and sulfur. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridinyl, oxetyl, and thioheptyleyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirbutyl, oxetyl, and thioheptyleyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolyl, dihydropyrrolyl, and pyrrolyl-2,5-diketone. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxopentyl, oxetyl-thiophentyl, and dithiophentyl. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, diazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thiaalkyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiaalkyl, and dialkyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazineyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptanyl, oxetaneheptyl, and thioheptanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptanyl, oxetaneheptyl, and thioheptanyl. Exemplary bicyclic heterocyclic groups include, but are not limited to, indololinyl, isoindololinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthidyl, decahydro-1,8-naphthidyl, octahydropyrrolo[3,2-b]pyrrole, indololinyl, phthalimide, naphthalimide, chromenyl, 1H-benzo[e][1,4]diazayl, 1,4,5,7-tetrahydropyranolo[3,4-b]pyrrole, 5 6-Dihydro-4H-furano[3,2-b]pyrrolithyl, 6,7-Dihydro-5H-furano[3,2-b]pyrrolithyl, 5,7-Dihydro-4H-thieno[2,3-c]pyrrolithyl, 2,3-Dihydro-1H-pyrroli[2,3-b]pyridyl, 2,3-Dihydrofuran[2,3-b]pyridyl, 4,5,6,7-Tetrahydro-1H-pyrroli[2,3-b]pyridyl, 4,5,6,7-Tetrahydrofuran[3,2-c]pyridyl, 4,5,6,7-Tetrahydrothieno[3,2-b]pyridyl, 1,2,3,4-Tetrahydro-1,6-naphthidyl, etc.

[0362] As used herein, the term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the ring array) having 0 heteroatoms and 6 to 14 ring carbon atoms provided in the aromatic ring system ("C6-14 aryl"). In some embodiments of the invention, the aryl group has 6 ring carbon atoms ("C6 aryl"; for example, phenyl). In some embodiments of the invention, the aryl group has 10 ring carbon atoms ("C10 aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments of the invention, the aryl group has 14 ring carbon atoms ("C14 aryl"; for example, anthracene). "Aryl" also includes ring systems in which the aryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the connecting point or group is on the aryl ring, and in such cases, the number of carbon atoms continues to indicate the number of carbon atoms in the aryl ring system. Unless otherwise stated, each instance of an aryl group is independently either unsubstituted (“unsubstituted aryl”) or substituted with one or more substituents (“substituted aryl”). In some embodiments of the invention, the aryl group is an unsubstituted C6-14 aryl group. In some embodiments of the invention, the aryl group is a substituted C6-14 aryl group.

[0363] The term "heteroaryl" refers to a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the ring array), having 1 to 4 cyclic heteroatoms and cyclic carbon atoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 14-membered heteroaryl"). When valence permits, in a heteroaryl containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom. A heteroaryl polycyclic ring system can contain one or more heteroatoms in one or two rings. "Heteroaryl" includes ring systems in which the heteroaryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the bonding point is on the heteroaryl ring, and in such cases, the number of ring members continues to indicate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups, wherein the connection point is on the aryl or heteroaryl ring, and in such cases, the number of ring members indicates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. A polycyclic heteroaryl (e.g., indolyl, quinolinyl, carbazolyl, etc.) in which one ring does not contain a heteroatom can have the connection point on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl). In some embodiments of the invention, the heteroaryl is a 5- to 10-membered aromatic ring system having 1 to 4 cyclic heteroatoms and cyclic carbon atoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In some embodiments of the invention, the heteroaryl group is a 5- to 8-membered aromatic ring system having 1 to 4 cyclic heteroatoms and cyclic carbon atoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 8-membered heteroaryl”). In some embodiments of the invention, the heteroaryl group is a 5- to 6-membered aromatic ring system having 1 to 4 cyclic heteroatoms and cyclic carbon atoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 6-membered heteroaryl”). In some embodiments of the invention, the 5- to 6-membered heteroaryl group has 1 to 3 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments of the invention, the 5- to 6-membered heteroaryl group has 1 to 2 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments of the invention, the 5- to 6-membered heteroaryl group has 1 cyclic heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise stated, each instance of a heteroaryl group is independently either unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In some embodiments of the invention, the heteroaryl group is an unsubstituted 5- to 14-membered heteroaryl group. In some embodiments of the invention, the heteroaryl group is a substituted 5- to 14-membered heteroaryl group. Exemplary 5-membered heteroaryl groups comprising one heteroatom include, but are not limited to, pyrroleyl, furanyl, and thiophenyl groups.Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, azole, isozolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, but are not limited to, triazolyl, diazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetraazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, azirheptatrienyl, oxaheptatrienyl, and thioheptatrienyl. Exemplary 5-6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzozozolyl, benzoisozolyl, benzodiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indoleyl, and purine. Exemplary 6-6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridyl, quinolinyl, isoquinolinyl, cenolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridine, dibenzofuranyl, carbazoleyl, acridineyl, phenothiazinyl, phenothiazinyl, and phenothiazinyl.

[0364] As used herein, unless explicitly stated otherwise, groups are optionally substituted. As used herein, the terms “optionally substituted” or “optionally substituted” refer to substituted or unsubstituted groups. In some embodiments of the invention, alkyl, alkenyl, ynyl, heteroalkyl, heteroalkenyl, heteroynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups are optionally substituted. "Optionally substituted" means a group that may or may not be substituted (e.g., a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted heteroalkenyl group, a substituted or unsubstituted heteroalkynyl group, a substituted or unsubstituted carbocyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group). Generally, the term "substituted" means that at least one hydrogen atom present on a group is replaced by a permitted substituent (e.g., a substituent that, after substitution, produces a stable compound), in a compound that, for example, will not spontaneously transform, for example, through rearrangement, cyclization, elimination, or other reactions. Unless otherwise stated, a “substituted” group has substituents at one or more substituted positions of that group, and when more than one position in any given structure is substituted, the substituents at each position are either the same or different. The intended term “substituted” includes substitution by all permissible substituents of an organic compound, and includes substitution by any substituent described herein that results in the formation of a stable compound. This disclosure contemplates any and all such combinations to obtain a stable compound. For the purposes of this disclosure,

[0365] Heteroatoms, such as nitrogen, may have hydrogen substituents and / or satisfy the valence of the heteroatom, resulting in the formation of any suitable substituents as described herein. This disclosure is not intended to be limited in any way by the exemplary substituents described herein.

[0366] In this invention, the "azide" in the sequence has the following structural units:

[0367] In this invention, the “amine-C6” in the sequence has the following structural units:

[0368] In this invention, the “BCN” in the sequence has the following structural units:

[0369] In this invention, "M01" in the sequence has the following structural units:

[0370] As used in this article, the connection direction of the listed linking groups is arbitrary when no connection direction is specified. For example, in RA-L-RB, the linking group L has a structure of -MW-. In this case, -MW- can connect RA and RB in the same direction as the reading order from left to right, thus forming RA-MW-RB, or connect RA and RB in the opposite direction to the reading order from left to right, thus forming RA-MW-RB.

[0371] 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.

[0372] 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.

[0373] As used herein, “subject” refers to any animal, such as a mammal or marsupial. Subjects of this invention 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.

[0374] 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.

[0375] 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.

[0376] The compositions of the present invention 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 the present invention, such as preservatives, antioxidants, and stabilizers. However, when added, such substances should not unduly interfere with the bioactivity of the components of the compositions of the present invention. The formulation may be sterilized, and if desired, 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. Attached Figure Description

[0377] Figure 1 shows the results of liver homogenization of the test substance ds99. The nucleobase sequences involved in Figure 1 are shown in SEQ ID NO:57-60.

[0378] Figure 2 shows the effect of the test substance on inhibiting the target protein in NHP.

[0379] Figure 3 shows the effect of the test substance on inhibiting the target protein in NHP.

[0380] Figure 4 shows the effect of the test substance on inhibiting the target protein in NHP.

[0381] Figure 5 shows the effect of the test substance on inhibiting the target protein in NHP.

[0382] Figure 6 shows the effect of the test substance on inhibiting target proteins in different tissues of mice.

[0383] Figure 7 shows the activation of PBMC cytokines by the test substance. Detailed Implementation

[0384] 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 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 the present invention.

[0385] The following examples are provided to illustrate the present invention and are intended to better understand it, but are not intended to limit the scope of the invention. Modifications or variations made to the elements of the invention without departing from its spirit and essence are all within the scope of the invention. Unless otherwise specified, the reagents, kits, and biological materials used in this invention are commercially available. Unless otherwise specified, the kits are used according to the kit instructions.

[0386] Example 1: Preparation of phosphorus amide monomer MO1

[0387] Step A: M01-1 (4 g, 14.217 mmol), M01-2 (2.02 g, 14.217 mmol), and triethylamine (3.952 mL, 28.434 mmol) were dissolved in methanol (20 mL) and stirred overnight (15 hours). After vacuum concentration, the solution was dissolved in ethyl acetate (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered to obtain M01-3, which was used directly in the next step.

