Poly-oligonucleotide capable of triggering gene silencing

Abstract

Disclosed are a poly-oligonucleotide capable of triggering gene silencing and a use thereof, and specifically disclosed are a compound represented by formula (I) and the use thereof.

Description

Polymeric oligonucleotides capable of triggering gene silencing TECHNICAL FIELD

[0001] The present application relates to a class of polymeric oligonucleotides capable of triggering gene silencing and their use. BACKGROUND

[0002] RNA interference (RNAi) technology refers to a highly conserved phenomenon in the evolutionary process, which is induced by double-stranded RNA (dsRNA) and is the efficient and specific degradation of homologous mRNA. RNA molecules inhibit the expression of certain genes by destroying specific mRNA. Because the use of RNAi technology can specifically eliminate or close the expression of specific genes, this technology has rapidly become one of the most concerned research tools in the field of gene function research and gene therapy research, and has been widely used in the field of exploring gene function, metabolic diseases and infectious diseases and cancer treatment.

[0003] However, inhibition by a single target sometimes cannot achieve the desired therapeutic effect, and it is necessary to simultaneously regulate two or more pathways in the same cell. In this case, the practice of administering two siRNA drugs targeting the same patient usually fails to achieve the desired therapeutic effect. This may be due to the influence of PK, which causes the drug concentrations of the two siRNA drugs in the same cell to be different, and cannot achieve effective simultaneous inhibition of two targets in the same cell to achieve synergistic effect.

[0004] One of the solutions to the above problems is to develop chemically coupled double-siRNA trigger siRNA drug molecules. The general method of implementation is to connect two siRNA triggers through a linker. When entering the cell, the change in the cell environment (pH) or the enzyme ability (hydrolysis, oxidation reduction, etc.) is used to realize the separation of the two triggers, and then the effect is achieved.

[0005] Double-target and multi-target RNAi technology can simultaneously target multiple genes, and has a wider range of applications, and can also show great potential in clinical treatment. SUMMARY

[0006] The present application provides a compound represented by formula (I),

[0007] wherein:

[0008] DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, which form a first double-stranded portion of 15-27 nucleotides in length, and a first 5' extension in the first antisense strand upstream of the first double-stranded portion, which is at least 3 nucleotides in length and cleavable at the 3' most nucleotide of the first 5' extension to yield a cleaved DS1 product comprising the first double-stranded portion, which is capable of silencing a first target RNA or inhibiting expression of a first target gene by RNA interference;

[0009] DS2 is a double-stranded oligonucleotide comprising a second sense strand and a second antisense strand, which form a second double-stranded portion of 15-27 nucleotides in length, and a second 5' extension in the second antisense strand upstream of the second double-stranded portion, which is at least 3 nucleotides in length and cleavable at the 3' most nucleotide of the second 5' extension to yield a cleaved DS2 product comprising the second double-stranded portion, which is capable of silencing a second target RNA or inhibiting expression of a second target gene by RNA interference;

[0010] L is a linker, one end of L is linked to the first sense strand, and the other end of L is linked to the second sense strand;

[0011] The first target RNA or first target gene is the same as or different from the second target RNA or second target gene.

[0012] In some embodiments of the present application, L is a linker, one end of L is linked to the 5' end or 3' end of the first sense strand, and the other end of L is linked to the 5' end or 3' end of the second sense strand. In some embodiments, L is a linker, one end of L is linked to the 5' end of the first sense strand, and the other end of L is linked to the 5' end of the second sense strand. In some embodiments, L is a linker, one end of L is linked to the 3' end of the first sense strand, and the other end of L is linked to the 3' end of the second sense strand. In some embodiments, L is a linker, one end of L is linked to the 3' end of the first sense strand, and the other end of L is linked to the 5' end of the second sense strand. In some embodiments, L is a linker, one end of L is linked to the 5' end of the first sense strand, and the other end of L is linked to the 3' end of the second sense strand.

[0013] In some embodiments of the present application, one end of L is linked to the 5' end of the first sense strand, the other end of L is linked to the 5' end of the second sense strand, and the second sense strand comprises the second 5' extension.

[0014] In some embodiments of the application, the first antisense strand further comprises a first 3' extension downstream of the 3' end of the first double-stranded portion.

[0015] In some embodiments of the application, the second antisense strand further comprises a second 3' extension downstream of the 3' end of the second double-stranded portion.

[0016] In some embodiments of the application, the first antisense strand further comprises a first 3' extension downstream of the 3' end of the first double-stranded portion, and the second antisense strand further comprises a second 3' extension downstream of the 3' end of the second double-stranded portion.

[0017] In some embodiments of the application, 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, and the first segment and the second segment have 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, and the fourth segment and the fifth segment have the same length.

[0018] In some embodiments of the application, the first double-stranded portion and / or the second double-stranded portion has a length independently selected from the group consisting of:

[0019] 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.

[0020] In some embodiments of the application, the first double-stranded portion and / or the second double-stranded portion has a length independently selected from the group consisting of: 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide pairs.

[0021] In some embodiments of the application, the first sense strand and / or the second sense strand has a length independently selected from the group consisting of: 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.

[0022] In some embodiments of the application, the first sense strand and / or the second sense strand is / are independently 25, 24, 24, 23, 22, or 21 nucleotides in length.

[0023] In some embodiments of the application, the first 5' extension and / or the second 5' extension is / are independently at least 3, 4, 5, 6, 7, or more nucleotides in length.

[0024] In some embodiments of the application, the first 3' extension and / or the second 3' extension is / are independently at least 1, 2, or more nucleotides in length.

[0025] In some embodiments of the application, the first 5' extension and / or the second 5' extension is / are independently at least 3, 4, 5, 6, 7, or more nucleotides in length, and the first 3' extension and / or the second 3' extension is / are independently at least 1, 2, or more nucleotides in length.

[0026] In some embodiments of the application, the first segment comprises a first targeting region, the first targeting region is sufficiently complementary to the first target RNA or a target mRNA encoding the first target gene, the fourth segment comprises a second targeting region, the second targeting region is sufficiently complementary to the second target RNA or a target mRNA encoding the second target gene, and the first targeting region and the second targeting region are the same or different.

[0027] In some embodiments of the application, the cleavage is an enzyme-catalyzed cleavage, optionally a specific cleavage by an endonuclease, optionally a specific cleavage by a ribonuclease (RNase).

[0028] In some embodiments of the application, the compound, wherein:

[0029] a) the first antisense strand comprises a first cleavage region, the first cleavage region comprises a nucleotide sequence represented by Formula A-1,

[0030] Formula A-1: (3'-5') X2-Y-Z,

[0031] the cleavage occurs between X2and Y, X2is the 5'-most nucleotide of the first segment, Y and Z are the 3'-most two nucleotides of the first 5' extension; and

[0032] b) the second antisense strand comprises a second cleavage region, the second cleavage region comprises a nucleotide sequence represented by Formula A-2,

[0033] Formula A-2: (3'-5') X2'-Y'-Z',

[0034] 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.

[0035] In some embodiments of the present invention, the above-described compound, wherein:

[0036] 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.

[0037] Formula B-1:(3'-5')X2-YZ-N1,

[0038] The above-mentioned splitting occurs between X2 and Y; and

[0039] 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.

[0040] Formula B-2: (3'-5')X2'-Y'-Z'-N1',

[0041] The above-mentioned splitting occurs between X2' and Y'.

[0042] In some embodiments of the present invention, the above-described compound, wherein:

[0043] 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.

[0044] Equation B'-1:(3'-5')X2-YZN,

[0045] 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

[0046] 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.

[0047] Formula B'-2:(3'-5')X2'-Y'-Z'-N',

[0048] 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.

[0049] In some embodiments of the application, the above-mentioned compound, wherein:

[0050] a) the above-mentioned DS1 comprises a double-stranded oligonucleotide of formula C-1,

[0051] C-1,

[0052] the above-mentioned first segment and the above-mentioned second segment form the above-mentioned first double-stranded portion by base pairing, and the above-mentioned first segment and the above-mentioned second segment are of the same length, the above-mentioned first 5' extension comprises at least 3 nucleotides, the above-mentioned first antisense strand comprises the above-mentioned first cleavage region, the above-mentioned first cleavage region comprises the nucleotide (X2) of the 5'-most end of the above-mentioned first segment and the two 3'-most nucleotides (Y-Z) of the above-mentioned first 5' extension, the above-mentioned first cleavage region comprises a nucleotide sequence of formula A-1,

[0053] A-1: (3'-5') X2-Y-Z,

[0054] the above-mentioned cleavage occurs between X2 and Y, formula A-1 is as defined in any one of the preceding claims, the above-mentioned first sense strand has a length of 15-35, 15-23, 15-22 or 15-21 nucleotides, the above-mentioned first antisense strand has a length of 25-35, 26-35, 26-30, 25-27 or 26-27 nucleotides, and the 3' or 5' end of the above-mentioned first sense strand is linked to one end of L; and / or

[0055] b) the above-mentioned DS2 comprises a double-stranded oligonucleotide of formula C-2,

[0056] C-2,

[0057] the above-mentioned fourth segment and the above-mentioned fifth segment form the above-mentioned second double-stranded portion by base pairing, and the above-mentioned fourth segment and the above-mentioned fifth segment are of the same length, the above-mentioned second 5' extension comprises at least 3 nucleotides, the above-mentioned second antisense strand comprises the above-mentioned second cleavage region, the above-mentioned second cleavage region comprises the nucleotide (X2') of the 5'-most end of the fourth segment and the two 3'-most nucleotides (Y'-Z') of the above-mentioned second 5' extension, the above-mentioned second cleavage region comprises a nucleotide sequence of formula A-2,

[0058] A-2: (3'-5') X2'-Y'-Z',

[0059] the cleavage occurs between X2' and Y', formula A-2 is as defined in any of the preceding claims, the second sense strand has a length of 15 to 35, 15 to 23, 15 to 22, or 15 to 21 nucleotides, the second antisense strand has a length of 25 to 35, 26 to 35, 26 to 30, 25 to 27, or 26 to 27 nucleotides, and the 3' or 5' end of the second sense strand is linked to one end of L.

[0060] In some embodiments of the application, the compound is as defined above, wherein:

[0061] a) DS1 comprises a double-stranded oligonucleotide of formula D-1,

[0062] D-1,

[0063] the first and second fragments form the first double-stranded portion by base pairing, and the first and second fragments have the same length, the first 5' extension comprises at least 3 nucleotides, the first antisense strand comprises a first cleavage region, the first cleavage region comprises the nucleotide (X2) at the 5' most end of the first fragment and the two nucleotides (Y-Z) at the 3' most end of the first 5' extension, and the first cleavage region comprises a nucleotide sequence of formula A-1:

[0064] A-1: (3'-5') X2-Y-Z,

[0065] the cleavage occurs between X2 and Y, formula A-1 is as defined in any of the preceding claims, the first sense strand has a length of 15 to 35, 15 to 23, 15 to 22, 15 to 21, 16 to 25, 17 to 23, 18 to 23, 19 to 23, 19 to 21, 20 to 23, 20 to 21, 21 to 23, 17, 18, 19, 20, 21, 22, or 23 nucleotides, the first antisense strand has a length of 25 to 35, 25 to 30, 26 to 35, 26 to 30, 25 to 27, 26 to 27, 25, 26, 27, 28, 29, or 30 nucleotides, and the 3' or 5' end of the first sense strand is linked to one end of L;

[0066] b) DS2 comprises a double-stranded oligonucleotide of formula D-2:

[0067] D-2,

[0068] the fourth segment and the fifth segment form the second double-stranded portion by base pairing, and the fourth segment and the fifth segment are identical in length, the second 5' extension comprises at least 3 nucleotides, the second antisense strand comprises the second cleavage region, the second cleavage region comprises the nucleotide (X2') at the 5' most end of the fourth segment and the two nucleotides (Y'-Z') at the 3' most end of the second 5' extension, the second cleavage region comprises a nucleotide sequence represented by Formula A-2,

[0069] Formula A-2: (3'-5') X2'-Y'-Z',

[0070] the cleavage occurs between X2' and Y', Formula A-2 is as defined in any of the preceding claims, 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 in length, 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 in length, and the 3' or 5' end of the second sense strand is linked to one end of L.

[0071] In some embodiments of the application, the first sense strand and the second sense strand are not linked to the same end of L.

[0072] In some embodiments of the application, Z and Z' are each 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.

[0073] In some embodiments of the application, Z and Z' are each independently G, a natural analog of G, or a non-natural analog of G.

[0074] In some embodiments of the application, X2 and X2' are each 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.

[0075] In some embodiments of the application, Formula A-1 and Formula A-2 are each independently of sequence (3'-5') selected from the group consisting of UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, and AGA, or a natural or non-natural analog thereof.

[0076] In some embodiments of the application, Y and Y' above are each 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.

[0077] In some embodiments of the application, Formula A-1 and Formula A-2 above are each independently have a sequence (3'-5') selected from the group consisting of: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, IUG, IAG, IUA, and IAA.

[0078] In some embodiments of the application, Formula B-1 and Formula B-2 above are each independently have a sequence (3'-5') selected from the group consisting of: 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 a natural or non-natural analog thereof.