[0388] Step B: Dissolve MO1-3 (5.36 g, 14.204 mmol) in dichloromethane (100 mL), add 4,5-dicyanimidazole (0.84 g, 7.102 mmol) and MO4-4 (4.28 g, 14.204 mmol), and stir at room temperature for 2 hours under nitrogen protection. Add saturated sodium bicarbonate (200 mL), and extract with dichloromethane (100 mL x 3). Wash the organic phase successively with water (100 mL x 3) and saturated brine (100 mL), dry with anhydrous sodium sulfate, filter, and concentrate to obtain the phosphorus amide monomer of MO1. 1 H NMR(400MHz, CDCl3)δ7.38(s,1H),3.88-3.77(m,3H),3.72-3.68(m,1H),3.65-3 .61(m,20H),3.60-3.51(m,4H),2.64(6.4Hz,2H),1.16(dd,J=6.8,4.8Hz,12H). 31P NMR (160MHz, CDCl3) δ 148.51.

[0389] Example 2: Synthesis of DO2 precursor (diisopropylaminophosphite-2-cyanoethylhexadecyl ester)

[0390] The synthesis method for preparing MO1 from MO1-3 is similar to that in Example 1.

[0391] 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).

[0392] 31 P NMR: (400MHz, CDCl3) δ 147.24.

[0393] Example 3: Synthesis of siRNA molecules

[0394] 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 25 min using 5-ethylthio-1H-tetrazole (ETT) as an activator. Phosphophosphate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazol-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for 10 min, or oxidized using a 0.05 M solution of iodine in pyridine / water (v / v = 9 / 1) for 4 min. All sequences were synthesized after the final removal of the DMT group.

[0395] 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 at 45°C for 20 hours in an ammonia / ethanol (v / v = 3 / 1) system. 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.

[0396] 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. Oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was 0.1 M ammonium acetate in 15% acetonitrile aqueous solution, and Buffer B consisted of 1.5 M sodium bromide + 0.1 M ammonium acetate in 15% acetonitrile aqueous solution. The target product was separated and desalted using a reversed-phase C18 column.

[0397] Amino-coupled click linker: An oligonucleotide carrying an amino group at the 5' end is added to a centrifuge tube and dissolved in sterile, enzyme-free water. A DMF solution of BCN-OSU active ester or azide-C4 carboxylic acid active ester is added, along with 50 equivalents of DIPEA. The mixture is stirred overnight at room temperature, purified by ion exchange column chromatography, then purified by reversed-phase liquid chromatography, desalted, and lyophilized to obtain the oligonucleotide fragment suitable for click linker formation.

[0398] Long single-chain oligonucleotides are formed by click chemistry. (1) SPAAC method: Oligonucleotide fragments linked with equivalent amounts of BCN-OSU active ester and azide-C4 carboxylic acid active ester are dissolved in sterile enzyme-free water, 1M HEPES solution is added, and the mixture is stirred at 40°C for 3 hours. The mixture is then purified by reversed-phase liquid chromatography, purified by ion exchange column, desalted, and lyophilized to obtain long single-chain oligonucleotides coupled by linkers. (2) CuAAC method: CuSO4 / THPTA solution is prepared. 15mM copper sulfate solution and 30mM THPTA solution are mixed and vortexed for 15 minutes. Oligonucleotide fragments linked with equivalent amounts of terminal alkyne oligonucleotide fragments and oligonucleotide fragments linked with azide-C4 carboxylic acid active ester are dissolved in sterile enzyme-free water, 1M HEPES solution is added, and the air is replaced with nitrogen. Then, freshly prepared CuSO4 / THPTA (1.5 equivalents / 3 equivalents) and sodium ascorbate solution (45mM, 9 equivalents) are quickly added, and the mixture is purged with nitrogen again and stirred overnight at room temperature. Copper ions were captured by adding 0.5 mol / L EDTA-Na solution, purified by reversed-phase liquid chromatography, purified by ion exchange column, desalted, and lyophilized to obtain long single-chain oligonucleotides coupled by linkers.

[0399] Annealing of single-stranded oligonucleotides to produce siRNA: The single-stranded oligonucleotides to be annealed are dissolved in sterile, enzyme-free water. Complementary strands are formed by combining equimolar amounts of three single-stranded oligonucleotide solutions. The annealing reaction system is set up as follows: the mixture is placed in a 95°C water bath for 2 minutes, cooled to room temperature, and freeze-dried to obtain the final product, siRNA.

[0400] The mass spectra of the detected compounds are shown in Table 87 below:

[0401] Table 87

[0402] Test Example 1: psiCHECK-2 plasmid transfection cell viability test

[0403] Research Objective

[0404] The purpose of this study was to evaluate the in vitro inhibitory activity of the compound against LPA.

[0405] Materials and Methods

[0406] 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.

[0407] Test methods

[0408] Day 0, psiCHECK-2 plasmid transfection

[0409] 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).

[0410] Day 1, siRNA transfection

[0411] 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.

[0412] Detection of dual fluorescence (firefly and sea kidney)

[0413] 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.

[0414] Data Analysis

[0415] Detection ratio = (Sample luminescence value of sea buckthorn - background luminescence value of sea buckthorn) / (Sample firefly luminescence value of firefly - background firefly luminescence value of firefly)

[0416] Inhibition % = (1 - Sample ratio / RNAiMAX control ratio) × 100%

[0417] Test Example 2: Huh7 Cell Viability Assay

[0418] Research Objective

[0419] The purpose of this experiment was to evaluate the in vitro inhibitory activity of the test substance against the target genes PCSK9 & LPAmRNA in Huh-7 cells.

[0420] Materials and Methods

[0421] Material

[0422] Test compound

[0423] Test compound: Prepared as a 100 uM siRNA stock solution using RNase-free H2O.

[0424] cell lines

[0425] 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).

[0426] Main instruments

[0427] The main instruments used in this experiment included a fluorescence qPCR instrument (Roche 480), a centrifuge (ThermoFisher SORVALL ST4Plus), a cell counter (Countstar Rigel2), a PCR instrument (Dongshenglong ETC811), and a carbon dioxide incubator (ThermoFisher HERACELL240i).

[0428] Main reagents and consumables

[0429] The main reagents used in this experiment included Lipofectamine. TM RNAiMAX transfection reagent (INVITROGEN, catalog number 13778150), SuperReal PreMix Plus SYBR Green (Tiangen catalog number FP205), RNA extraction kit (Qiagen, catalog number 74182), FastKing RT Kit With gDNase (TianGen, catalog number KR116), 96-well plate (Costar 3799), qPCR-specific primers for GAPDH, and qPCR-specific primers for PCSK9 & ANGPTL3.

[0430] Experimental methods

[0431] 1. Compound transfection plate

[0432] Huh-7 cells (2 × 10⁴ cells / well) were seeded into 96-well cell culture plates. Simultaneously, siRNA was transfected into the cells using RNAiMAX. Seven siRNA concentrations were set up for assays (10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.0391 nM, 0.0098 nM, 0.0024 nM). Cells were incubated at 37°C in a 5% CO₂ incubator for 24 hours, with two replicates per well. A control group containing RNAiMAX and without the compound was also included.

[0433] 2. RNA extraction and reverse transcription

[0434] 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 (QIAGEN-74182). cDNA was synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116) according to the manufacturer's instructions.

[0435] 3qPCR detection of target gene mRNA expression levels

[0436] The target cDNA will be detected using SYBR Green qPCR, while GAPDH cDNA will be detected as an internal control for parallel testing. The cDNA was first diluted 2.5-fold with RNase-free H2O, and 7.5 μL of the prepared PCR reaction solution and 2.5 μL of the sample cDNA were added to each of 384 wells. The qPCR program was as follows: heat at 50°C for 2 min, heat at 95°C for 10 min, then enter cycling mode, heat at 95°C for 15 sec, followed by 60°C for 1 min, for a total of 40 cycles.

[0437] Data Analysis

[0438] 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.

[0439] The calculation formula is as follows:

[0440] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0441] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0442] Relative expression levels of target genes PCSK9 and LPA = 2 - ΔΔCT

[0443] PCSK9 & LPA inhibition rate % = (1 - value of sample / Ave. value of RNAiMAX Control) * 100

[0444] GraphPad Prism software is used for graphing and analysis.

[0445] The results of Test Examples 1 and 2 are shown in Tables E2-1, E2-2, E2-3, E2-4, E2-5, and E2-6. (Note: The inhibitory activity against PCSK9 was detected using the method in Test Example 2, and the inhibitory activity against LPA was detected using the method in Test Example 1.)

[0446] Table E2-1 Cell viability test results

[0447] Table E2-2 Cell viability test results

[0448] Table E2-3 Cell viability test results

[0449] Table E2-4 Cell viability test results

[0450] Table E2-5 Cell Viability Test Results

[0451] Table E2-6 Cell Viability Test Results

[0452] in conclusion:

[0453] The compounds of this invention exhibit significant inhibitory activity against both the first and second target genes.

[0454] Test Example 3: PHH Cell Viability Assay

[0455] Test compound: Prepared into a stock solution of the corresponding concentration using Nuclease-Free Water.

[0456] Cell lines:

[0457] PHH cells were provided by WuXi AppTec Co., Ltd. in Shanghai or Chengdu. PHH cells were cultured in InvitroGRO CP medium containing 10% fetal bovine serum.

[0458] Main instruments:

[0459] The main instruments used in this experiment included a fluorescence qPCR instrument (Quanstudio 7flex), a centrifuge (Beckman Allegra-X15RCentrifuge), and a cell counter (Countstar Rigel S2).

[0460] Main reagents and consumables:

[0461] The main reagents and consumables used in this experiment included: RNA extraction kit (Qiagen-74182), FastKing cDNA first-strand synthesis kit (TIANGEN-KR116-02), and FastStart Universal kit. Probe Master (Roche-04914058001), AceQ Universal U Probe Master Mix V2 (Vazyme-Q513-02), FasStart Universal SYBR Green Master (Roche-4913914001) and 96-well plate (Costar 3599).

[0462] The target gene PCSK9 and the internal reference gene primers and probes were purchased from Thermo Fisher. Other reagents and consumables were provided by WuXi AppTec.

[0463] Experimental methods:

[0464] Compound free uptake cladding

[0465] Dilute the test compound to a final concentration of 10 times with Nuclease-Free Water (e.g., if the final concentration is 25 nM, dilute to 250 nM).

[0466] PHH cells (5.4 × 10⁴ cells / well) were seeded into collagen-coated 96-well cell culture plates, and different concentrations of siRNA were added to the corresponding wells during plate formation. A compound-free control group containing nuclease-free water was also included.

[0467] RNA extraction and reverse transcription

[0468] After 48 hours of free uptake, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using the RNA Extraction Kit (Qiagen-74182) according to the kit instructions. cDNA was synthesized using the FastKing cDNA First-Strand Synthesis Kit (TIANGEN-KR116-02) according to the kit instructions.

[0469] qPCR detection of target gene mRNA expression levels

[0470] The target cDNA will be detected by qPCR, with parallel detection of the corresponding internal reference gene (such as GAPDH cDNA or β-actin cDNA). qPCR reaction program (TapMan Probe): Heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, then at 60°C for 1 minute, for a total of 40 cycles. qPCR reaction program (SYBR Green): Heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, then at 60°C for 1 minute, for a total of 40 cycles.

[0471] Data Analysis

[0472] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT quantification method. The relative expression level of the target gene was expressed using 2-ΔΔCT.

[0473] The calculation formula is as follows:

[0474] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0475] ΔCT = ΔCT (drug-treated group) - ΔCT (Nuclease-Free Water control group)

[0476] Relative expression level of target gene = 2 - ΔΔCT

[0477] Target gene inhibition rate % = (1 - value of sample / Nuclease-Free Water Control) * 100

[0478] The GraphPad Prism software was used for graphical analysis, and the inhibition rate results are expressed as mean ± SD.

[0479] Test results: see Tables E3-1, E3-2, E3-3 and E3-4.

[0480] Table E3-1 Results of PCSK9 activity assay in PHH cells

[0481] Table E3-2 Results of PCSK9 Activity Assay in PHH Cells

[0482] Conclusion: The compounds of this invention exhibit significant inhibitory activity against both the first and second target genes. In particular, compounds with two GalNAc delivery systems show even higher inhibitory activity under free uptake conditions.

[0483] Table E3-3 Results of PCSK9 / ANGPTL3 Activity Assay in Cells

[0484] Table E3-4 Results of PCSK9 / LPA Activity Assay in PHH Cells

[0485] Conclusion: The compounds of this invention exhibit significant inhibitory activity against a variety of first and second target genes.

[0486] Test Example 4: Cell activity assay targeting different genes

[0487] Experimental Objective

[0488] Using Hep3B and Huh7 cell lines, the in vitro activity of siRNA molecules was evaluated by assessing the degree of silencing of target genes by various candidate molecules.

[0489] Hep3B and Huh7 cells were provided by Nanjing Kebai Biotechnology Co., Ltd.

[0490] 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).

[0491] 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).

[0492] Main instruments

[0493] 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).

[0494] Main reagents and consumables

[0495] The main reagents used in this experiment included Lipofectamine. TM iRNAiMAX 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.

[0496] Experimental methods

[0497] 1. Compound transfection plate

[0498] Refer to the table below to seed the corresponding cells (1.5 × 10⁻⁶). 4Cells were plated into 96-well cell culture plates, and siRNA was transfected into the cells using RNAiMAX. Seven concentration points were set for the siRNA assay (dosing range is shown in Table 88 below). The cells were incubated overnight at 37°C in a 5% CO2 incubator for 24 hours. A compound-free control group containing RNAiMAX was also set up.

[0499] Table 88

[0500] 2. RNA extraction and reverse transcription

[0501] 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.

[0502] 3. qPCR detection of target gene mRNA expression levels

[0503] 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.

[0504] Primer information is shown in Table 89 below:

[0505] Table 89

[0506] Data Analysis

[0507] 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.

[0508] The calculation formula is as follows:

[0509] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0510] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0511] Relative expression level of target gene = 2 - ΔΔCT

[0512] 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)%.

[0513] Experimental results: IC50 of each candidate molecule on the target gene 50 As shown in Tables E4-1, E4-2, E4-3, E4-4, E4-5, E4-6, E4-7 and E4-8 below.

[0514] Table E4-1 Cell viability test results

[0515] Table E4-2 Cell viability test results

[0516] Table E4-3 Cell viability test results

[0517] Table E4-4 Cell Viability Test Results

[0518] Table E4-5 Cell Viability Test Results

[0519] Table E4-6 Cell Viability Test Results

[0520] Table E4-7 Cell Viability Test Results

[0521] Table E4-8 Cell Viability Test Results

[0522] Experimental conclusion: This invention has broad sequence and target applicability, and shows significant inhibitory activity against a variety of first and second target genes.

[0523] Test Example 5: ARPE-19 and G401 Cell Viability Assay

[0524] Experimental Objective

[0525] Using ARPE-19 and G401 cell lines, the in vitro activity of siRNA molecules was evaluated by assessing the degree of silencing of target genes by various candidate molecules.

[0526] G401 cells were provided by Nanjing Kebai Biotechnology Co., Ltd.

[0527] ARPE-19 cells were provided by Shanghai Fuheng Biotechnology Co., Ltd.

[0528] G401 cells were cultured in McCoy's 5A medium (Gibco catalog number 16600-082) containing 10% fetal bovine serum (FBS, Gibco catalog number 10099-141C).

[0529] ARPE-19 cells were cultured in DMEM medium (Gibco catalog number 11965-092) containing 10% fetal bovine serum (FBS, Gibco catalog number 2279804CP).

[0530] Main instruments

[0531] 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).

[0532] Main reagents and consumables

[0533] The main reagents used in this experiment included Lipofectamine. TM iRNAiMAX 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.

[0534] Primer information is shown in Table 90:

[0535] Table 90

[0536] Experimental methods

[0537] 1. Compound transfection plate

[0538] 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 transfected into the cells using RNAiMAX. Seven concentration points were set for the siRNA assay (dosing range is shown in Table 91 below). The cells were incubated overnight at 37°C in a 5% CO2 incubator for 24 hours. A compound-free control group containing RNAiMAX was also set up.

[0539] Table 91

[0540] 2. RNA extraction and reverse transcription

[0541] 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.

[0542] 3. qPCR detection of target gene mRNA expression levels

[0543] 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.

[0544] Data Analysis

[0545] 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 calculated using a 2-1T / T ratio. -ΔΔCT express.

[0546] The calculation formula is as follows:

[0547] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0548] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0549] relative expression level of target gene = 2 -ΔΔCT

[0550] 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)%.

[0551] Experimental results: IC50 of each candidate molecule on the target gene 50 See Tables E5-1 and E5-2 below.

[0552] Table E5-1 Cell viability test results

[0553] Table E5-2 Cell Viability Test Results

[0554] Experimental conclusion: This invention has broad sequence and target applicability, and shows significant inhibitory activity against a variety of first and second target genes.