[0079] In some embodiments of the application, Formula B-1 and Formula B-2 above are each independently have a sequence (3'-5') selected from the group consisting of: UUGC, UAGC, AUGC, AAGC, AAGG, UUAC, UAAC, AUAC, and AAAC.

[0080] In some embodiments of the application, the first cleavage region and the second cleavage region above each independently comprise at least one modified nucleotide, preferably all of the nucleotides of the first cleavage region and / or the second cleavage region above are modified nucleotides. In some embodiments of the application, Formula A-1, Formula A-2, Formula B-1, and Formula B-2 above each independently comprise at least one modified nucleotide, preferably all of the nucleotides of Formula A-1, Formula A-2, Formula B-1, and Formula B-2 above are modified nucleotides.

[0081] In some embodiments of the application, the modified nucleotide above comprises a modified base, a modified sugar, and / or a modified internucleoside linkage.

[0082] In some embodiments of the application, the first cleavage region above is not sufficiently complementary to the first target RNA or a target mRNA encoding the first target gene, and / or the second cleavage region above is not sufficiently complementary to the second target RNA or a target mRNA encoding the second target gene.

[0083] In some embodiments of the application, the compound above, wherein:

[0084] (a) the first cleavage region above comprises at least one internucleoside linkage that is not a phosphorothioate linkage;

[0085] (b) the internucleoside linkage between X2and Y is not a phosphorothioate linkage;

[0086] (c) the internucleoside linkage between Y and Z is not a phosphorothioate linkage;

[0087] (d) all of the internucleoside linkages in the first cleavage region described above are not phosphorothioate linkages;

[0088] (e) the first cleavage region described above comprises at least one internucleoside linkage that is a phosphodiester linkage;

[0089] (f) the internucleoside linkage between X2and Y is a phosphodiester linkage;

[0090] (g) the internucleoside linkage between Y and Z is a phosphodiester linkage;

[0091] (h) all of the internucleoside linkages in the first cleavage region described above are phosphodiester linkages;

[0092] (i) the internucleoside linkage between the first cleavage region described above and the first segment described above is a phosphodiester linkage;

[0093] (a') the second cleavage region described above comprises at least one internucleoside linkage that is not a phosphorothioate linkage;

[0094] (b') the internucleoside linkage between X2' and Y' is not a phosphorothioate linkage;

[0095] (c') the internucleoside linkage between Y' and Z' is not a phosphorothioate linkage;

[0096] (d') all of the internucleoside linkages in the second cleavage region described above are not phosphorothioate linkages;

[0097] (e') the second cleavage region described above comprises at least one internucleoside linkage that is a phosphodiester linkage;

[0098] (f') the internucleoside linkage between X2' and Y' is a phosphodiester linkage;

[0099] (g') the internucleoside linkage between Y' and Z' is a phosphodiester linkage;

[0100] (h') all of the internucleoside linkages in the second cleavage region described above are phosphodiester linkages; and / or

[0101] (i') the internucleoside linkage between the second cleavage region described above and the fourth segment described above is a phosphodiester linkage.

[0102] In some embodiments of the application, the compound described above, wherein:

[0103] (a) the first cleavage region described above comprises at least one nucleotide selected from a 2'-OMe modified nucleotide or a 2'-F modified nucleotide;

[0104] (b) each nucleotide of the first cleavage region described above is a 2’-OMe modified nucleotide or a 2’-F modified nucleotide;

[0105] (c) the first cleavage region described above comprises at least one 2’-F modified nucleotide;

[0106] (d) the first cleavage region described above comprises no more than two 2’-F modified nucleotides;

[0107] (e) Z in Formula A-1, Formula B-1, or Formula B’-1 is a 2’-F modified nucleotide, and optionally X2 in Formula A-1, Formula B-1, or Formula B’-1 is a 2’-F modified nucleotide;

[0108] (f) N1 in Formula B-1, or Formula B’-1 is a 2’-F modified nucleotide;

[0109] (g) X2 and Y in Formula A-1, Formula B-1, or Formula B’-1 are both 2’-OMe modified nucleotides, and Z is a 2’-F modified nucleotide, and further N1 in Formula B-1, or Formula B’-1 is a 2’-F modified nucleotide;

[0110] (h) X2, Y, and Z in Formula A-1, Formula B-1, or Formula B’-1 are all 2’-OMe modified nucleotides, and further N1 in Formula B-1, or Formula B’-1 is a 2’-F modified nucleotide;

[0111] (a’) the second cleavage region described above comprises at least one nucleotide selected from a 2’-OMe modified nucleotide or a 2’-F modified nucleotide;

[0112] (b’) each nucleotide of the second cleavage region described above is a 2’-OMe modified nucleotide or a 2’-F modified nucleotide;

[0113] (c’) the second cleavage region described above comprises at least one 2’-F modified nucleotide;

[0114] (d’) the second cleavage region described above comprises no more than two 2’-F modified nucleotides;

[0115] (e’) Z’ in Formula A-2, Formula B-2, or Formula B’-2 is a 2’-F modified nucleotide, and optionally X2’ in Formula A-2, Formula B-2, or Formula B’-2 is a 2’-F modified nucleotide;

[0116] (f’) N1’ in Formula B-2, or Formula B’-2 is a 2’-F modified nucleotide;

[0117] (g') X2' and Y' in Formula A-2, Formula B-2, or Formula B'-12 are both 2'-OMe modified nucleotides, and Z' is a 2'-F modified nucleotide, and further N1' in Formula B-2 or Formula B'-2 is a 2'-F modified nucleotide; and / or

[0118] (h') X2', Y', and Z' in Formula A-2, Formula B-2, or Formula B'-2 are all 2'-OMe modified nucleotides, and further N1' in Formula B-2 or Formula B'-2 is a 2'-F modified nucleotide.

[0119] In some embodiments of the application, the above DS1 or the above first double- stranded portion in the above DS1 further comprises at least one internucleoside linkage selected from a phosphorothioate linkage or a methylphosphonate linkage, and / or the above DS2 or the above second double-stranded portion in the above DS2 further comprises at least one internucleoside linkage selected from a phosphorothioate linkage or a methylphosphonate linkage.

[0120] In some embodiments of the above compound, wherein:

[0121] (a) the 1st and / or 2nd internucleoside linkage from the 5' end of the above first segment and / or fourth segment is a phosphorothioate linkage or a methylphosphonate linkage; and / or

[0122] (b) the 1st and / or 2nd internucleoside linkage from the 3' end of the above first segment and / or fourth segment is a phosphorothioate linkage or a methylphosphonate linkage.

[0123] In some embodiments of the above compound, wherein: the length of the above first sense strand and / or second sense strand is independently 17-23, 17-22, or 17-21 nucleotides, respectively; and the length of the above first antisense strand and / or second antisense strand is independently 22-28, 22-27, or 22-26 nucleotides, respectively.

[0124] In some embodiments of the above compound, wherein:

[0125] (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

[0126] (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.

[0127] In some embodiments of the application, the compound described above, wherein L is selected from a bond, a degradable linker, or a non-degradable linker.

[0128] In some embodiments of the application, the compound described above, wherein L is selected from DNA, RNA, a functionalized monosaccharide, or an oligosaccharide.

[0129] In some embodiments of the application, the compound described above, wherein L is a non-degradable linker.

[0130] In some embodiments of the application, the compound described above, wherein the compound is capable of cleaving within a cell, but is sufficiently stable outside of a cell.

[0131] In some embodiments of the application, the compound described above, wherein L is sufficiently stable in a biological or in vitro environment.

[0132] In some embodiments of the application, the compound described above, wherein L remains un-cleaved for a period of time in the corresponding environment before the compound described above contacts the target mRNA, for example, remains un-cleaved for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, or 72 hours in plasma, with no more than 5%, 10%, 20%, 30%, 40%, or 50% of the compound cleaving.

[0133] In certain embodiments, the compound can remain linked between DS1 and DS2 outside of a cell. In certain embodiments, the compound can resist degrading factors (e.g., enzymes, pH, etc.) in an in vitro or in vivo environment for a sufficient period of time to maintain the linkage between DS1 and DS2. In certain embodiments, the compound has a half-life of no less than 55 hours (e.g., no less than 54 hours, no less than 53 hours, no less than 52 hours) after mixing with human plasma. In certain embodiments, the compound degrades no more than 15% (e.g., no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, or no more than 8%) within 24 hours after mixing with human plasma.

[0134] In some embodiments, a compound provided herein is sufficiently stable in a biological or in vitro environment, more optionally L remains unbroken in the corresponding environment (e.g., outside a cell or in blood circulation) for a period of time before the compound contacts the target mRNA (e.g., before entering a cell), for example, no more than 5%, 10%, 20%, 30%, 40%, or 50% of the compound is broken down in plasma for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, or 72 hours. In some embodiments, a compound provided herein does not comprise a deoxyribonucleoside, a disulfide bond, a pH-sensitive cleavable moiety, a polypeptide hydrolase substrate, or a sugar. In some embodiments, a compound provided herein does not comprise a deoxyribonucleotide, an abasic ribonucleic acid, an abasic deoxyribonucleic acid, an inverted abasic ribonucleic acid, or an inverted abasic deoxyribonucleic acid. In some embodiments, a compound provided herein does not comprise a deoxyribonucleoside (e.g., dT), a disulfide bond, a pH-sensitive cleavable moiety, a polypeptide hydrolase substrate, or a sugar in or adjacent to the adjoining 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 adjacent thereto). In some embodiments, a compound provided herein does not comprise a deoxyribonucleotide, an abasic ribonucleic acid, an abasic deoxyribonucleic acid, an inverted abasic ribonucleic acid, or an inverted abasic deoxyribonucleic acid in or adjacent to the adjoining region. In some embodiments, L is a bond or a chemical linker.

[0135] In some aspects of the application, the compound described above, wherein the molar ratio of the cleaved DS1 product that acts on the first target RNA or a target mRNA encoding the first target gene described above and the cleaved DS2 product that acts on the second target RNA or a target mRNA encoding the second target gene described above is about 1.

[0136] In some aspects of the application, the compound described above, wherein L is as shown in Formula (I-1’) or Formula (I-1),

[0137] wherein:

[0138] X is O or S;

[0139] Y is a single bond, -O-, or -S-;

[0140] 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 -CH2-NH-; L2, L3, L4, L6, L7, L8, and L9are each independently 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-, -C(=0)0-, -NHC(=0)0-, -NHC(=0)NH-, -OC(=0)0-, -OC(=0)NH-, -S(0)2NH-, -NHS(0)2-, and -CH2-NH-;

[0141] L5is 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-, carbocycle, and heterocycle; one of the above L is connected to the 3' end or 5' end of the above first sense strand of the above DS1, and the other is connected to the 3' end or 5' end of the above second sense strand of the above DS1.

[0142] In some embodiments of the application, the above compound, wherein the above L is as shown in formula (I-2') or formula (I-2),

[0143] wherein:

[0144] Ring A is absent and L6is directly connected to the triazole;

[0145] Alternatively, Ring A is selected from a 5-16 membered heterocycle.

[0146] In some embodiments of the application, the above compound, the above L comprises 1, 2, or 3 dT, and optionally, the above L further comprises a structure as shown in the preceding formula (I-1') or formula (I-1) or formula (I-2') or formula (I-2).

[0147] In some embodiments of the application, the above compound, the above L is as shown in (I-3), (I-4), or (I-5):

[0148] In some embodiments of the application, the above compound, wherein the above L is as shown in formula (I-6), (I-7), or (I-8):

[0149] In some embodiments of the application, the above compound, wherein: the structural unit is selected from the group consisting of optionally substituted​

[0150] In some embodiments of the application, the above-mentioned compound, wherein 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).

[0151] In some embodiments of the application, the above-mentioned compound, wherein L is selected from

[0152] In some embodiments of the application, the above-mentioned compound, the 5' or 3' terminal nucleotide of the first sense strand is linked to the 5' or 3' terminal nucleotide of the second sense strand by L, preferably the 5' terminal nucleotide of the first sense strand is linked to the 5' terminal nucleotide of the second sense strand by L.

[0153] In some embodiments of the application, the above-mentioned compound, wherein one end of L is linked to the nucleotide at position 6 from the 5' end of the first sense strand, and the other end of L is linked to the 5' end of the second sense strand.

[0154] In some embodiments of the application, the above-mentioned compound, wherein at least one nucleotide is a modified nucleotide, preferably all nucleotides are modified nucleotides, and the modification comprises 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, modification to enhance the affinity of double-stranded ribonucleic acid to ARGO protein.

[0155] In some embodiments of the application, the above-mentioned compound, wherein the first and / or second antisense strand further comprises a capping group at the 5' end, optionally the capping group is linked to the 5' end of the first 5' extension and / or the second 5' extension.

[0156] In some embodiments of the application, the above-mentioned compound, wherein the capping group comprises an inverted abasic deoxynucleotide or M06.

[0157] In some embodiments of the application, the compound above, wherein the capping group is attached to the 5' end of the first 5' extension and / or second 5' extension via an internucleoside linkage, which is optionally modified or unmodified.