[0555] Test Example 6: Response to Rat Liver Homogenization

[0556] 1.00 mg of the test substance ds99 was dissolved in 0.961 mL of water to obtain a working solution with a corrected concentration of 1,000,000 ng / mL. The working solution was mixed with 20% rat liver homogenate and incubated at 37 °C for 48 h to obtain a final sample with a concentration of 10,000 ng / mL. The cultured sample was subjected to liquid-liquid extraction with 50.0 μL of working extraction buffer (phenol / chloroform / isoamyl alcohol = 25 / 24 / 1, v / v / v). After centrifugation, 300 μL of the supernatant was used for solid-phase extraction. The sample was then transferred to an equilibrated solid-phase extraction 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)

[0557] Test substance ds99:

[0558] SS(5'→3'):g*u*caucCfaCfAfAfugagagUfacu[L96](SEQ ID NO:90)

[0559] AS(5'→3'):(M06)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a(SEQ ID NO:57)

[0560] The results are shown in Figure 1. After 48 h of incubation, the observed products included undigested test substance (31%), test substance with one nucleotide cleavage at the 3' end (15%), test substance with two nucleotide cleavages at the 3' end and M06 cleavage (18%), test substance with three nucleotide cleavages at the 5' end (15% + 5% + 10%), and other small amounts of metabolites (5%).

[0561] Therefore, (1) the only 5′ cleavage product is the product of the cleavage between the 3rd and 4th nucleotides at the 5' end of the test substance; (2) the 3' cleavage products include the test substance with one or two nucleotides cleaved at the 3' end; (3) among all cleavage products, the product of the cleavage between the 3rd and 4th nucleotides at the 5' end is the major product.

[0562] Test Example 7: In vivo efficacy study in non-human primates (NHP)

[0563] Dosage regimen: On the day of administration, the animals were weighed, and the prepared formulation was drawn according to their weight and dosage volume. The selected administration site (back) was disinfected, followed by administration of Z8-1 (dosage: 6 mg / kg) and Z10-1 (dosage: 6 mg / kg). Hemostasis was achieved using dry cotton balls after administration. On the seventh day after administration, blood was collected from the animals. The blood collection site was disinfected with alcohol swabs before collection. Hemostasis was achieved using dry cotton balls after collection. The collected whole blood was sent to the clinical laboratory for centrifugation to obtain serum. The levels of ANGPTL3 and PCSK9 proteins were measured in the serum.

[0564] (I) Detection of ANGPTL3 protein levels in cynomolgus monkey serum

[0565] reagents

[0566] Human ANGPTL3 ELISAKit, Brand - Cat No.: R&D-DANL30.

[0567] instrument

[0568] Microplate reader (Molecular Devices-SpectraMax 3841, Molecular Devices-SpectraMax M5, Molecular Devices-SpectraMax M2e), centrifuge eppendorf 5910R.

[0569] Experimental steps

[0570] Reagent preparation

[0571] (1) 1× Washing Buffer

[0572] Add 10 mL of 25× wash buffer to 240 mL of deionized water to prepare 250 mL of 1× wash buffer.

[0573] (2) 1× dilution buffer RD6Q:

[0574] Add 5 mL of 5× dilution buffer to 20 mL of deionized water to prepare 25 mL of 1× dilution buffer RD6Q.

[0575] (3) Colorimetric solution:

[0576] Mix equal amounts of color developer A and B together 15 minutes before use, taking care not to contaminate the color developer and keeping it away from light.

[0577] Prepare samples

[0578] Thaw the sample at room temperature, centrifuge at 2000 rpm for 2 minutes, and dilute the sample by a certain factor during the test.

[0579] Preparation of standard curves

[0580] Add 1 mL of deionized water to the lyophilized standard and briefly vortex to completely dissolve and mix. This is the standard stock solution (100 ng / mL). Let it stand for 15 minutes before use. Add 100 μL of the standard stock solution (100 ng / mL) to 900 μL of 1× dilution buffer RD6Q and mix to obtain 1000 μL of the highest concentration standard (10 ng / mL). Using 500 μL of 1× dilution buffer RD6Q as the diluent, perform six consecutive 2-fold dilutions of the 10 ng / mL highest concentration standard in six separate test tubes: after mixing the 10 ng / mL highest concentration standard, transfer 500 μL of the dilution to the next test tube, and so on. The 1-fold dilution buffer is used as a blank standard (0 ng / mL).

[0581] Experimental Procedure

[0582] (1) Before testing, take out an appropriate amount of ELISA test plate according to the number of samples and bring it to room temperature;

[0583] (2) Add 100 μL of diluent RD1-76 to each test well;

[0584] (3) Add 50 μL of standard, reference, or sample to each well, and seal the plate with sealing film.

[0585] Incubate at room temperature on a microplate shaker for 2 hours;

[0586] (4) Add 300 μL of 1× washing buffer to each well, then pour out the liquid and pat dry on a paper towel. Repeat this step to wash the plate 4 times.

[0587] (5) Add 200 μL of detection antibody working solution to each well. Seal the plate with sealing film and incubate on a microplate shaker at room temperature for 1 hour;

[0588] (6) Remove the liquid from the hole and repeat the washing operation in step (4);

[0589] (7) Add 200 μL of colorimetric solution (equal volumes of colorimetric solutions A and B) to each well and mix gently. Incubate at room temperature in the dark for 30 minutes;

[0590] (8) Add 50 μL of stop solution to each well, gently tap the plate to ensure it is fully mixed, and read the absorbance of the entire plate at 450 nm wavelength within 30 minutes.

[0591] Data Analysis

[0592] Calculate the average absorbance of each standard and sample, subtract the average standard optical density of the blank control, and obtain the standard curve equation using the four-parameter curve fitting (4PL) method. Substitute the OD value of the detection well into the standard curve equation to calculate the corresponding ANGPTL3 content. Finally, multiply the value by the dilution factor to obtain the ANGPTL3 content in the sample.

[0593] (II) Detection of PCSK9 protein levels in cynomolgus monkey serum

[0594] reagents

[0595] Human PCSK9 ELISAKit(Sino Biological-KIT10594)

[0596] instrument

[0597] Microplate reader (Molecular Devices-SpectraMax 3841, Molecular Devices-SpectraMax M5, Molecular Devices-SpectraMax M2e), centrifuge (Eppendorf-5724R)

[0598] Experimental steps

[0599] Reagent preparation

[0600] (1) 1× Washing Buffer

[0601] Add 10 mL of 20× wash buffer to 190 mL of deionized water to prepare 200 mL of 1× wash buffer.

[0602] (2) 1× dilution buffer:

[0603] Add 2.5 mL of 20× dilution buffer to 47.5 mL of deionized water to prepare 50 mL of 1× dilution buffer.

[0604] (3) Antibody detection:

[0605] Dilute with 1× dilution buffer at a ratio of 1:660. Prepare 10 minutes before use and use as needed.

[0606] (4) Colorimetric solution:

[0607] Developing solutions A and B should be mixed in equal amounts within 10 minutes before use. Take care not to contaminate the developing solutions and keep them away from light.

[0608] Prepare samples

[0609] Thaw the sample at room temperature, centrifuge at 2000 rpm for 2 minutes, and dilute the sample by a certain factor during the test.

[0610] To prepare the standard curve, add 1 mL of 1× dilution buffer to the lyophilized standard, let it stand for 10 minutes, and then briefly vortex to completely dissolve and mix. This is the standard stock solution (610,000 pg / mL). Add 49 μL of the standard stock solution to 951 μL of 1× dilution buffer and mix to obtain 1000 μL of the highest concentration standard of 30,000 pg / mL. Using 500 μL of 1× dilution buffer as a diluent, perform six consecutive 2-fold dilutions of the 30,000 pg / mL highest concentration standard in six separate test tubes: after mixing the 30,000 pg / mL highest concentration standard, transfer 500 μL of the dilution to the next test tube, and so on. The 1-fold dilution buffer is used as a blank standard (0 pg / mL).

[0611] Experimental Procedure

[0612] (1) Before testing, take out an appropriate amount of ELISA test plate according to the number of samples, bring it to room temperature, add 300 μL of 1× washing buffer to each test well, let it stand for about 2 minutes, then pour out the washing buffer and pat it dry on a paper towel. Repeat this step twice.

[0613] (2) Add 100 μL of standard, test sample and control sample to each well, seal the plate with sealing film and incubate at room temperature for 2 hours;

[0614] (3) Add 300 μL of 1× washing buffer to each well, let stand for about 2 minutes, then pour out the liquid and pat dry on a paper towel. Repeat this step to wash the plate 3 times.

[0615] (4) Add 100 μL of detection antibody working solution to each well, mix gently, seal the plate with sealing film and incubate at room temperature for 1 hour;

[0616] (5) Remove the liquid from the hole and repeat the washing operation in step (3);

[0617] (6) Add 100 μL of colorimetric solution (equal volumes of colorimetric solutions A and B) to each well and mix gently. Incubate at room temperature in the dark for 25 minutes;

[0618] (7) Add 100 μL of stop solution to each well, gently tap the plate to ensure it is fully mixed, and read the absorbance of the entire plate at a wavelength of 450 nm within 10 minutes.

[0619] Data Analysis

[0620] Calculate the average absorbance of each standard and sample, subtract the average standard optical density of the blank control, and obtain the standard curve equation using the four-parameter curve fitting (4PL) method. Substitute the OD value of the detection well into the standard curve equation to calculate the corresponding PCSK9 content. Finally, multiply the value by the dilution factor to obtain the PCSK9 content in the sample.

[0621] Experimental results: See Figure 2.

[0622] Experimental conclusion: The compound of this invention can significantly downregulate the target protein on NHP.

[0623] Test Example 8: In vivo efficacy test of NHP

[0624] Dosage regimen: On the day of administration, the animals were weighed, and the prepared formulation was drawn according to their weight and dosage volume. The selected administration site (back) was disinfected, followed by administration of Z1-1 (dosage: 6 mg / kg) and Z2-1 (dosage: 6 mg / kg). Hemostasis was achieved using dry cotton balls after administration. Blood was collected from the animals on day 7 after administration. The blood collection site was disinfected with alcohol swabs before collection. Hemostasis was achieved using dry cotton balls after collection. The collected whole blood was sent to the clinical laboratory for centrifugation to obtain serum. Serum was used to detect blood lipids and PCSK9 protein levels.

[0625] After centrifugation, serum samples were 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 met operational requirements, the sample was placed on the sample tray, the animal tattoo number on the sample label was verified, and lipid (LPa) testing began.

[0626] (I) Detection of PCSK9 protein levels in cynomolgus monkey serum

[0627] reagents

[0628] Human PCSK9 ELISA Kit (Sino Biological-KIT10594).

[0629] instrument

[0630] Microplate reader (Molecular Devices-SpectraMax 3841, Molecular Devices-SpectraMax M5, Molecular Devices-SpectraMax M2e), centrifuge eppendorf 5724R.

[0631] Experimental steps

[0632] Reagent preparation

[0633] (1) 1× Washing Buffer

[0634] Add 10 mL of 20× wash buffer to 190 mL of deionized water to prepare 200 mL of 1× wash buffer.

[0635] (2) 1× dilution buffer:

[0636] Add 2.5 mL of 20× dilution buffer to 47.5 mL of deionized water to prepare 50 mL of 1× dilution buffer.

[0637] (3) Antibody detection:

[0638] Dilute with 1× dilution buffer at a ratio of 1:660. Prepare 10 minutes before use and use as needed.

[0639] (4) Colorimetric solution:

[0640] Developing solutions A and B should be mixed in equal amounts within 10 minutes before use. Take care not to contaminate the developing solutions and keep them away from light.

[0641] Prepare samples

[0642] Thaw the sample at room temperature, centrifuge at 2000 rpm for 2 minutes, and dilute the sample by a certain factor during the test.

[0643] Preparation of standard curves

[0644] Add 1 mL of 1× dilution buffer to the lyophilized standard, let stand for 10 minutes, and then briefly vortex to completely dissolve and mix. This is the standard stock solution (610,000 pg / mL). Add 49 μL of the standard stock solution to 951 μL of 1× dilution buffer and mix to obtain 1000 μL of the highest concentration standard of 30,000 pg / mL. Using 500 μL of 1× dilution buffer as a diluent, perform six consecutive 2-fold dilutions of the 30,000 pg / mL highest concentration standard in six separate test tubes: after mixing the 30,000 pg / mL highest concentration standard, transfer 500 μL of the dilution to the next test tube, and so on. The 1× dilution buffer is used as a blank standard (0 pg / mL).

[0645] Experimental Procedure

[0646] (1) Before testing, take out an appropriate amount of ELISA test plate according to the number of samples, bring it to room temperature, add 300 μL of 1× washing buffer to each test well, let it stand for about 2 minutes, then pour out the washing buffer and pat it dry on a paper towel. Repeat this step twice.

[0647] (2) Add 100 μL of standard, test sample and control sample to each well, seal the plate with sealing film and incubate at room temperature for 2 hours;

[0648] (3) Add 300 μL of 1× washing buffer to each well, let stand for about 2 minutes, then pour out the liquid and pat dry on a paper towel. Repeat this step to wash the plate 3 times.

[0649] (4) Add 100 μL of detection antibody working solution to each well, mix gently, seal the plate with sealing film and incubate at room temperature for 1 hour;

[0650] (5) Remove the liquid from the hole and repeat the washing operation in step (3);

[0651] (6) Add 100 μL of colorimetric solution (equal volumes of colorimetric solutions A and B) to each well and mix gently. Incubate at room temperature in the dark for 25 minutes;

[0652] (7) Add 100 μL of stop solution to each well, gently tap the plate to ensure it is fully mixed, and read the absorbance of the entire plate at a wavelength of 450 nm within 10 minutes.

[0653] Data Analysis

[0654] Calculate the average absorbance of each standard and sample, subtract the average standard optical density of the blank control, and obtain the standard curve equation using the four-parameter curve fitting (4PL) method. Substitute the OD value of the detection well into the standard curve equation to calculate the corresponding PCSK9 content. Finally, multiply the value by the dilution factor to obtain the PCSK9 content in the sample.

[0655] Experimental results: See Figure 3.

[0656] Experimental conclusion: The compound of this invention can significantly downregulate the target protein on NHP.

[0657] Test Example 9: Long-term plasma target gene knockout model of NHP

[0658] The in vivo target gene knockout effect of modified siRNA was evaluated in cynomolgus monkeys. On the day of administration, the animals were weighed, and the prepared formulation was drawn according to the weight and volume of the drug. The selected administration site (lower back) of the animals was disinfected. For the mixed administration group (N=2), Inclisiran and R1 were mixed and administered subcutaneously at the same site, with each drug in the mixture at a dose of 3 mg / kg, for a total dose of 6 mg / kg. For the other group (N=2), a single subcutaneous injection of 6 mg / kg of Z1-1 reagent was administered. Hemostasis was achieved using dry cotton balls after administration. Blood samples were collected before administration and on days 7, 14, 21, 28, 56, and 85 after administration. 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.

[0659] 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, and the animal tattoo number on the sample label was verified. The analysis of LPa, LDL-c, and ApoB was then initiated. Data are expressed as a percentage of baseline values ​​and presented as the mean plus / minus the standard error of the mean.

[0660] Following the manufacturer's experimental procedures, the circulating PCSK9 protein content in cynomolgus monkeys was quantified using an ELISA kit (Sino Biological-KIT10594) that is specific for human PCSK9 (and cross-reactive with cynomolgus monkeys). Data are expressed as a percentage of baseline values ​​and presented as the mean plus / minus the standard error of the mean.

[0661] R1:

[0662] Table 92

[0663] Experimental results: See Figure 4.

[0664] Experimental conclusion: The dual-targeting compound produced by this invention can inhibit the target on NHP and has significantly better persistence than single-target compound mixtures.

[0665] Test Example 10: Long-term plasma target gene knockout model of NHP

[0666] The in vivo target gene knockout effect of 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) of the animals was disinfected, and multiple groups of animals (N=4) were subcutaneously injected with different doses of Z1-1 reagent. Hemostasis was achieved using dry cotton balls after administration. Blood samples were collected before administration and on days 7, 14, 21, 28, 56, and 85 post-administration. 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.

[0667] 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, and the animal tattoo number on the sample label was verified. The analysis of LPa, LDL-c, and ApoB was then initiated. Data are expressed as a percentage of baseline values ​​and presented as the mean plus / minus the standard error of the mean.

[0668] Following the manufacturer's experimental procedures, the circulating PCSK9 protein content in cynomolgus monkeys was quantified using an ELISA kit (Sino Biological-KIT10594) that is specific for human PCSK9 (and cross-reactive with cynomolgus monkeys). Data are expressed as a percentage of baseline values ​​and presented as the mean plus / minus the standard error of the mean.

[0669] Experimental results: See Figure 5.

[0670] Experimental conclusion: The compound of this invention exhibits dose-dependent inhibitory effects in NHP and can significantly inhibit two targets simultaneously.

[0671] Test Example 11: Mouse IVT-induced ocular knockout model (mSOD-1+mTTR)

[0672] Male C57 / 6J mice, 6-8 weeks old; bilateral single intravitreal injection (IVT), optic cups harvested on day 7.

[0673] Experimental grouping and dosage design: Group 1 was PBS, Group 2 was a mixture of Z53 and Z54 (10 μg + 10 μg), and Group 3 was Z52 (20 μg), as shown in Table 93.

[0674] Samples were collected from both eyes: retina, RPE layer samples, and choroid / scleral complex. RPE layer samples or choroid / scleral complex samples from all four eyes were combined as a single sample for RNA extraction and qPCR experiments. The left eye retina was used as a separate sample for qPCR experiments. The right eye retina was treated with RNAlater and then cryopreserved. qPCR experiments were conducted; the target gene (mouse SOD1 + mouse TTR + internal control) was detected by qPCR. Ocular surface and fundus examinations were performed before and after drug administration to observe for any abnormalities. If abnormalities were found, OCT imaging was performed (to observe inflammatory responses).