[0158] In some embodiments of the application, the compound above, wherein the capping group is attached to the 5' end of the first 5' extension and / or second 5' extension via a phosphorothioate linkage.

[0159] In some embodiments of the application, the compound above, further comprising 1 or more (e.g., 1, 2, 3, 4 or more) delivery systems, optionally each of the delivery systems above is independently linked to DS1, DS2 or L.

[0160] In some embodiments of the application, each of the delivery systems above is independently a ligand, preferably the ligand above alters the distribution, targeting or lifetime of the compound above, more preferably the ligand above provides enhanced affinity to a target, such as a molecule, a cell or cell type, a compartment, a receptor, such as a cellular or organ compartment, a tissue, an organ or a body region, more preferably the ligand above enables the delivery of the compound above to a target tissue and produces an RNA interference effect.

[0161] In some embodiments of the application, each of the ligands above is independently selected from a GalNAc ligand, a lipophilic ligand, or other ligands that target receptors to facilitate endocytosis of the compound above, such as a TfR-targeting ligand, a LDL-R-targeting ligand or an integrin-targeting ligand.

[0162] In some embodiments of the application, the compound above, wherein the ligand above is a GalNAc ligand and the number of GalNAc ligands is at least 1; further, the number of GalNAc ligands above is 1 or 2. In some embodiments of the application, the number of GalNAc ligands above is 1. In some embodiments of the application, the number of GalNAc ligands above is 2.

[0163] In some embodiments of the application, the compound above, wherein: a) the number of GalNAc ligands above is 2, each linked to the most 3' nucleotide of the first sense strand and the second sense strand, or b) the number of GalNAc ligands above is 1, linked to the most 3' nucleotide of the first sense strand or the second sense strand; or c) the number of GalNAc ligands above is 2, one linked to the most 3' nucleotide of the first sense strand and the other linked to the most 5' nucleotide of the second sense strand.

[0164] In some embodiments of the present application, the compound described above, wherein the GalNAc ligands described above are independently L96 or NAG37, respectively; further, a) the number of GalNAc ligands described above is 2, one of which is L96 and is linked to the most 3' end nucleotide of the first sense strand, and the other of which is NAG37 and is linked to the most 5' end nucleotide of the second sense strand, or b) the number of GalNAc ligands described above is 2 and both are L96, or c) the GalNAc ligand described above is one L96 and is linked to the most 3' end nucleotide of the first sense strand or the second sense strand.

[0165] In some embodiments of the present application, the compound described above, 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 is a different fragment of the same RNA or the same gene as the second target RNA or the second target gene; or the first target RNA or the first target gene is a different RNA or a different gene from the second target RNA or the second target gene.

[0166] In some embodiments of the present application, the compound described above or a pharmaceutically acceptable salt thereof is a compound as shown in any one of Tables 1-85 or a pharmaceutically acceptable salt thereof.

[0167] Table 1 Structure of polymeric oligonucleotide S1-1

[0168] Table 2 Structure of polymeric oligonucleotide S1-2

[0169] Table 3 Structure of polymeric oligonucleotide S2-1

[0170] Table 4 Structure of polymeric oligonucleotide S2-2

[0171] Table 5 Structure of polymeric oligonucleotide S3-1

[0172] Table 6 Structure of polymeric oligonucleotide S3-2

[0173] Table 7 Structure of polymeric oligonucleotide S4-1

[0174] Table 8 Structure of polymeric oligonucleotide S4-2

[0175] Table 9 Structure of polymeric oligonucleotide S5-1

[0176] Table 10 Structure of polymeric oligonucleotide S5-2

[0177] Table 11 Structure of polymeric oligonucleotide S6-1

[0178] Table 12 Structure of polymeric oligonucleotide S6-2

[0179] Table 13 Structure of polymeric oligonucleotide S7-1

[0180] Table 14 Structure of polymeric oligonucleotide S7-2

[0181] Table 21 Structure of polymeric oligonucleotide conjugate S11-1

[0182] Table 22 Structure of polymeric oligonucleotide conjugate S11-2

[0183] Table 23 Structure of polymeric oligonucleotide conjugate S12-1

[0184] Table 24 Structure of polymeric oligonucleotide conjugate S12-2

[0185] Table 25 Structure of polymeric oligonucleotide conjugate Z1-1

[0186] Table 26 Structure of polymeric oligonucleotide conjugate Z1-2

[0187] Table 27 Structure of polymeric oligonucleotide conjugate Z2-1

[0188] Table 28 Structure of polymeric oligonucleotide conjugate Z2-2

[0189] Table 29 Structure of polymeric oligonucleotide conjugate Z3-1

[0190] Table 30 Structure of polymeric oligonucleotide conjugate Z3-2

[0191] Table 31 Polymeric oligonucleotide conjugate Z4-1 structure

[0192] Table 32 Polymeric oligonucleotide conjugate Z4-2 structure

[0193] Table 33 Polymeric oligonucleotide conjugate Z5-1 structure

[0194] Table 34 Polymeric oligonucleotide conjugate Z5-2 structure

[0195] Table 35 Polymeric oligonucleotide conjugate Z6-1 structure

[0196] Table 36 Polymeric oligonucleotide conjugate Z6-2 structure

[0197] Table 37 Polymeric oligonucleotide conjugate Z7-1 structure

[0198] Table 38 Polymeric oligonucleotide conjugate Z7-2 structure

[0199] Table 45 Polymeric oligonucleotide conjugate Z11-1 structure

[0200] Table 46 Polymeric oligonucleotide conjugate Z11-2 structure

[0201] Table 47 Polymeric oligonucleotide conjugate Z12-1 structure

[0202] Table 48 Polymeric oligonucleotide conjugate Z12-2 structure

[0203] Table 49 Polymeric oligonucleotide conjugate Z13 structure

[0204] Table 50 Polymeric oligonucleotide conjugate Z14 structure

[0205] Table 51 Polymeric oligonucleotide conjugate Z15 structure

[0206] Table 52 Polymeric oligonucleotide conjugate Z16 structure

[0207] Table 53 Polymeric oligonucleotide conjugate Z17 structure

[0208] Table 54 Polymeric oligonucleotide conjugate Z18 structure

[0209] Table 55 Polymeric oligonucleotide conjugate Z19 structure

[0210] Table 56 Polymeric oligonucleotide conjugate Z20 structure

[0211] Table 57 Polymeric oligonucleotide conjugate Z21 structure

[0212] Table 58 Polymeric oligonucleotide conjugate Z22 structure

[0213] Table 59 Polymeric oligonucleotide conjugate Z23 structure

[0214] Table 60 Polymeric oligonucleotide conjugate Z24 structure

[0215] Table 61 Polymeric oligonucleotide conjugate Z25 structure

[0216] Table 62 Polymeric oligonucleotide conjugate Z26 structure

[0217] Table 63 Polymeric oligonucleotide conjugate Z27 structure

[0218] Table 64 Polymeric oligonucleotide conjugate Z28 structure

[0219] Table 65 Polymeric oligonucleotide conjugate Z29 structure

[0220] Table 66 Polymeric oligonucleotide conjugate Z30 structure

[0221] Table 67 Polymeric oligonucleotide conjugate Z31 structure

[0222] Table 68 Polymeric oligonucleotide conjugate Z32 structure

[0223] Table 69 Structure of polymeric oligonucleotide conjugate Z33

[0224] Table 70 Structure of polymeric oligonucleotide conjugate Z34

[0225] Table 71 Structure of polymeric oligonucleotide conjugate Z35

[0226] Table 72 Structure of polymeric oligonucleotide conjugate Z36

[0227] Table 79 Structure of polymeric oligonucleotide conjugate Z43

[0228] Table 81 Structure of polymeric oligonucleotide conjugate Z55

[0229] Table 82 Structure of polymeric oligonucleotide conjugate Z56

[0230] Table 83 Structure of polymeric oligonucleotide conjugate Z57

[0231] Table 84 Structure of polymeric oligonucleotide conjugate Z58

[0232] Table 85 Structure of molecule targeting multiple genes

[0233] The present application also provides a pharmaceutical composition comprising the above-mentioned compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0234] The present application also provides a method for inhibiting the expression of a target gene in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.

[0235] The present application also provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition, optionally the above-mentioned disease or disorder is related to a first target gene and / or a second target gene.

[0236] The present application also provides the following non-limiting embodiments.

[0237] Embodiment 1. A compound represented by formula (I),

[0238] wherein:

[0239] DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, which form a first double-stranded portion of 15-27 nucleotide pairs in length, and a first 5’ extension in the first antisense strand upstream of the 5’ of the first double-stranded portion, which is at least 3 nucleotides in length and cleavable at the 3’ most nucleotide of the first 5’ extension to yield a cleaved DS1 product comprising the first double-stranded portion, which is capable of silencing a first target RNA or inhibiting expression of a first target gene by RNA interference;

[0240] DS2 is a double-stranded oligonucleotide comprising a second sense strand and a second antisense strand, which form a second double-stranded portion of 15-27 nucleotide pairs in length, and a second 5’ extension in the second antisense strand upstream of the 5’ of the second double-stranded portion, which is at least 3 nucleotides in length and cleavable at the 3’ most nucleotide of the second 5’ extension to yield a cleaved DS2 product comprising the second double-stranded portion, which is capable of silencing a second target RNA or inhibiting expression of a second target gene by RNA interference;

[0241] L is a linking structural unit, one end of L is connected to 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;

[0242] the first target RNA or first target gene is PCSK9, and the second target RNA or second target gene is LPA;

[0243] the first antisense strand comprises the following sequence (5’-3’): a*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a;

[0244] the second antisense strand comprises the following sequence (5’-3’): a*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g (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 deoxyribonucleoside; (dC): cytosine deoxyribonucleoside; *: phosphorothioate bond).

[0245] Embodiment 2. The compound according to embodiment 1, wherein the first antisense strand comprises the following sequence (5’-3’): CfGfaa*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a.

[0246] Embodiment 3. The compound according to embodiment 1 or 2, wherein the second antisense strand comprises the following sequence (5’-3’): CfGfaa*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g.

[0247] Embodiment 4. The compound according to any one of embodiments 1-3, wherein wherein the first antisense strand and / or second antisense strand further comprises a capping group at the 5’ end, optionally the capping group is linked to the 5’ end of the first 5’ extension and / or second 5’ extension.

[0248] Embodiment 5. The compound according to embodiment 4, wherein the first antisense strand comprises or has the following sequence (5’-3’): (invAB)*CfGfaa*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a.

[0249] Embodiment 6. The compound according to embodiment 4 or 5, wherein the second antisense strand comprises or has the following sequence (5’-3’): (invAB)*CfGfaa*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g.

[0250] Embodiment 7. The compound according to any one of embodiments 1-6, wherein the first sense strand comprises or has the following sequence (5’-3’): c*uagacCfuGfu(dT)uugcuuuu*g*u.

[0251] Embodiment 8. The compound according to any one of embodiments 1-7, wherein the second sense strand comprises or has the following sequence (5’-3’): a*augguaaUfgGfaCfagaguuau.

[0252] Embodiment 9. The compound according to any one of embodiments 1-8, wherein L is

[0253] Embodiment 10. The compound according to any one of embodiments 1-9, wherein further comprises 1 or more delivery systems, optionally the delivery systems are each independently linked to DS1 or DS2; preferably, the compound comprises 1 delivery system

[0254] Embodiment 11. The compound according to Embodiment 10, wherein the delivery system is each independently attached to the 5’ end of the first sense strand, to the 3’ end of the first sense strand, to the 5’ end of the second sense strand, or to the 3’ end of the second sense strand; preferably, the delivery system is attached to the 3’ end of the second sense strand. 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 of, more preferably the ligand provides enhanced affinity to a target, such as a molecule, a cell or cell type, a compartment, a receptor, such as a cellular or organ compartment, a tissue, an organ or a body region, more preferably the ligand enables the compound to be delivered to a target tissue and produce an RNA interference effect.

[0255] Embodiment 13. The compound according to any one of Embodiments 10-12, wherein the ligand is each independently selected from a GalNAc ligand, a lipophilic ligand, or other ligand targeting a receptor to facilitate endocytosis of the compound, such as a TfR-targeting ligand, a LDL-R-targeting ligand or an integrin-targeting ligand; optionally the ligand is each independently NAG37 or L96; preferably, the ligand is L96.

[0256] Embodiment 14. The compound according to any one of Embodiments 10-13, wherein the second sense strand comprises or has the sequence (5’-3’): a*augguaaUfgGfaCfagaguuau[L96].

[0257] Embodiment 15. The compound according to any one of the preceding embodiments, selected from Z1-1, or a pharmaceutically acceptable salt thereof.

[0258] Embodiment 16. A pharmaceutical composition comprising a compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0259] Embodiment 17. A method of inhibiting expression of a target gene in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound according to any one of Embodiments 1-15, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to Embodiment 16.

[0260] Embodiment 18. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound according to any one of Embodiments 1-15, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to Embodiment 16, optionally the disease or disorder is related to a first target RNA or first target gene and / or a second target RNA or second target gene.

[0261] In some embodiments, the disease or condition comprises, but is not limited to, atherosclerotic cardiovascular disease (e.g., coronary artery disease (CAD), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD)), calcific aortic valve stenosis (CAVS), familial hypercholesterolemia (FH), diabetes mellitus with CVD, chronic kidney disease (CKD) with cardiovascular disease, etc.

[0262] As known in the art, there are three free hydroxyl groups on the sugar ring of ribonucleosides, which can form three different nucleotides: 2'-ribonucleotides, 3'-ribonucleotides, and 5'-ribonucleotides, and there are two free hydroxyl groups on the sugar ring of deoxynucleosides, which can form two nucleotides: 3'-deoxyribonucleotides and 5'-deoxyribonucleotides. Based on this, all other embodiments obtained by those of ordinary skill in the art without creative labor on the basis of the embodiments of the present application belong to the technical solutions of the present application and fall within the protection scope of the present application.

[0263] Definitions

[0264] As used herein and unless otherwise indicated, the following terms and phrases are intended to have the following meanings. No particular term or phrase should be construed as being indefinite or unclear unless specifically defined, but should be construed in accordance with the ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0265] As used herein, "optionally," "optional" or "optionally" mean the same and refer to the possibility that the event or circumstance described after the term can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" or "alkyl optionally substituted" includes "alkyl" (H on the alkyl is not replaced / substituted by a non-H substituent) and "substituted alkyl" (H on the alkyl is replaced / substituted by a non-H substituent). As used herein, those of ordinary skill in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically non-feasible, and / or inherently unstable. For example, "optionally modified" includes unmodified and modified, further, "nucleotides optionally modified" includes nucleotides unmodified and nucleotides modified.

[0266] As used herein, when any variable (e.g., substituent R, e.g., nucleic acid modification) occurs more than one time in a composition or structure, its definition on each occurrence is independent of its definition at every other occurrence. For example, if a group is substituted with 0-2 R groups, then the group is optionally substituted with up to two R groups, and the R groups are independently selected at each occurrence. As another example, when multiple nucleotides are modified, each nucleotide is independently optionally modified, and each nucleotide can be modified the same or differently.

[0267] As used herein, “comprising,” “including,” “at least,” “has,” “having,” “at least having,” “containing,” or the like are open-ended terms that mean that other elements, components, or steps are optional and not excluded from the methods or compositions described herein.

[0268] As used herein, the term “nucleotide” refers to a five-carbon sugar (ribose or deoxyribose), a phosphate group, and a base (natural or unnatural base), and is intended to include 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 can be optionally further modified.

[0269] Natural nucleotides are composed of natural bases, natural ribose, and phosphate. Natural nucleotides as used herein refer to an adenine ribonucleotide, an adenine deoxyribonucleotide, a guanine ribonucleotide, a guanine deoxyribonucleotide, a cytosine ribonucleotide, a cytosine deoxyribonucleotide, a uracil ribonucleotide, a thymine ribonucleotide, or a thymine deoxyribonucleotide. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2’ position of the sugar moiety of the nucleotide. “Deoxyribonucleotide” refers to a nucleotide having a hydrogen at the 2’ position of the sugar moiety of the nucleotide.

[0270] Natural bases of RNA include A (adenine), G (guanine), C (cytosine), U (uracil), and T (thymine).

[0271] Nucleotides can be substituted by their analogs, including natural and unnatural analogs. Examples of guanosine analogs include, but are not limited to, 6-thioguanosine, 8-azaguanosine, 8-oxoguanosine, 2-aminopurine nucleosides, and the like. Examples of adenosine analogs include, but are not limited to, vermixine (3'-deoxyadenosine), n6-benzyladenosine, 2-chloroadenosine, and the like. Examples of cytidine analogs include, but are not limited to, gemcitabine (2',2'-difluoro-2'-deoxycytidine), cytarabine (1-β-d-arabinofuranosylcytosine), decitabine (5-aza-2'-deoxycytidine), and the like. Examples of uracil analogs include, but are not limited to, 5-fluorouracil, pseudouracil, 5-bromouracil, 4-thiouracil, 5-azouracil, and the like.

[0272] As used herein, "ribonucleic acid" (RNA) is a carrier of genetic information found in cells and some viruses and viroids. RNA is a long chain-like molecule formed from ribonucleotides connected by internucleosidic linkages, including single-stranded and double-stranded RNA. The natural internucleosidic linkage is a phosphodiester bond. In some embodiments, the ribose, the base, and the internucleosidic linkage in the RNA are independently optionally modified.

[0273] As used herein, "double-stranded ribonucleic acid" is a complex comprising two nucleic acid strands comprising antiparallel and substantially complementary sequences, substantially meaning that the two nucleic acid strands can comprise a number of base mismatches and mispairs in their reverse complementary regions while maintaining functionality, bonded from natural or unnatural nucleotides. In some embodiments, the number refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the number refers to 1, 2, 3, 4, or 5. In some embodiments, the number refers to 1, 2, or 3.

[0274] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) of, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0275] As used herein, "bonded" refers to the connection between the residues (e.g., nucleosides) of two monomers (e.g., nucleotides) by a single bond or a group (e.g., by a phosphodiester bond, a phosphorothioate bond, or a phosphorodithioate bond). In some embodiments, the bonded refers to the connection between the residues of two nucleotides by a phosphodiester bond, a phosphorothioate bond, or a phosphorodithioate bond.

[0276] As used herein, a "monomer" is a class of compounds that can be assembled into a ribonucleic acid strand and are capable of performing a certain function. As used herein, "monomer" includes, but is not limited to, a ligand (e.g., a GalNAc ligand, such as L96 or NAG37), a natural nucleotide, a non-natural nucleotide (e.g., a modified nucleotide, a nucleotide analog, an inverted abasic deoxyribonucleotide, GNA, LNA, etc.), a capping group (e.g., M06).

[0277] As used herein, the term "internucleoside linkage" refers to a bond (e.g., a bond or linking group) between two moieties of an oligonucleotide disclosed herein (e.g., between two monomers), including bonds between a nucleoside, between a nucleoside and a ligand, between a nucleoside and a capping group, and between a nucleoside and an abasic nucleoside disclosed herein.

[0278] In the present application, the term "capping group" refers to a group that can be selectively associated with a compound described herein, which capping group is located on the sense strand, the antisense strand, or both strands. The capping group can be attached to the 3' end, the 5' end, or both ends of the sense strand, the antisense strand, or both strands. In certain embodiments, the capping group is associated with the antisense strand, particularly the 5' end of the antisense strand. In certain embodiments, the capping group is associated with the oligonucleotide (e.g., the 5' end of the antisense strand) through an internucleotide linkage, which internucleotide linkage is optionally modified as described above. In certain embodiments, the capping group is linked to a double-stranded oligonucleotide through a phosphorothioate linkage. In certain embodiments, the capping group is linked to a double-stranded oligonucleotide through a phosphodiester linkage. In the present application, to reduce or inhibit degradation by exonucleases, a "capping group" can be associated with the 5' end of the first antisense strand and / or the second antisense strand of a compound described herein. In certain embodiments, the capping group can reduce or inhibit the RNA interference effect of the oligonucleotide. In certain embodiments, the capping group is cleaved from the oligonucleotide prior to providing the RNA interference effect. Examples of capping groups include, but are not limited to, abasic nucleotides, inverted abasic nucleotides, and (M06), for example.

[0279] As used herein, "inhibit" means to reduce gene expression when a given gene is expressed when a cell, cell population, or tissue is treated with a single / double-stranded ribonucleic acid, single / double-stranded ribonucleic acid conjugate, or pharmaceutical composition comprising one or more of them described herein, as compared to a cell, cell population, or tissue that has not been so treated. The terms "inhibit," "reduce," "silence," "down-regulate," "suppress," and other similar terms are used interchangeably herein and include inhibition at any level. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0280] As used herein, "conjugate" refers to the covalent linkage between two or more chemical moieties each having a particular function; correspondingly, "conjugate" refers to the compound formed by the covalent linkage between the respective chemical moieties. For example, "double-stranded ribonucleic acid conjugate" refers to a compound or complex formed by covalent linkage of one or more chemical moieties having a particular function (e.g., a delivery system, a ligand group, or a conjugation group) to a double-stranded ribonucleic acid. In some embodiments, the delivery system, ligand group, or conjugation group can be linked to a phosphate group, a sugar ring (including covalent linkage of the delivery system, ligand group, or conjugation group to an atom at the 3' or 5' position of the nucleotide via a phosphodiester bond), a 2'-position hydroxyl, a 5'-position hydroxyl, or a base of any nucleotide of the double-stranded ribonucleic acid. In some embodiments, the delivery system, ligand group, or conjugation group can also be linked to the 2'-position of a nucleotide, in which case the nucleotides are linked via 2'-5' phosphodiester bonds. In some embodiments, the delivery system, ligand group, or conjugation group can also be linked to the 3'-position of a nucleotide, in which case the nucleotides are linked via 3'-5' phosphodiester bonds.

[0281] As used herein, "complementary" or "reverse complementary" can be used interchangeably to refer to the structural relationship between two nucleotides (e.g., on two opposite nucleic acid strands or on opposite regions of a single nucleic acid strand) that allows the two nucleotides to form base pairs with each other (e.g., purine nucleotides of one nucleic acid complementary to pyrimidine nucleotides of the opposite nucleic acid can form base pairs together by hydrogen bonding to each other). In some aspects of the application, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows for the formation of a stable duplex. In some aspects of the application, two nucleic acid strands can have multiple regions that form complementary duplexes. In some aspects of the application, 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 aspects of the application, complementary nucleotides can also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing. In some aspects of the application, nucleotides containing hypoxanthine as their base can base pair with nucleotides containing adenine, cytosine, or uracil. In some aspects of the application, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the application with nucleotides containing, for example, inosine (in the application, the capital letter "I" can represent a hypoxanthine base, inosine, or an inosine-containing nucleotide depending on its context) (this replacement is referred to as an I modification). In some aspects of the application, adenine and cytosine anywhere in an oligonucleotide can be replaced with guanine and uracil, respectively, to form G-U wobble base pairs with a target mRNA.

[0282] The degree of complementarity between one oligonucleotide and another oligonucleotide is referred to as complementarity and is measured by the percentage of bases in each strand that can hydrogen bond with one another, according to the standard base pairing rules. The oligonucleotide sequence need not be 100% (i.e., "perfectly") complementary to its corresponding nucleic acid sequence. In some embodiments, a first nucleotide sequence can be 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% degree of sequence complementarity with the second nucleotide sequence. In an exemplary embodiment, 18 out of 20 nucleobases of a first nucleotide sequence pair with the corresponding region of a second nucleotide sequence, achieving 90% complementarity. Non-complementary nucleobases, also referred to as "mismatches," can be clustered or interspersed with complementary bases, and need not be adjacent to one another or to complementary nucleobases.

[0283] As used herein, "mismatch" includes, but is not limited to:

[0284] 1) two opposing (independent natural or unnatural) nucleotides pair (other than A-T, A-U or G-C);

[0285] 2) no hydrogen bond is formed between two opposing (independent natural or unnatural) nucleotides;

[0286] 3) absence of a base between two opposing (independent natural or unnatural) nucleotides.

[0287] In some embodiments, a mismatch includes wobble base pairing and Hoogstein base pairing.

[0288] The term "fully complementary" refers to a first nucleotide sequence and a second nucleotide sequence forming a hybrid in a region of complete complementarity consisting only of Watson-Crick base pairs. An oligonucleotide that is "sufficiently complementary" can include an internal region (e.g., at least 7, 8, 9, or 10 nucleotides) that is fully complementary to the target RNA. In some embodiments, a first targeting region contained in a first fragment provided herein is sufficiently complementary to a portion of a target mRNA encoding a first target gene. In some embodiments, the first targeting region has at least 80%, 85%, 90%, or 95% complementarity to a portion of a target mRNA encoding a first target gene. In some embodiments, the first targeting region has 100% complementarity (is fully complementary) to a portion of a target mRNA encoding a first target gene. In some embodiments, a second targeting region contained in a third fragment provided herein is sufficiently complementary to a portion of a target mRNA encoding a second target gene. In some embodiments, the second targeting region has at least 80%, 85%, 90%, or 95% complementarity to a portion of a target mRNA encoding a second target gene. In some embodiments, the second targeting region has 100% complementarity (is fully complementary) to a portion of a target mRNA encoding a second target gene.

[0289] As used herein, the term "modified nucleotide" refers to a nucleotide having a modified internucleoside linkage, and / or a modified base, and / or a modified sugar. In some embodiments, a modified nucleotide comprises one, two, three, or more modifications. In some embodiments, a nucleotide comprises one modification. In some embodiments, a nucleotide comprises two modifications. In some embodiments, a nucleotide comprises three modifications.