[0675] Table 93

[0676] Total RNA extraction (pipe tips and centrifuge tubes are all enzyme-free and aseptically packaged; used in a laminar flow hood):

[0677] a. Quickly transfer the frozen retinal / RPE cell / choroid + scleral tissue samples to a centrifuge tube containing 1 mL of Trizol lysis buffer and homogenization beads, and rapidly homogenize by vortexing on a homogenizer. Insoluble matter will still remain after tissue homogenization. Centrifuge at 12,000 × g for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube.

[0678] b. Incubate the lysate at room temperature for 5 minutes to allow complete separation of the nucleic acid-protein complex. Add 0.2 mL of chloroform to each 1 mL of Trizol, tighten the cap, shake vigorously for 15 seconds, and incubate at room temperature for 2-3 minutes.

[0679] c. Centrifuge at 12,000×g for 15 min at 4℃. The sample will separate into three layers: an orange-yellow lower organic phase, an intermediate layer, and a colorless upper aqueous phase. Transfer the upper aqueous phase containing total RNA to a new centrifuge tube, being careful not to aspirate the intermediate protein layer.

[0680] d. Add 0.5 mL of isopropanol to each 1 mL of Trizol used initially, invert several times to mix, and incubate at room temperature for 10 min. Centrifuge at 12,000 × g for 10 min at 4 °C, discard the supernatant, and a gel-like RNA precipitate will be visible.

[0681] e. Add 1 mL of 75% ethanol to each 1 mL of Trizol used initially, invert several times to mix, and wash the precipitate. Incubate at 4°C, 12,000 × g for 5 min, discard the supernatant, and air dry at room temperature for 5-10 min or vacuum dry. Add an appropriate amount (e.g., 25 μL) of Nuclease-free Water (DEPC-treated) or TE buffer, and pipette several times to dissolve the RNA.

[0682] f. Use Nanodrop 2000 to determine RNA concentration and purity. Use the obtained RNA immediately or aliquot and store at -80°C, avoiding repeated freeze-thaw cycles.

[0683] Reverse transcription:

[0684] a. Prepare the reaction system shown in Table 94 in RNase-free centrifuge tubes: Table 94

[0685] After gently mixing with a pipette, briefly centrifuge.

[0686] b. The reaction conditions are shown in Table 95 below: Table 95

[0687] After the reaction is complete, place the resulting cDNA on ice for subsequent experiments or freeze it for storage.

[0688] Quantitative real-time PCR:

[0689] a. Take 0.2 ml PCR tubes and prepare the reaction system as shown in Table 96 below. Prepare 3 tubes for each reverse transcription product. Table 96:

[0690] b. The reaction conditions are shown in Table 97 below: Table 97

[0691] Result analysis and processing: ΔΔCT method:

[0692] A = CT(target gene, sample to be tested) - CT(internal standard gene, sample to be tested)

[0693] B = CT(target gene, control sample) - CT(internal standard gene, control sample)

[0694] K = AB

[0695] Expression multiple = 2 - K

[0696] Experimental results:

[0697] No abnormal reactions were observed during the experiment.

[0698] The results of target mRNAKD in various tissues (retina, RPE layer) are shown in Figure 6.

[0699] Experimental conclusion: The compound of this invention can significantly inhibit two extrahepatic targets simultaneously in vivo, and exhibits significant effects on different targets in various tissues in vivo, demonstrating broad applicability.

[0700] Test Example 12: PBMC Cytokine Activation Assay

[0701] Experimental materials:

[0702] Table 98

[0703] Experimental steps:

[0704] PBMC cell treatment:

[0705] 1) Fresh whole blood (10 mL) is drawn from healthy volunteers into a vacuum blood collection tube containing an EDTA anticoagulant tube. Immediately after the whole blood sample is collected, it should be slowly and repeatedly inverted to mix evenly to ensure full contact with the anticoagulant.

[0706] 2) Dilute the blood sample twice with the same volume of PBS. Add 10 mL of Lymphoprep (lymphocyte separation solution) to a 50 mL centrifuge tube. Slowly add 20 mL of the diluted blood sample onto the Lymphoprep, being careful not to disrupt the interface.

[0707] 3) After adding the sample, centrifuge the centrifuge tube at 1000×g for 25 minutes at room temperature without using the brake.

[0708] 4) Collect the intermediate white membrane layer containing PBMCs into a new 50mL centrifuge tube, wash twice with 40mL PBS, centrifuge at 350×g for 10min at room temperature. Resuspend the cells in complete culture medium to a density of 1.6×10^6 cells / mL.

[0709] 5) Spread PBMCs into 96-well plates at a density of 2E5 cells per well (125 μL).

[0710] 6) Add the diluted test drug Z1-1 (125 μL) to the 96-well plate according to the plate map, with 2 replicates for each well; the blank group is made by adding the same amount of RNase-free water medium and DMSO medium.

[0711] 7) After gently mixing the well plate, incubate it in an incubator for 18-20 hours.

[0712] 8) Centrifuge to collect the supernatant, and use ELISA to detect the secretion levels of IL-6, TNF-α, IL-1β, and IFN-γ.

[0713] 9) Analyze the data using GraphPad Prism 8 software.

[0714] Experimental results: See Figure 7.

[0715] Experimental conclusion: The compounds of this invention have a low risk of immunogenicity.