[0290] As used herein, a "modification" of a nucleotide includes, but is not limited to: a 2'-OMe (2'-O-methyl) modification, a 2'-F (2'-deoxy-2'-fluoro) modification, a 2'-O-MOE (2'-O-methoxyethyl) modification, a 2'-deoxy (2'-d) modification, a 5'-morpholino (5'-Mo) modification, an unlocked nucleic acid (UNA) modification, a glycol nucleic acid (GNA) modification, a locked nucleic acid (LNA) modification, a tricyclo-DNA (tcDNA) modification, a (S)-constrained ethyl bicyclic nucleic acid ((S)-cEt-BNA) modification, a phosphorothioate (PS) modification, a phosphorodithioate (PS2) modification, a methylphosphonate (MP) modification, a methoxypropylmethylphosphonate (MOP) modification, a phosphoroselenoate modification, a phosphorodiselenoate modification, a phosphoramidothioate modification, a phosphoramidate modification, a phosphoramide

[0291] As used herein, in some embodiments, "G," "A," "C," "U," and "T" refer to guanine ribonucleotides, adenine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, and thymine ribonucleotides, respectively. Exemplary structures are as follows:

[0292] As used herein, some embodiments are synthesized according to phosphoramidite solid-phase synthesis techniques, in which: "G," "A," "C," "U," and "T" are linked at the 5' end 1 position of the chain with the structure

[0293] "G", "A", "C", "U", and "T" are connected at the 1 position of the 3' end of the strand

[0294] As used herein, the designation "d" preceding a monomer (e.g., nucleotides A, U, C, G, and T, etc.) indicates that the monomer is 2'-deoxy-modified. Exemplary, 2'-deoxy-modified nucleotide structures are as follows:

[0295] As used herein, the designation "f" following a monomer (e.g., nucleotides A, U, C, G, and T, etc.) indicates that the monomer is 2'-deoxy-2'-fluoro-modified (2'-F modified). Exemplary, 2'-F modified nucleotide structures are as follows:

[0296] As used herein, the designation "GNA-" preceding a monomer (e.g., nucleotides A, U, C, G, and T, etc.) indicates that the monomer is glycol nucleic acid modified (GNA modified). As used herein, Tgn is the GNA-T designation, and is intended to be equivalent. Exemplary, GNA modified nucleotide structures are as follows:

[0297] As used herein, lower case letters (a, u, c, g, t, etc.) indicate that the nucleotide represented by its corresponding upper case letter (A, U, C, G, and T, etc.) is 2'-O-methyl (2'-OMe) modified. Exemplary, 2'-OMe modified nucleotide structures are as follows:

[0298] As used herein, the designation "invAB" modification refers to the inversion of an abasic deoxy nucleotide linked (e.g., at the 5' end or 3' end of a nucleotide) to a monomer. Exemplary, the structure of an invAB modification:

[0299] As used herein, the designation "invAb" modification refers to the replacement of a monomer (e.g., a nucleotide) with an inverted abasic nucleotide (invAb) modification. Exemplary, the structure of an invAb modification:

[0300] As used herein, the designation "VP" modification refers to the modification of a monomer (e.g., a nucleotide 5' position) with an (E)-vinyl phosphate (e.g., 5'-(E)-vinyl phosphate). Exemplary, the structure of a modified U, u, and dU are as follows:

[0301] As used herein, the designation "M06" modification refers to the linkage of a 5' or 3' end of a monomer (e.g., a nucleotide) Exemplary, Structure after modification by M06: In some embodiments, M06 is a modification by a precursor bonded to a nucleotide.

[0302] As used herein, Uhd represents a 2’-O-C16alkyl modified uracil ribonucleotide:

[0303] As used herein, the notation “*” between monomers (such as A, U, C, G, and T, etc.) indicates that the two monomers are connected by a phosphorothioate bond (i.e., a phosphorodithioate bond), i.e., are modified by phosphorothioate (PS).

[0304] As used herein, the absence of the notation “*” between monomers (such as A, U, C, G, and T, etc.) indicates that the two nucleotides are connected by a phosphodiester bond.

[0305] As used herein, represents a chemical bond (such as a single bond) by which the site is connected to other groups.

[0306] Exemplary, structural unit combined together represent the structure as

[0307] Exemplary, structural unit combined together represent the structure as “ug*cucaac*u*(dT)”.

[0308] Unless otherwise specified, sequences of the present application are written in the 5’-3’ order, e.g., refers to the 5’ end of a 2’-F modified cytosine ribonucleotide being connected to the indicated by refers to the 3’ end of a 2’-OMe modified uracil ribonucleotide being connected to the indicated by

[0309] Exemplary, structure as structure as structure as structure as structure as structure as structure as structure as

[0310] For example, "5'-A(dU)gCf*(dT)-3'" means: the sequence is from the 5' end, 1 is an adenine ribonucleotide, 2 is a uracil deoxyribonucleotide, 3 is a 2'-methoxy modified guanine ribonucleotide, 4 is a 2'-fluoro modified cytosine ribonucleotide, and 5 is a thymine deoxyribonucleotide linked between 4 by a phosphorothioate bond.

[0311] For example, "5'-(M06)*A(dT)gCf*(invAB)-3'" means the structure is:

[0312] As used herein, "small interfering RNA," "siRNA," "RNAi agent," or "iRNA agent" are used interchangeably to refer to a double-stranded ribonucleic acid molecule (e.g., an siRNA agent or a cleavage product thereof can down-regulate a target gene by, for example, inducing RNAi with respect to a target RNA, wherein the target can include an endogenous or pathogenic target RNA) 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 deleterious interferon response in human cells. In some embodiments of the application, the siRNA is at least partially complementary to a coding sequence in a target gene expressed in a cell. In some embodiments of the application, the siRNA is capable of inhibiting or blocking expression of a gene in vitro or in vivo after the siRNA is delivered to a cell expressing the gene. Typically, the siRNA comprises a double-stranded region of less than 60, 50, 40, or 30 complementary base pairs; preferably, it comprises 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 application, the sense and antisense strands of the siRNA are 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 application, the sense and antisense strands of the siRNA are perfectly complementary, 15-30 base pairs in length. In some embodiments of the application, the sense and antisense strands of the siRNA are perfectly complementary, 17, 18, 19, 20, 21, or 22 base pairs in length.

[0313] In some embodiments of the application, the length of a ribonucleic acid strand is calculated in units of nt (nucleotides), 1 nt (1 nucleotide) includes, but is not limited to, 1 natural nucleotide, 1 modified nucleotide.

[0314] In some embodiments, the compound disclosed herein (e.g., the first 5’ extension segment and / or the second 5’ extension segment) undergoes cleavage mediated by an enzyme (e.g., an endonuclease) prior to mediating RNAi. In some embodiments, the cleavage is mediated by a ribonuclease (RNase). In some embodiments, the cleavage is mediated by a Dicer endonuclease. In some embodiments, the cleavage is specific cleavage.

[0315] As used herein, the term “specific” or “specifically” with respect to cleavage of a compound (e.g., enzyme-mediated cleavage) refers to a controlled or selective cleavage at a particular location (i.e., between two desired nucleotides of an oligonucleotide strand undergoing specific cleavage). The product of specific cleavage can include a plurality of cleavage products, wherein the desired cleavage product (i.e., the product obtained from cleavage at a particular or desired location) is the most predominant (i.e., produced in the highest amount). In some embodiments, the desired cleavage product comprises at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% (by moles or weight) of all cleavage products. In some embodiments, the amount of the desired cleavage product (by moles or weight) is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold of any other cleavage product. While not wishing to be bound by theory, specific cleavage can be based on recognition by the enzyme mediating the cleavage of a particular sequence, feature, motif, or combination thereof of the compound (e.g., oligonucleotide strand, specifically the first 5’ extension segment and / or the second 5’ extension segment) undergoing cleavage. Without being bound by any theory, it is reasonable to expect that specific types and modifications of certain nucleotides at certain positions of the cleavage region (e.g., the first cleavage region and / or the second cleavage region) can provide the desired superior effect.

[0316] In some embodiments, the specific cleavage occurs at the 3’-most nucleotide of the first 5’ extension segment. In some embodiments, the specific cleavage occurs at the 3’-most nucleotide of the second 5’ extension segment. In some embodiments, the specific cleavage occurs at the 3’-most nucleotide of the first 5’ extension segment and at the 3’-most nucleotide of the second 5’ extension segment.

[0317] 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’.

[0318] Without being bound by any theory, the compounds disclosed herein are capable of being specifically cleaved between X2and Y and between X2and Y, such that the first 5' stretch and the second 5' stretch are removed, thereby generating a cleaved DS1 product and a cleaved DS2 product. The cleaved DS1 product and / or the cleaved DS2 product are capable of mediating RNAi.

[0319] 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 X2and the nucleotide in the first sense strand that is complementary thereto. In some embodiments, the first base pair at the 5' end of the first antisense strand is an AU base pair.

[0320] 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 X2and the nucleotide in the second sense strand that is complementary thereto. In some embodiments, the first base pair at the 5' end of the second antisense strand is an AU base pair.

[0321] As used herein, the NAG37 monomer and the L96 monomer structures are

[0322] [NAG37] and [L96] represent the residues thereof, respectively. Exemplarily, the structure of 5'-[NAG37]AfGfu*[L96]-3' is

[0323] Also provided herein is a delivery system D02, (D02) represents the residues thereof, and (D02)* represents the residues thereof linked to a nucleotide via a phosphorothioate bond, the structures of which are shown below, respectively.

[0324] As used herein, the term "carbocyclyl" or "carbocyclic" refers to a radical of a non-aromatic ring system having from 3 to 20 ring carbon atoms ("C3-20 carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In some embodiments of the application, a carbocyclyl group has from 3 to 18 ring carbon atoms ("C3-18 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 3 to 16 ring carbon atoms ("C3-16 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 3 to 12 ring carbon atoms ("C3-12 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 3 to 10 ring carbon atoms ("C3-10 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 3 to 8 ring carbon atoms ("C3-8 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 3 to 7 ring carbon atoms ("C3-7 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 4 to 6 ring carbon atoms ("C4-6 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 5 to 6 ring carbon atoms ("C5-6 carbocyclyl"). In some embodiments of the application, a carbocyclyl group has from 5 to 10 ring carbon atoms ("C5-10 carbocyclyl"). Some exemplary C3-6 carbocyclyl 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), and the like. Some exemplary C3-8 carbocyclyl groups include, but are not limited to, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), and the like. Some exemplary C3-10 carbocyclyl groups include, but are not limited to, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-lH-indenyl (C9), bicyclo[6.1.0]non-4-enyl (C9), bicyclo[6.1.0]nonyl (C9), bicyclo[6.1.0]non-4-ynyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decyl (C10), and the like. As noted previously, in some embodiments of the application, a carbocyclyl group is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spiro ring system such as a bicyclic ring system ("bicyclic carbocyclyl") or a tricyclic ring system ("tricyclic carbocyclyl")) and can be saturated or can contain one or more carbon-carbon double or triple bonds."Cycloalkyl" also includes ring systems in which a cycloalkyl ring as defined above is fused with one or more aryl or heteroaryl groups, where the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons continues to designate the number of carbons in the cycloalkyl ring system. Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In some embodiments of the application, a cycloalkyl group is an unsubstituted C3-14 cycloalkyl group. In some embodiments of the application, a cycloalkyl group is a substituted C3-14 cycloalkyl group. In some embodiments of the application, a cycloalkyl group is an unsubstituted C5-16 cycloalkyl group. In some embodiments of the application, a cycloalkyl group is a substituted C5-16 cycloalkyl group. In some embodiments of the application, "cycloalkyl" is a monocyclic saturated carbocyclic ring having from 3 to 14 ring carbon atoms ("C3-14 cycloalkyl"). In some embodiments of the application, a cycloalkyl group has from 3 to 10 ring carbon atoms ("C3-10 cycloalkyl"). In some embodiments of the application, a cycloalkyl group has from 3 to 8 ring carbon atoms ("C3-8 cycloalkyl"). In some embodiments of the application, a cycloalkyl group has from 3 to 6 ring carbon atoms ("C3-6 cycloalkyl"). In some embodiments of the application, a cycloalkyl group has from 4 to 6 ring carbon atoms ("C4-6 cycloalkyl"). In some embodiments of the application, a cycloalkyl group has from 5 to 6 ring carbon atoms ("C5-6 cycloalkyl"). In some embodiments of the application, a cycloalkyl group has from 5 to 10 ring 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 aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Some examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In some embodiments of the application, a cycloalkyl group is an unsubstituted C3-14 cycloalkyl group. In some embodiments of the application, a cycloalkyl group is a substituted C3-14 cycloalkyl group.