Claims

A compound capable of inhibiting the expression of a first target gene and a second target gene, having a structure according to Formula (I): in: DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, the first sense strand and the first antisense strand forming a first double-stranded portion of 15-27 nucleotide pairs in length, and a first 5' extension in the first antisense strand located upstream of the 5' of the first double-stranded portion. The first 5' extension is at least 3 nucleotides in length and can be cleaved at the 3' end nucleotide of the first 5' extension to obtain a cleaved DS1 product containing the first double-stranded portion. The cleaved DS1 product can silence a first target RNA or inhibit the expression of a first target gene through RNA interference. DS2 is a double-stranded oligonucleotide containing a second sense strand and a second antisense strand, which together form a second double-stranded portion of 15-27 nucleotide pairs in length. L is a structural unit that serves as a connector. One end of L is connected to the first positive chain, and the other end of L is connected to the second positive chain. The first target gene and the second target gene may be the same or different. The compound according to claim 1, wherein: The first antisense chain further includes a first 3' extension located downstream of the 3' of the first double-stranded portion. The compound according to claim 1 or 2, wherein: The first double-stranded portion is formed by base pairing of a first segment of the first antisense strand and a second segment of the first sense strand, wherein the first segment and the second segment are of the same length; and / or the second double-stranded portion is formed by base pairing of a fourth segment of the second antisense strand and a fifth segment of the second sense strand, wherein the fourth segment and the fifth segment are of the same length. The compound according to any of the preceding claims, wherein the lengths of the first double-chain portion and / or the second double-chain portion are independently: a) 15-25, 15-24, 15-23, 16-24, 16-23, 16-22, 16-21, 16-20, 17-23, 17-22, 17-21, 17-20, 18-23, 18-22, 18-21, 18-20, 19-23, 19-22, 19-21, or 19-20 nucleotide pairs; or b) 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotide pairs. The compound according to any of the preceding claims, wherein the lengths of the first and / or second sense chains are independently: a) 15-35, 16-35, 16-30, 16-27, 16-26, 16-25, 16-21, 17-35, 17-30, 17-25, 17-21, 18-35, 18-30, 18-25, 18-23, 18-21, 19-35, 19-30, 19-25, 19-21, 20-35, 20-30, 20-25, 20-23, 21-35, 21-30, 21-25, or 21-23 nucleotides; or b) 25, 24, 24, 23, 22, or 21 nucleotides. The compound according to any of the preceding claims, wherein: The length of the first 5' extension is at least 3, 4, 5, 6, 7 or more nucleotides, and / or the length of the first 3' extension is at least 1, 2 or more nucleotides. The compound according to any of the preceding claims, wherein: The first fragment contains a first target region that is fully complementary to the mRNA encoding the first target gene, and the fourth fragment contains a second target region that is fully complementary to the mRNA encoding the second target gene. The first target region and the second target region may be the same as or different from each other. The compound according to any of the preceding claims, wherein: The cleavage is enzymatic cleavage, optionally by specific cleavage of a nuclease, optionally by specific cleavage of a ribonuclease (RNase). The compound according to any of the preceding claims, wherein: The first antisense strand includes a first cleavage region, which contains the nucleotide sequence shown in Formula A-1. Equation A-1: ​​(3'-5')X2-YZ The cleavage occurs between X2 and Y, where X2 is the nucleotide at the 5' end of the first fragment, and Y and Z are the two nucleotides at the 3' end of the first 5' extension. The compound according to claim 9, wherein: The first cleavage region further comprises a nucleotide (N1), where N1 is the third nucleotide from the 3' end of the first 5' extension, and the first cleavage region comprises the nucleotide sequence shown in Formula B-1. Formula B-1:(3'-5')X2-YZ-N1. The compound according to claim 9, wherein: a) The first cleavage region further comprises a third fragment (N), the third fragment comprising at least one nucleotide, wherein the nucleotide at the 3' end of the third fragment is N1, and the first cleavage region comprises the nucleotide sequence shown in Formula B'-1. Equation B'-1:(3'-5')X2-YZN, The length of N is 1-10 nucleotides, preferably 1-5 nucleotides, and more preferably 1 nucleotide. The compound according to any of the preceding claims, wherein: a) the DS1 comprises a double-stranded oligonucleotide of Formula C-1, The first fragment and the second fragment form the first double-stranded portion through base pairing, and the first fragment and the second fragment are of the same length. The first 5' extension contains at least 3 nucleotides, the first antisense strand contains the first cleavage region, the first cleavage region contains the nucleotide at the 5' end of the first fragment (X2) and the two nucleotides at the 3' end of the first 5' extension (YZ), and the first cleavage region contains the nucleotide sequence shown in Formula A-1. Equation A-1: ​​(3'-5')X2-YZ The cleavage occurs between X2 and Y, as defined in Formula A-1 as in any of the preceding claims, wherein the length of the first sense strand is 15-35, 15-23, 15-22, or 15-21 nucleotides, the length of the first antisense strand is 25-35, 26-35, 26-30, 25-27, or 26-27 nucleotides, and the 3' or 5' end of the first sense strand is attached to one end of L; and / or b) the DS2 comprises a double-stranded oligonucleotide of Formula C-2, The fourth and fifth fragments form the second double-stranded portion through base pairing, and the fourth and fifth fragments are of the same length. The length of the second sense strand is 15-35, 15-23, 15-22, or 15-21 nucleotides, and the length of the second antisense strand is 15-35, 17-23, 17-22, 17-21, 19-35, 20-35, 19-30, 19-23, or 21-23 nucleotides. The 3' or 5' end of the second sense strand is connected to one end of L. The compound according to any of the preceding claims, wherein: a) the DS1 comprises a double-stranded oligonucleotide of Formula D-1, The first fragment and the second fragment form the first double-stranded portion through base pairing, and the first fragment and the second fragment are of the same length. The first 5' extension contains at least 3 nucleotides, and the first antisense strand contains a first cleavage region. The first cleavage region contains the nucleotide (X2) at the 5' end of the first fragment and the two nucleotides (YZ) at the 3' end of the first 5' extension. The first cleavage region contains the nucleotide sequence shown in Formula A-1. Equation A-1: ​​(3'-5')X2-YZ The cleavage occurs between X2 and Y, as defined in Formula A-1 as in any of the preceding claims, wherein the length of the first sense strand is 15-35, 15-23, 15-22, 15-21, 16-25, 17-23, 18-23, 19-23, 19-21, 20-23, 20-21, 21-23, 17, 18, 19, 20, 21, 22, or 23 nucleotides, and the length of the first antisense strand is 25-35, 25-30, 26-35, 26-30, 25-27, 26-27, 25, 26, 27, 28, 29, or 30 nucleotides, and the 3' or 5' end of the first sense strand is attached to one end of L; and b) the DS2 comprises a double-stranded oligonucleotide of Formula D-2: The fourth and fifth segments form the second double-stranded portion through base pairing, and the fourth and fifth segments are of the same length. The second 3' protrusion and the second 5' protrusion each independently contain 0, 1, 2, or 3 nucleotides. The length of the second sense strand is 15-35, 15-23, 15-22, 15-21, 16-25, 17-23, 18-23, 19-23, 19-21, 20-23, 20-21, or 21-23, 17, 18, 19, 20, 21, 22, or 23 nucleotides. The length of the second antisense strand is 15-35, 19-30, 21-35, 21-30, 19-23, 21-23, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. The 3' or 5' end of the second sense strand is connected to one end of L. The compound according to any of the preceding claims, wherein: The first justice chain and the second justice chain are not connected to the same end of L. The compound according to any one of claims 9-14, wherein Z is G, a natural analog of G, a non-natural analog of G, A, a natural analog of A, or a non-natural analog of A. The compound according to claim 15, wherein Z is G, a natural analog of G, or a non-natural analog of G. The compound according to any one of claims 9-16, wherein X2 and X2' are independently A, a natural analog of A, a non-natural analog of A, U, a natural analog of U, or a non-natural analog of U. The compound according to any one of claims 9-17, wherein the formula A-1 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 analogues thereof. The compound according to any one of claims 9-18, wherein Y is A, a natural analog of A, a non-natural analog of A, U, a natural analog of U, or a non-natural analog of U. The compound according to any one of claims 9-19, wherein the formula A-1 has a sequence (3'-5') selected from the following: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IUG, IAG, IUA and IAA. The compound according to any one of claims 9-20, wherein the first cleavage region comprises at least one modified nucleotide, preferably, all nucleotides in the first cleavage region are modified nucleotides. The compound of claim 21, wherein the modified nucleotide comprises a modified base, a modified glycoside, and / or a modified nucleoside bond. The compound according to any one of claims 9-22, wherein the first cleavage region is not sufficiently complementary to the mRNA encoding the first gene. The compound of any one of claims 9-23, wherein, The compound satisfies one or more of the following: (a) The first 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 first cleavage region are not thiophosphate bonds; (e) The first 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 internucleotide bond between Y and Z is a phosphodiester bond; (h) All internucleotide bonds in the first cleavage region are phosphodiester bonds; and (i) The nucleoside bond between the first cleavage region and the first fragment is a phosphodiester bond. The compound of any one of claims 9-24, wherein, The compound satisfies one or more of the following: (a) The first cleavage region contains at least one nucleotide selected from 2'-OMe modified nucleotides or 2'-F modified nucleotides; (b) Each nucleotide in the first cleavage region is a nucleotide modified with 2'-OMe or a nucleotide modified with 2'-F; (c) The first cleavage region contains at least one 2'-F modified nucleotide; (d) The first cleavage region contains no more than two 2'-F modified nucleotides; (e) Z in formula A-1, formula B-1 or formula B'-1 is a nucleotide modified by 2'-F, and optionally X2 in formula A-1, formula B-1 or formula B'-1 is a nucleotide modified by 2'-F; (f) N1 in formula B-1 or formula B-1 is a nucleotide modified with 2'-F; (g) In formula A-1, B-1, or B'-1, X2 and Y are both nucleotides modified with 2'-OMe, and Z is a nucleotide modified with 2'-F, and further, N1 in formula B-1 or B'-1 is a nucleotide modified with 2'-F; and (h) X2, Y and Z in formula A-1, formula B-1 or formula B'-1 are nucleotides modified with 2'-OMe, and further N1 in formula B-1 or formula B'-1 is a nucleotide modified with 2'-F. The compound according to any of the preceding claims, wherein: The DS1 or the first double-stranded portion of the DS1 further comprises at least one nucleoside inter-bond selected from thiophosphate bonds or methylphosphonate bonds, and / or the DS2 or the second double-stranded portion of the DS2 further comprises at least one nucleoside inter-bond selected from thiophosphate bonds or methylphosphonate bonds. The compound according to any of the preceding claims, wherein: (a) The first and / or second nucleotide internucleotide bond from the 5' end of the first and / or fourth segments is a thiophosphate bond or a methylphosphonate bond; and / or (b) The first and / or second internucleotide bond from the 3' end of the first and / or fourth segments is a thiophosphate bond or a methylphosphonate bond. The compound according to any of the preceding claims, wherein: The lengths of the first sense strand and / or the second sense strand are independently 17-23, 17-22, or 17-21 nucleotides, respectively; and the lengths of the first antisense strand and / or the second antisense strand are independently 19-28, 19-27, or 19-26 nucleotides, respectively. The compound according to any of the preceding claims, wherein: a) The lengths of the first sense strand and the first antisense strand are (a) 19 and 25 nucleotides, (b) 20 and 25 nucleotides, (c) 21 and 25 nucleotides, (d) 19 and 26 nucleotides, (e) 20 and 26 nucleotides, (f) 21 and 26 nucleotides, or (g) 21 and 27 nucleotides, respectively; and / or b) The lengths of the second sense strand and the second antisense strand are (a) 19 and 23 nucleotides, (b) 20 and 23 nucleotides, (c) 21 and 23 nucleotides, (d) 19 and 21 nucleotides, (e) 20 and 21 nucleotides, (f) 21 and 21 nucleotides, or (g) 20 and 22 nucleotides, respectively. The compound according to any of the preceding claims, wherein L is selected from bonds, degradable linkers or non-degradable linkers. The compound according to any of the preceding claims, wherein L is selected from DNA, RNA, functionalized monosaccharides, or oligosaccharides. The compound according to any of the preceding claims, wherein L is a non-degradable linker. The compound according to any of the preceding claims, wherein L has sufficient stability in vivo or in vitro. The compound according to any of the preceding claims, wherein L remains intact in a suitable environment for a period of time before the compound contacts the mRNA, for example, in plasma for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48 or 72 hours, with a breakage of no more than 5%, 10%, 20%, 30%, 40% or 50%. The compound according to any of the preceding claims, wherein the molar ratio of the cleaved DS1 product that acts on the mRNA encoding the first gene to the DS2 product that acts on the mRNA encoding the second gene is about 1 (e.g., about 0.8, 0.9, 1, 1.1 or 1.2). The compound according to any one of the preceding claims, wherein L is as indicated in formula (I-1') or formula (I-1), in: X is either O or S; Y represents a single bond, -O-, or -S-; L1is selected from the group consisting of a single bond, -0-, -S-, -S-S-, -(C=0)-, -NH-, -NH-(C=0)-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -0-CH2-, -S-CH2-, -0-CH2CH2-, -S-CH2CH2-, -CH2-0-CH2CH2-, -CH2CH2-0-CH2CH2-, -CH2CH2-0-CH2CH2-0-, -CH2-0-CH2-0-, -CH2CH2CH2-0-, -CH2CH2CH2-S-, -CH2-(C=0)-, -CH2-NH-(C=0)-, -0-(C=0)-NH-, -CH2-NH-, -C(=0)0-, -NHC(=0)0-, -NHC(=0)NH-, -OC(=0)0-, -OC(=0)NH-, -S(0)2NH-, -NHS(0)2-, and L2, L3, L4, L6, L7, L8, and L9 are independently selected from single bonds, -O-, -S-, -SS-, -(C=O)-, -NH-, -NH-(C=O)-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -O-CH2-, -S-CH2CH2-, -O-CH2CH2-, -S-CH2CH2-, -CH2-O-CH2CH2-, - CH2CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-O-, -CH2-O-CH2-O-, -CH2CH2CH2-O-, -CH2CH2CH2-S-, -CH2-(C=O)-, -CH2-NH-(C =O)-, -O-(C=O)-NH-, -C(=O)O-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)O-, -OC(=O)NH-, -S(O)2NH-, -NHS(O)2-, and -CH2-NH-; L5 is selected from a single bond, -O-, -S-, -S-S-, -(C=O)-, -NH-, -NH-(C=O)-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -O-CH2-, -S-CH2-, -O-CH2CH2-, -S-CH2CH2-, -CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-, -CH2CH2-O-CH2CH2-O-, -CH2-O-CH2-O-, -CH2CH2CH2-O-, -CH2CH2CH2-S-, -CH2-(C=O)-, -CH2-NH-(C=O)-, -O-(C=O)-NH-, -CH2-NH-, -C(=O)O- The second justice chain is connected to the 3' or 5' end. The compound according to any of the preceding claims, wherein the L is as shown in formula (I-2') or formula (I-2), in: Ring A is absent and L6 is directly connected to triazole; Alternatively, ring A can be selected from 5-16 membered heterocycles. According to the compound of claim 37, the L comprises 1, 2 or 3 dT, and optionally, the L further comprises the structure shown in the foregoing formula (I-1') or formula (I-1) or formula (I-2') or formula (I-2). The compound according to any one of claims 1-36, wherein L is as shown in (I-3), (I-4), or (I-5): The compound according to any one of claims 1-36, wherein the L is as shown in formula (I-6), (I-7) or (I-8): The compound according to claim 40, wherein: Structural unit Selected from the arbitrarily replaced locations The compound according to any one of claims 1-36, wherein the L is as shown in formula (I-9), (I-10), (I-11), (I-12), (I-13), (I-14), (I-15), (I-16), (I-17) or (I-18), The compound according to any of the preceding claims, wherein... The L is selected from According to any of the preceding claims, in the compound, the 5' or 3' end nucleotide of the first sense strand is connected to the 5' or 3' end nucleotide of the second sense strand via an L link, preferably, the 5' end nucleotide of the first sense strand is connected to the 5' end nucleotide of the second sense strand via an L link, or preferably, the 5' end nucleotide of the first sense strand is connected to the 3' end nucleotide of the second sense strand via an L link. The compound according to any one of claims 1-43, wherein one end of L is connected to a nucleotide at position 6 from the 5' end of the first sense strand, and the other end of L is connected to the 5' end of the second sense strand. The compound according to any of the preceding claims, wherein at least one nucleotide is a modified nucleotide, preferably all nucleotides are modified nucleotides, the modification comprising one, two or more of the following: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, C16 modification, DO2 modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAB modification, invAb modification, and modification enhancing the affinity of double-stranded ribonucleic acid for ARGO protein; optionally, the modification comprises one, two or more of the following: 2'-OMe modification, 2'-F modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAB modification, and modification enhancing the affinity of double-stranded ribonucleic acid for ARGO protein; further optionally, the modification comprises one, two or more of the following: 2'-OMe modification, 2'-F modification, VP modification, PS modification, and invAB modification. The compound according to any of the preceding claims, wherein the first antisense chain further includes a capping group at the 5' end, optionally said capping group being connected to the 5' end of the first 5' extension. The compound of claim 47, wherein the end-capping group comprises an inverted non-base deoxynucleotide or MO6. The compound according to claim 47 or 48, wherein the capping group is connected to the 5' end of the first 5' extension via a nucleoside interbond, the nucleoside interbond optionally being modified or unmodified. The compound according to claim 49, wherein the capping group is connected to the 5' end of the first 5' extension via a thiophosphate bond. The compound according to any of the preceding claims further comprises one or more (e.g., one, two, three, four or more) delivery systems, optionally each of which is independently connected to DS1, DS2 or L. According to the compound of claim 51, wherein the delivery system is each independently a ligand, preferably the ligand alters the distribution, targeting, or lifetime, more preferably the ligand provides enhanced affinity to a target, such as a molecule, cell or cell type, compartment, receptor, such as a cell or organ compartment, tissue, organ, or body region, and more preferably the ligand causes the compound to be delivered to the target tissue and produce an RNA interference effect. The compound according to claim 51 or 52, wherein each of the ligands is independently selected from GalNAc ligands, lipophilic ligands, or other ligands that target receptors to promote the endocytosis of the compound, such as TfR-targeting ligands, LDL-R-targeting ligands, or integrin-targeting ligands. The compound according to any one of claims 51-53, wherein the ligand is a GalNAc ligand and the number is at least 1; further, the number of GalNAc ligands is 1 or 2. The compound according to claim 54 has one GalNAc ligand. The compound according to claim 54 has two GalNAc ligands. The compound according to claim 54, wherein: a) There are two GalNAc ligands, each linked to the 3' end nucleotide of the first and second sense strands, respectively; or b) There is one GalNAc ligand, linked to the 3' end nucleotide of either the first or second sense strand; or c) There are two GalNAc ligands, one linked to the 3' end nucleotide of the first sense strand and the other linked to the 5' end nucleotide of the second sense strand. The compound according to any one of claims 53-57, wherein the GalNAc ligand is independently L96 or NAG37; further, a) the number of GalNAc ligands is two, one of which is L96 and linked to the 3' end nucleotide of the first sense strand, and the other is NAG37 and linked to the 5' end nucleotide of the second sense strand, or b) the number of GalNAc ligands is two and both are L96, or c) the GalNAc ligand is one L96 and linked to the 3' end nucleotide of the first sense strand or the second sense strand. According to any of the preceding claims, the first target RNA or the first target gene is the same as the second target RNA or the second target gene; or the first target RNA or the first target gene and the second target RNA or the second target gene are different segments of the same RNA or the same gene; or the first target RNA or the first target gene and the second target RNA or the second target gene are different RNAs or different genes. The compound according to any of the preceding claims, wherein the compound is any one of the compounds shown in Tables 1-85 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A method for inhibiting the expression of a target gene in a subject in need, comprising administering to the subject a pharmaceutically effective amount of a compound according to any one of claims 1-60 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 61. A method of treating a disease or condition in a subject in need, comprising administering to the subject a pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-60, or a pharmaceutical composition according to claim 61, wherein the disease or condition is optionally related to a first gene and / or a second gene. The use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-60, or the pharmaceutical composition according to claim 61, in the preparation of an agent for inhibiting the expression of a target gene, or in the preparation of a medicament for treating a disease or condition; optionally, the disease or condition is related to a first gene and / or a second gene. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-60, used to inhibit the expression of a target gene, or for the treatment of a disease or condition, or a pharmaceutical composition according to claim 61; optionally, the disease or condition is related to a first gene and / or a second gene.

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