[0325] As used herein, the term “heterocyclyl” or “heterocycle” refers to a radical of a 3- to 20-membered nonaromatic ring system having ring carbon atoms and 1 to 8 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3- to 20-membered heterocyclyl”). The point of attachment can be a carbon or nitrogen atom, when valence permits, in a heterocyclyl group comprising one or more nitrogen atoms. The heterocyclyl group can be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused ring, bridged ring, or spiro ring system, such as a bicyclic ring system (“bicyclic heterocyclyl”), a tricyclic ring system (“tricyclic heterocyclyl”), or a tetracyclic ring system (“tetracyclic heterocyclyl”)) and can be saturated or can comprise one or more carbon-carbon double bonds or triple bonds. The ring system of a heterocyclyl polycycle can comprise one or more heteroatoms in one, two, or three rings. “Heterocyclyl” also includes ring systems in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl rings, wherein the point of attachment is on either the carbocyclyl ring or the heterocyclyl ring; or includes ring systems in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases the number of ring members continues to indicate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of a heterocyclyl group is independently unsubstituted (“unsubstituted heterocyclyl”) or substituted with one or more substituents (“substituted heterocyclyl”). In some aspects of the application, the heterocyclyl group is an unsubstituted 3- to 20-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is a substituted 3- to 20-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is an unsubstituted 5- to 18-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is a substituted 5- to 18-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is an unsubstituted 5- to 16-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is a substituted 5- to 16-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is an unsubstituted 5- to 12-membered heterocyclyl. In some aspects of the application, the heterocyclyl group is a substituted 5- to 12-membered heterocyclyl. In some embodiments of the application, the heterocyclyl group is a 5- to 10-membered nonaromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 10-membered heterocyclyl”). In some aspects of the application, the heterocyclyl group is a 5- to 8-membered nonaromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 8-membered heterocyclyl”). In some aspects of the application, the heterocyclyl group is a 5- to 6-membered nonaromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 6-membered heterocyclyl”). In some aspects of the application, the 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects of the application, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.In some embodiments of the application, 5- to 6-membered heterocyclyl groups have 1 ring heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur. Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, but are not limited to, triazolinyl, diazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azocinyl, oxocinyl, and thiocinyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthalenyl, decahydro-l,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, lH-benzo[e][l,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-lH-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-lH-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, l,2,3,4-tetrahydro-l,6-naphthyridinyl, and the like.

[0326] As used herein, the term "aryl" refers to a radical of 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 a cyclic array) having zero heteroatoms in the aromatic ring system and 6 to 14 ring carbon atoms ("C6-i4 aryl"). In some aspects of the application, aryl has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some aspects of the application, aryl has 10 ring carbon atoms ("C10 aryl"; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some aspects of the application, aryl has 14 ring carbon atoms ("C14 aryl"; e.g., anthryl). "Aryl" also includes ring systems in which an aryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment 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 specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In some aspects of the application, aryl is unsubstituted C6-i4 aryl. In some aspects of the application, aryl is substituted C6-i4 aryl.

[0327] The term “heteroaryl” refers to a radical of a 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) that is a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) ring system, having from 1 to 4 ring heteroatoms and ring 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 heteroaryls containing one or more nitrogen atoms, the point of attachment can be a carbon or a nitrogen atom. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclic or heterocyclic rings, wherein the point of attachment is on the heteroaryl ring, and in such cases the number of ring members continues to designate 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 to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryls in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., the ring having a heteroatom (e.g., 2-indolyl) or the ring not containing a heteroatom (e.g., 5-indolyl). In some embodiments of the application, a heteroaryl is a 5- to 10-membered aromatic ring system having from 1 to 4 ring heteroatoms and ring 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 aspects of the application, a heteroaryl is a 5- to 8-membered aromatic ring system having from 1 to 4 ring heteroatoms and ring 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 aspects of the application, a heteroaryl is a 5- to 6-membered aromatic ring system having from 1 to 4 ring heteroatoms and ring 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 aspects of the application, a 5- to 6-membered heteroaryl has from 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects of the application, a 5- to 6-membered heteroaryl has from 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects of the application, a 5- to 6-membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In some aspects of the application, a heteroaryl is an unsubstituted 5- to 14-membered heteroaryl. In some aspects of the application, a heteroaryl is a substituted 5- to 14-membered heteroaryl. Exemplary 5-membered heteroaryls containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl.Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, 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, pyridyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5-6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzodiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazyolyl, indolizinyl, and purinyl. Exemplary 6-6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, phenoxazinyl, phenothiazinyl, phenazinyl, and phenoxazinyl.

[0328] As used herein, unless explicitly provided otherwise, a group is optionally substituted. As used herein, the term "optionally substituted" or "optionally substituted" means substituted or unsubstituted. In some aspects of the present disclosure, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionally substituted. "Optionally substituted" means a group that can be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). In general, the term "substituted" means that at least one hydrogen present on the group is replaced with an allowed substituent (e.g., a substituent that results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, or other reaction). Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure can be substituted, the substituent is the same or different at each position. The term "substituted" is contemplated to include all allowed substituents of organic compounds and includes substitution by any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all combinations in order to arrive at a stable compound. For purposes of the present disclosure, a heteroatom such as nitrogen can have a hydrogen substituent and / or any suitable substituent as described herein that satisfies the valence of the heteroatom and results in the formation of a stable moiety. The present disclosure is not intended to be limited in any way by the exemplary substituents described herein.

[0329] A heteroatom such as nitrogen can have a hydrogen substituent and / or any suitable substituent as described herein that satisfies the valence of the heteroatom and results in the formation of a stable moiety. The present disclosure is not intended to be limited in any way by the exemplary substituents described herein.

[0330] In the present disclosure, "azide" in a sequence has the following structural element:

[0331] In the present disclosure, "amine-C6" in a sequence has the following structural element:

[0332] In the present disclosure, "BCN" in a sequence has the following structural element:

[0333] In the present invention, "M01" in a sequence has the following structural unit:

[0334] As used herein, when the listed linking groups do not indicate their direction of attachment, their direction of attachment is arbitrary. For example, in RA-L-RB, the linking group L has the structure -M-W-, which can be attached to RAand RBin the same direction as the reading order from left to right, i.e., to form RA-M-W-RB, or in the opposite direction as the reading order from left to right, i.e., to form RA-M-W-RB.

[0335] As used herein, when a certain sequence region is 0 nucleotides, it includes the case where the region does not exist and the two regions adjacent to it on the left and right are directly connected.

[0336] As used herein, "pharmaceutically acceptable carrier" can 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 for delivering one or more nucleic acids to an animal. Such agents are well known in the art.

[0337] As used herein, "subject" refers to any animal, such as a mammal or a marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, cows, rats, or any kind of poultry.

[0338] As used herein, the words "treat," "treatment," or "treating," relate to the management and / or elimination, reduction, or amelioration of a disease, as well as symptoms associated therewith, and refer to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the improvement of one or more established physiological signs or symptoms of the underlying disorder being treated, even though the subject can still be afflicted with the disorder. While it is possible for a disease or associated symptoms to be completely eliminated, treatment of a disease does not require that the disease or associated symptoms be completely eliminated. As used herein, the word "treat" also includes "prophylactic treatment," i.e., application prior to the manifestation of symptoms or disease to reduce the likelihood of occurrence or recurrence of a disease, or to reduce the likelihood of recurrence of a disease that has already been controlled. This applies to individuals who have not yet been affected but are at risk, or are prone to relapse, or individuals who are at risk or susceptible to relapse. In the sense of the present invention, "treatment" also includes prevention of recurrence or prevention of the stage, as well as treatment of acute or chronic signs, symptoms, and / or dysfunction. Treatment can be directed at symptoms, such as suppressing symptoms. Treatment can be carried out in the short term, in the medium term, or as a long-term treatment, such as maintenance treatment.

[0339] As used herein, "effective amount" means the amount of a drug that will elicit the desired local or systemic therapeutic effect at a reasonable benefit / risk ratio, applicable to any treatment alone or in conjunction with further doses. The desired local or systemic therapeutic effect in the treatment of a particular disease generally relates to the inhibition of the disease process. This includes the slowing of disease progression, particularly the interruption or reversal of disease processes. When used to prevent disease, the amount will be sufficient to avoid or delay occurrence of the disease. An effective amount of a drug need not have a curative effect or completely prevent the occurrence of the disease. The effective amount of a drug described above will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of accompanying therapy, if any, the specific route of administration and similar factors. Accordingly, the dosage of the drug can vary according to these parameters. If the initial dosage is insufficient to achieve the desired effect, a higher dosage (or an effective higher dosage achieved by a different, more localized route of administration) can be employed. In certain instances, the effective amount of a drug will also depend on its therapeutic index, solubility, and the like.

[0340] The compositions of the present application can additionally contain other auxiliary components conventionally present in pharmaceutical compositions at levels established in the art. Thus, for example, the compositions can contain additional, compatible pharmaceutical active agents, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or can contain additional substances useful in the physical formulation of the various dosage forms of the compositions of the present application, such as preservatives, antioxidants, and stabilizers. However, when added, such substances should not unduly interfere with the biological activities of the components of the compositions of the present application. The preparations can be sterilized and, if desired, mixed with auxiliary agents that do not deleteriously interact with the nucleic acids of the preparation, such as preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0341] Figure 1 is a liver homogenate treatment result for subject ds99.

[0342] Figure 2 shows the effect of a subject on inhibition of a target protein in NHP.

[0343] Figure 3 shows the effect of a subject on inhibition of a target protein in NHP.

[0344] Figure 4 shows the effect of a subject on inhibition of a target protein in NHP.

[0345] Figure 5 shows the activation of PBMC cytokines by a subject. DETAILED DESCRIPTION

[0346] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by a combination of the specific embodiments set forth below with other chemical or biological synthetic methods well known in the art, and equivalents thereof as will be apparent to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present application.

[0347] The following examples are intended to illustrate the present application and to better enable a person skilled in the art to make and use the application. The examples are not intended to limit the scope of the application. Modifications or variations of the present application, which are obvious to those skilled in the art, are intended to be within the scope of the application. Unless otherwise indicated, reagents, kits, and biological materials used in the present application are available from commercial sources. Unless otherwise indicated, kits are used according to the manufacturer's instructions.

[0348] Example 1 Preparation of Phosphoramidite Monomer M01

[0349] 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 concentration in vacuo, it was dissolved in ethyl acetate (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered to give M01-3, which was used directly in the next step. Step B: M01-3 (5.36 g, 14.204 mmol) was dissolved in dichloromethane (100 mL), 4,5-dicyanoimidazole (0.84 g, 7.102 mmol) and M04-4 (4.28 g, 14.204 mmol) were added, and stirred at room temperature for 2 hours under nitrogen protection. Saturated sodium bicarbonate (200 mL) was added, and extracted with dichloromethane (100 mL x 3). The organic phase was washed with water (100 mL x 3) and saturated brine (100 mL) in turn, dried over anhydrous sodium sulfate, filtered, and concentrated to give the phosphoramidite monomer of M01. 1 H NMR (400 MHz, CDC13) δ 7.38 (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 (6.4 Hz, 2H), 1.16 (dd, J = 6.8, 4.8 Hz, 12H). 31 P NMR (160 MHz, CDC13) δ 148.51.

[0350] Example 2 Synthesis of Precursor of D02 (diisopropylamino phosphorodithioate 2-cyanoethyl hexadecyl ester)

[0351] The synthesis method for preparing M01-3 from M01-2 in Example 1 was similarly applied.

[0352] 1 H NMR (400 MHz, CDC13) δ 3.90-3.72 (m, 2H), 3.68-3.52 (m, 4H), 2.63 (t, J = 6.8 Hz, 2H), 1.59 (p, J = 6.8 Hz, 2H), 1.28-1.24 (m, 26H), 1.17 (d, J = 6.8, 4.2 Hz, 12H), 0.87 (t, J = 6.8 Hz, 3H).

[0353] 31 P NMR: (400 MHz, CDC13) δ 147.24.

[0354] Example 3: Synthesis of siRNA molecules

[0355] Oligoribonucleotides were synthesized according to phosphoramidite solid phase synthesis techniques. Synthesis was performed on universal controlled pore glass CPG. All 2'-modified RNA phosphoramidites and ancillary reagents were commercially available reagents. All phosphoramidites were dissolved in anhydrous acetonitrile and molecular sieves were added, and the coupling time was 25 minutes using 5-ethylthio-lH-tetrazole (ETT) as activator. Phosphorothioate linkages were generated using 50 mM 3-((dimethylamino-methylene)amino)-3H-l,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1), reaction time 10 minutes, or oxidation of phosphodiester linkages using 0.05 M, iodine in pyridine / water (v / v = 9 / 1), reaction time 4 minutes. All sequences were synthesized without final DMT group removal.

[0356] Cleavage and deprotection of oligomers bound to CPG: After the termination of solid phase synthesis, the protecting groups were removed without cleaving the oligonucleotides from the CPG by treating with a solution of 20% diethylamine in acetonitrile for 30 minutes. Subsequently, the dried CPG was treated in an ammonia / ethanol (v / v = 3 / 1) system at 45°C for 20 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. Concentration of the combined solution gave a solid mixture.

[0357] Purification of single-stranded oligoribonucleotides: Purification by ion pair reverse phase using C18 column, buffer A, 0.1 M TEAA in 5% acetonitrile in water; buffer B, acetonitrile. After obtaining the target product, anion exchange was performed. The oligomer was purified by HPLC using NanoQ anion exchange. Buffer A was 0.1 M ammonium acetate 15% acetonitrile in water, and buffer B was 1.5 M sodium bromide + 0.1 M ammonium acetate 15% acetonitrile in water. The target product was isolated and desalted using a reverse phase C18 column.

[0358] Amino coupling click chemistry linker: Add the oligonucleotide with amino group at the 5' end in a centrifuge tube, and dissolve it using sterile enzyme-free water. Add a DMF solution of BCN-OSU active ester or azido-C4 carboxylic acid active ester, add 50 equivalents of DIPEA to the reaction solution, stir overnight at room temperature, perform ion exchange column purification, reverse phase liquid chromatography purification, desalination, and lyophilization to obtain a fragment oligonucleotide that can be used for click chemistry ligation.

[0359] Forming a long single strand by click chemistry. (1) SPAAC method: Dissolve the fragment oligonucleotides connected with equal amounts of BCN-OSU active ester and azido-C4 carboxylic acid active ester, respectively, in sterile enzyme-free water, add 1 M HEPES solution, stir at 40°C for 3 hours, perform reverse phase liquid chromatography purification, ion exchange column purification, desalination, and lyophilization to obtain a long single-stranded oligonucleotide coupled by a linker; (2) CuAAC method: Prepare a CuSO4 / THPTA solution, mix 15 mM copper sulfate solution and 30 mM THPTA solution, and vortex for 15 minutes. Dissolve the oligonucleotides connected with equal amounts of terminal alkyne oligonucleotide fragments and azido-C4 carboxylic acid active ester fragments, respectively, in sterile enzyme-free water, add 1 M HEPES solution, and replace the air with nitrogen. Then quickly add the prepared CuSO4 / THPTA (1.5 equivalents / 3 equivalents) and sodium ascorbate solution (45 mM, 9 equivalents), replace the air with nitrogen again, and stir overnight at room temperature. Add 0.5 mol / L EDTA-Na solution to capture copper ions, perform reverse phase liquid chromatography purification, ion exchange column purification, desalination, and lyophilization to obtain a long single-stranded oligonucleotide coupled by a linker.

[0360] Annealing of single-stranded oligoribonucleotides to produce siRNA: Dissolve the single-stranded oligoribonucleotides to be annealed in sterile enzyme-free water. Form complementary strands by combining equal molar amounts of single-stranded oligoribonucleotide solutions. Set up the annealing reaction system as follows, place the mixture in a 95°C water bath for 2 minutes, reduce to room temperature, and freeze-dry to obtain the final product siRNA.

[0361] The mass spectrometry of the compounds is as follows:

[0362] Test Example 1: psiCHECK-2 plasmid transfection cell activity test

[0363] Purpose of the study

[0364] The purpose of this study is to evaluate the in vitro inhibitory activity of the compound on LPA.

[0365] Materials and methods

[0366] The test compound was prepared into a 100 mM stock solution with RNase-free water. Huh7 was resuscitated and cultured in DMEM medium (Gibco catalog number 11965-092) containing 10% fetal bovine serum (FBS, Gibco catalog number 10091148), 1% penicillin-streptomycin (PS, HyClone catalog number SV30010). The main instruments used in the experiment include a fluorescent multi-mode microplate reader (Synergy HTX), a centrifuge (Beckman Allegra-X15R Centrifuge), and a cell counter (Countstar Rigel S2). The main reagents used in the experiment include 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. 2105), a centrifuge (Beckman Allegra-X15R Centrifuge), and a cell counter (Countstar Rigel S2). The main reagents used in the experiment include 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.

[0367] Test method

[0368] 1 On day 0, psiCHECK-2 plasmid transfection

[0369] Add 300 μL Opti MEM and 3 μg psiCHECK-2 plasmid to an RNase-free EP tube (mixing solution #1). Take one bottle of T15 cell bottle and add trypsin-digested Huh7 cells, count the cells using a cell counter, and adjust the cell density to 1*10^5 / ml. Transfer 12 μL Fugene-HD to the mixing solution #1 tube, mix the resulting solution and incubate for 15 minutes (mixing solution #2). Add the solution to 12 mL of cell suspension, mix well, and aliquot the suspension into a 96-well plate (100 μL / well).

[0370] 2 On day 1, siRNA transfection

[0371] Dilute the siRNA with Opti-MEM medium at a ratio of 4.7:0.3 Incubate RNAiMAX reagent at room temperature for 15 minutes. Dilute siRNA with water without RNA to make 20x stock solution (for example, if the final test concentration is 25nM, make this stock solution to 500nM). Mix equal volume of diluted RNAiMax and siRNA (v:v = 1:1). Incubate the mixture at room temperature for 15 minutes to form complex. Take 25 μl complex per well, mix with 225 μl fresh DMEM medium, discard the supernatant in assay plate, take 120 μl complex mixture to 96 well plate.

[0372] Dual fluorescence (Firefly and Renilla) detection

[0373] After 48 hours, remove the culture medium. Dilute the culture medium with 10% FBS at 1:1 ratio Reagent, add 120 μL, shake for 15 minutes to lyse the cells, transfer 60 μL to white 96 well plate. Detect Firefly fluorescence with Envision. Detect Renilla fluorescence with Stop and Reagent, prepared at 1:100 ratio, add 30 μL to white 96 well plate, detect Renilla fluorescence with Envision.

[0374] Data analysis

[0375] Detection ratio = (Renilla luminescence value of sample - Renilla luminescence value of background) / (Firefly luminescence value of sample - Firefly luminescence value of background)

[0376] Inhibition % = (1 - sample ratio / RNAiMAX control ratio) x 100%

[0377] Test Example 2: Huh7 cell activity test

[0378] Purpose of the study

[0379] The purpose of this experiment is to evaluate the in vitro inhibitory activity of the test substance on the target gene PCSK9 & ANGPTL3 mRNA in Huh-7 cells.

[0380] Materials and methods

[0381] Materials

[0382] Test compound

[0383] Test compound: prepare 100 uM siRNA stock solution with RNase free H2O.

[0384] Cell line

[0385] Huh-7 cells were cultured in DMEM medium (Gibco Cat# 11965-092) containing 10% fetal bovine serum (FBS, Gibco Cat# 2279804CP) and 1% NEAA (ThermoFisher Cat# 11140050).

[0386] Main instruments

[0387] The main instruments used in this experiment include fluorescent qPCR instrument (Roche 480), centrifuge (ThermoFisher SORVALL ST4 Plus), cell counter (Countstar Rigel2), PCR instrument (Dongshenglong ETC811), carbon dioxide incubator (ThermoFisher HERACELL240i).

[0388] Main reagents and consumables

[0389] The main reagents used in this experiment include Lipofectamine TM RNAiMAX transfection reagent (INVITROGEN, Cat# 13778150), SuperReal PreMix Plus SYBR Green (Tiangen Cat# FP205), RNA extraction kit (Qiagen Cat# 74182), FastKing RT Kit With gDNase (TianGen Cat# KR116), 96-well plate (Costar 3799), qPCR specific primers for GAPDH, qPCR specific primers for PCSK9 & ANGPTL3.

[0390] Experimental methods

[0391] 1 Compound transfection plating

[0392] Huh-7 cells were seeded (2x104cells / well) into 96-well cell plates, and siRNA was transfected into cells using RNAiMAX at the same time. The siRNA test was set at 7 concentration points (10nM, 2.5nM, 0.625nM, 0.156nM, 0.0391nM, 0.0098nM, 0.0024nM), and cultured in a 37°C, 5% CO2 incubator for 24 hours. Two replicate wells were measured in parallel. At the same time, a no-compound control group containing RNAiMAX was set.

[0393] 2 RNA extraction and reverse transcription

[0394] After 24 hours of transfection, the culture medium was removed and the cells were collected for RNA extraction. The RNA extraction kit was used according to the instructions. 96 Total RNA was extracted by RNeasy® Kit (QIAGEN-74182). cDNA was synthesized by FastKing RT Kit (With gDNase) (Tiangen-KR116) according to the instruction.

[0395] 3 qPCR to detect the expression level of target gene mRNA

[0396] The target cDNA will be detected by SYBR Green qPCR, with GAPDH cDNA as internal control. The cDNA was diluted 2.5 times with RNase free H2O, and 7.5 μL of prepared PCR reaction solution and 2.5 μL of sample cDNA were added to a 384-well plate. The qPCR reaction program was as follows: 50°C for 2 min, 95°C for 10 min, then enter the cycle mode, 95°C for 15 sec, then 60°C for 1 min, for a total of 40 cycles.

[0397] Data analysis

[0398] The expression level of target gene mRNA of each sample was calculated by ΔΔCt relative quantification method. The relative expression of target gene was represented by 2-ΔΔCT.

[0399] The calculation formula is as follows:

[0400] ΔCT = average Ct value of target gene - average Ct value of internal reference gene

[0401] ΔΔCT = ΔCT (drug group) - ΔCT (RNAiMAX control group)

[0402] Relative expression of target gene PCSK9 & ANGPTL3 = 2-ΔΔCT

[0403] Inhibition rate of PCSK9 & ANGPTL3 = (1-value of sample / Ave.value of RNAiMAX Control)*100

[0404] GraphPad Prism software was used for graph analysis.

[0405] Results of Test Examples 1 and 2: See Tables E2-1, E2-2, E2-3, E2-4 and Table E2-5. (Note: the inhibition activity of PCSK9 was detected by the method of Test Example 2, and the LPA inhibition activity was detected by the method of Test Example 1.)

[0406] Table E2-1 Cell activity test results

[0407] Table E2-2 Cell activity test results

[0408] Table E2-3 Cell Activity Test Results

[0409] Table E2-4 Cell Activity Test Results

[0410] Table E2-5 Cell Activity Test Results

[0411] Conclusion:

[0412] The compound of the present application shows significant inhibitory activity on both the first target gene and the second target gene.

[0413] Test Example 3: PHH Cell Activity Test Test Compound: Nuclease-Free Water was used to prepare a corresponding concentration stock solution.

[0414] Cell Strain:

[0415] The PHH cells were provided by Shanghai or Chengdu PharmaGenn Biopharma Development Co., Ltd. The PHH cells were cultured in InvitroGRO CP medium containing 10% fetal bovine serum.

[0416] Main Instruments:

[0417] The main instruments used in this experiment include a fluorescence qPCR instrument (Quanstudio 7flex), a centrifuge (Beckman Allegra-X15R Centrifuge), and a cell counter (Countstar Rigel S2).

[0418] Main Reagents and Consumables:

[0419] The main reagents and consumables used in this experiment include: an RNA extraction kit (Qiagen-74182), a FastKing cDNA first strand synthesis kit (TIANGEN-KR116-02), a FasStart Universal Probe Master (Roche-04914058001), an AceQ Universal U Probe Master Mix V2 (Vazyme-Q513-02), a FasStart Universal SYBR Green Master (Roche-4913914001), and a 96-well plate (Costar 3599).

[0420] ​Target gene PCSK9 and internal reference gene primers and probes were purchased from ThermoFisher. Other reagents and consumables were provided by WuXi.

[0421] Experimental methods:

[0422] Compound free uptake plating

[0423] Dilute the test compound to a final concentration of 10 times (e.g. a final concentration of 25 nM, corresponding to dilution to 250 nM) with Nuclease-Free Water.

[0424] Inoculate PHH cells (5.4 x 104cells / well) into collagen-coated 96-well cell plates, and at the same time, add different concentrations of siRNA to the corresponding cell wells. At the same time, set up a no-compound control group containing Nuclease-Free Water.

[0425] RNA extraction and reverse transcription

[0426] After 48 hours of free uptake, remove the culture medium and collect the cells for RNA extraction. Extract total RNA using the RNA extraction kit (Qiagen-74182) according to the kit instructions. Synthesize cDNA using the FastKing cDNA first-strand synthesis kit (TIANGEN-KR116-02) according to the instructions.

[0427] qPCR detection of target gene mRNA expression level

[0428] The target cDNA will be detected by qPCR, and the corresponding internal reference gene (such as GAPDH cDNA or β-actin cDNA) will be detected in parallel. qPCR reaction program (TapMan Probe): heat at 95°C for 10 minutes, then enter the cycle 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 the cycle mode, heat at 95°C for 15 seconds, then at 60°C for 1 minute, for a total of 40 cycles.

[0429] Data analysis

[0430] The expression level of the target gene mRNA of each sample is calculated by the ΔΔCT quantitative method. The relative expression of the target gene is represented by 2-ΔΔCT.

[0431] The calculation formula is as follows:

[0432] ΔCT = average Ct value of target gene - average Ct value of internal reference gene

[0433] ΔΔCT = ΔCT (drug group) - ΔCT (Nuclease-Free Water control group)

[0434] Target gene relative expression = 2-ΔΔCT

[0435] Target gene inhibition rate % = (1 - value of sample / Nuclease-Free Water Control) * 100

[0436] GraphPad Prism software was used for graphing analysis, and the inhibition rate results were expressed as mean ± SD.

[0437] Test results: see Table E3-1, Table E3-2 and Table E3-3.

[0438] Table E3-1 PHH cell test PCSK9 activity results

[0439] Table E3-2 PHH cell test PCSK9 activity results

[0440] Conclusion: The compounds of the present application show significant inhibitory activity on both the first target gene and the second target gene. In particular, the compounds with two GalNAc delivery systems show higher inhibitory activity under free uptake conditions.

[0441] Test Example 6 Rat liver homogenate treatment reaction

[0442] 1.00 mg of 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 10000 ng / mL. The culture sample was added with 50.0 μL of IS working extraction solution (phenol / chloroform / isopentanol = 25 / 24 / 1, v / v / v) for liquid-liquid extraction. After centrifugation, 300 μL of supernatant was subjected to solid phase extraction. Then, the sample was transferred to an equilibrated solid phase extraction plate, and the solid phase extraction plate was washed and eluted. The collected eluate was evaporated. LC-HRMA: liquid chromatography-high resolution mass spectrometer; LC: Shimadzu, LC-30AD; HRMA: Q Exactive Plus or Q Exactive Focus (Thermo San Jose, CA)

[0443] Test substance ds99:

[0444] SS (5'→ 3'): g*u*caucCfaCfAfAfugagagUfacu[L96]

[0445] AS (5'→ 3'): (M06)*CfGfaa*Gfuac(Tgn)cucauugUfgGfaugac*g*a

[0446] The results are shown in Figure 1. After 48h incubation, the observed products include un-digested test substance (31%), test substance with 1 nucleotide cleavage at 3' end (15%), test substance with 2 nucleotide cleavage at 3' end and M06 cleavage (18%), test substance with 3 nucleotide cleavage at 5' end (15%+5%+10%), and other minor metabolites (5%).

[0447] It can be seen that (1) the only 5' cleavage product is the product of cleavage between the 3rd and 4th nucleotides at 5' end of the test substance; (2) the 3' end cleavage products include the test substance with cleavage of one or two nucleotides at 3' end; (3) among all cleavage products, the product of cleavage between the 3rd and 4th nucleotides at 5' end is the major product.

[0448] Test Example 8: NHP in vivo efficacy test

[0449] Dosing regimen: On the day of dosing, the dosing animals were weighed, and the prepared formulation was drawn according to the weight information and the dosing volume. The dosing site (dorsal waist) of the selected animals was disinfected, and then Z1-1 (dose: 6 mg / kg) and Z2-1 (dose: 6 mg / kg) were administered, and dry cotton ball was used for hemostasis after completion of dosing. After dosing, the animals were bled on D7, and the animal bleeding site was disinfected using alcohol cotton ball, and dry cotton ball was used for hemostasis after bleeding. The collected whole blood was sent to the clinical laboratory department for centrifugation to obtain serum. The serum was detected for blood lipid and PCSK9 protein content.

[0450] The serum centrifuged after blood collection was detected for blood biochemistry using HITACHI 008AS biochemical instrument. After turning on the biochemical instrument, the reagent was replaced, and then quality control and / or calibration were performed. After determining that the instrument met the operation requirements, the sample to be tested was placed on the sample tray, then the animal tattoo number on the sample label was checked, and the detection of blood lipid (LPa) was started.

[0451] (I) Detection of PCSK9 protein level in serum of cynomolgus monkeys

[0452] Reagents

[0453] Human PCSK9 ELISA Kit (Sino Biological-KIT10594).

[0454] Instrument

[0455] Microplate reader (Molecular Devices-SpectraMax 3841, Molecular Devices-SpectraMax M5, Molecular Devices-SpectraMax M2e), centrifuge eppendorf 5724R.

[0456] Experimental procedure

[0457] Reagent configuration

[0458] (1) 1 x wash buffer

[0459] Add 10 mL of 20 x wash buffer to 190 mL of deionized water to make 200 mL of 1 x wash buffer.

[0460] (2) 1 x dilution buffer:

[0461] Add 2.5 mL of 20 x dilution buffer to 47.5 mL of deionized water to make 50 mL of 1 x dilution buffer.

[0462] (3) Detection antibody:

[0463] Dilute 1:660 with 1 x dilution buffer, configure 10 minutes before use, prepare extemporaneously as needed.

[0464] (4) Color reagent:

[0465] Mix equal amounts of color reagent A and B together 10 minutes before use, take care not to contaminate the color reagent, protect from light.

[0466] Prepare samples

[0467] Thaw samples at room temperature, centrifuge at 2000 rpm for 2 minutes, dilute samples in the assay by a certain number of folds.

[0468] Prepare the calibration curve

[0469] 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).

[0470] Experimental Procedure

[0471] (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.

[0472] (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;

[0473] (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.

[0474] (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;

[0475] (5) Remove the liquid from the hole and repeat the washing operation in step (3);

[0476] (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;

[0477] (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.

[0478] Data Analysis

[0479] 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.

[0480] Results: see Figure 2.

[0481] Conclusion: the compound of the present application can significantly down-regulate target protein in NHP

[0482] Test Example 9: NHP long-term plasma target gene knockout model

[0483] The in vivo target gene knockout effect of the modified siRNA was evaluated in cynomolgus monkeys. On the day of administration, the animals to be administered were weighed, and the prepared preparation was drawn according to the body weight information and the administration volume. The administration site (dorsal waist) of the selected animals was disinfected, and for the mixed administration group animals (N = 2), Inclisiran and R1 were mixed and prepared, and a single subcutaneous injection was performed at the same position of the animals, and the drug dose in the mixture was 3 mg / kg, and the total dose was 6 mg / kg; for another group of animals (N = 2), a single subcutaneous injection of Z1-1 reagent at a dose of 6 mg / kg was performed. After completing the administration, dry cotton balls were used for hemostasis. Blood sampling was performed before administration and on day 7, day 14, day 21, day 28, day 56 and day 85 after administration. After the animal blood sampling site was disinfected with alcohol cotton ball, blood sampling was performed, and dry cotton ball was used for hemostasis after blood sampling. The collected whole blood was sent to the laboratory department for centrifugation to obtain serum.

[0484] The serum centrifuged after blood sampling was subjected to blood biochemical detection using a HITACHI 008AS biochemical analyzer. After turning on the biochemical analyzer, the reagent was replaced, followed by quality control and / or calibration. After determining that the instrument meets the operation requirements, the sample to be tested is placed on the sample tray, then the animal tattoo number on the sample label is checked, and the detection of LPa, LDL-c and ApoB is started. The data is expressed as a percentage of the baseline value, and is presented as the mean value plus / minus the standard error of the mean value.

[0485] According to the experimental steps provided by the manufacturer, the circulating PCSK9 protein content of cynomolgus monkeys was quantified using an ELISA kit specific for human PCSK9 (and cross-reactive with cynomolgus monkeys) (Sino Biological-KIT10594). The data is expressed as a percentage of the baseline value, and is presented as the mean value plus / minus the standard error of the mean value.

[0486] R1:

[0487] Results: see Figure 3.

[0488] Conclusion: the double-targeting compound produced by the present technology can inhibit the target in NHP, and is significantly superior to the single-target compound mixture in terms of persistence.

[0489] Test Example 10: NHP long-term plasma target gene knockout model

[0490] The in vivo target gene knockdown effect of the modified siRNA was evaluated in cynomolgus monkeys. On the day of dosing, the dosing animals were weighed, and the prepared formulation was drawn up according to the body weight information and the dosing volume. The dosing sites (dorsal lumbar) of the selected animals were disinfected, and a plurality of different doses of Z1-1 reagent were subcutaneously injected into a plurality of groups of animals (N = 4). After the completion of dosing, a dry cotton ball was used for hemostasis. Blood sampling was performed before dosing and on day 7, day 14, day 21, day 28, day 56, and day 85 after dosing. After the blood sampling site of the animals was disinfected using an alcohol cotton ball, blood was collected, and a dry cotton ball was used for hemostasis after blood collection. The collected whole blood was sent to the clinical laboratory department for centrifugation to obtain serum.

[0491] The serum centrifuged after blood sampling was subjected to blood biochemical detection using a HITACHI 008AS biochemical analyzer. After the biochemical analyzer was turned on, the reagents were replaced, followed by quality control and / or calibration. After it was determined that the instrument met the operation requirements, the samples to be tested were placed on the sample tray, then the animal tattoo number on the sample label was checked, and the detection of LPa, LDL-c, and ApoB was started. The data were expressed as percentages of baseline values and presented as mean values plus / minus standard errors of the mean values.

[0492] The circulating PCSK9 protein content of cynomolgus monkeys was quantified using an ELISA kit (Sino Biological-KIT10594) specific for human PCSK9 (and cross-reactive with cynomolgus monkeys) according to the experimental steps provided by the manufacturer. The data were expressed as percentages of baseline values and presented as mean values plus / minus standard errors of the mean values.

[0493] Experimental results: see Figure 4.

[0494] Experimental conclusion: the compound of the present application showed dose-dependent inhibition in NHPs and was able to significantly inhibit two targets simultaneously.

[0495] Test Example 12: PBMC cytokine activation test

[0496] Experimental materials:

[0497] Experimental steps:

[0498] PBMC cell treatment:

[0499] 1) Freshly extracted whole blood (10 mL) of healthy volunteers was collected in an EDTA anticoagulant tube vacuum blood collection tube. The whole blood sample was mixed evenly by repeatedly inverting up and down immediately after collection to ensure sufficient contact with the anticoagulant.

[0500] 2) Blood sample was diluted 2-fold with PBS at the same volume, 10 mL Lymphoprep (lymphocyte separation medium) was added to a 50 mL centrifuge tube, and 20 mL of the diluted blood sample was slowly added on top of the Lymphoprep, taking care not to break the interface.

[0501] 3) The centrifuge tube after sample addition was centrifuged at 1000xg for 25 min at room temperature without brake.

[0502] 4) The middle white membrane layer where the PBMCs were collected was transferred to a new 50 mL centrifuge tube, washed twice with 40 mL PBS, centrifuged at 350xg for 10 min at room temperature, and the cells were resuspended in complete medium at a cell density of 1.6x10^6 cells / mL.

[0503] 5) The PBMCs were plated in a 96-well plate at a cell number of 2E5 (125 μL) per well.

[0504] 6) The diluted test drug Z1-1 (125 μL) was added to the 96-well plate according to the plate map, with 2 duplicate wells; the blank group was added with the same content of RNase-free water medium and DMSO medium.

[0505] 7) After gently mixing the well plate, it was incubated in an incubator for 18-20 h.

[0506] 8) The supernatant was collected by centrifugation, and the secretion amounts of IL-6, TNF-α, IL-1β, and IFN-γ were detected by ELISA.

[0507] 9) The data were analyzed using GraphPad Prism 8 software.

[0508] Experimental results: see Figure 5.

[0509] Experimental conclusion: the compound of the present application has a low immunogenicity risk.

Claims

1. The compound represented by formula (I), wherein: DS1 is a double-stranded oligonucleotide comprising a first sense strand and a first antisense strand, which form a first double-stranded portion of 15-27 nucleotide pairs in length, and a first 5' extension in the first antisense strand upstream of the first double-stranded portion, the first 5' extension being at least 3 nucleotides in length and cleavable at the 3' most nucleotide of the first 5' extension to yield a cleaved DS1 product comprising the first double-stranded portion, which is capable of silencing a first target RNA or inhibiting expression of a first target gene by RNA interference; DS2 is a double-stranded oligonucleotide comprising a second sense strand and a second antisense strand, which form a second double-stranded portion of 15-27 nucleotide pairs in length, and a second 5' extension in the second antisense strand upstream of the second double-stranded portion, the second 5' extension being at least 3 nucleotides in length and cleavable at the 3' most nucleotide of the second 5' extension to yield a cleaved DS2 product comprising the second double-stranded portion, which is capable of silencing a second target RNA or inhibiting expression of a second target gene by RNA interference; L is a linking structural unit, one end of L being linked to the 5' end of the first sense strand and the other end of L being linked to the 5' end of the second sense strand; the first target RNA or first target gene is PCSK9 and the second target RNA or second target gene is LPA; the first antisense strand comprises the following sequence (5'-3'): a*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a; the second antisense strand comprises the following sequence (5'-3'): a*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g (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 deoxyriboside; (dC): cytosine deoxyriboside; *: phosphorothioate bond).

2. The compound of claim 1, wherein the first antisense strand comprises the following sequence (5'-3'): CfGfaa*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a.

3. The compound of claim 1 or 2, wherein the second antisense strand comprises the following sequence (5'-3'): CfGfaa*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g.

4. The compound according to any one of claims 1-3, wherein wherein the first antisense strand and / or second antisense strand further comprises a capping group at the 5’ end, optionally the capping group is linked to the 5’ end of the first 5’ extension and / or second 5’ extension.

5. The compound according to claim 4, wherein the first antisense strand comprises or has the following sequence (5’-3’): (invAB)*CfGfaa*CfaAfAfAfgCfaAfaAfcAfgGfuCfuag*a*a.

6. The compound according to claim 4 or 5, wherein the second antisense strand comprises or has the following sequence (5’-3’): (invAB)*CfGfaa*Ufaa(dC)u(dC)uguc(dC)aUfuaccauu*g*g.

7. The compound according to any one of claims 1-6, wherein the first sense strand comprises or has the following sequence (5’-3’): c*uagacCfuGfu(dT)uugcuuuu*g*u.

8. The compound according to any one of claims 1-7, wherein the second sense strand comprises or has the following sequence (5’-3’): a*augguaaUfgGfaCfagaguuau.

9. The compound of any one of claims 1-8, wherein L is 10. The compound according to any one of claims 1-9, wherein further comprising 1 or more delivery systems, optionally each of the delivery systems is independently linked to DS1 or DS2; preferably, the compound comprises 1 delivery system.

11. The compound according to claim 10, wherein each of the delivery systems is independently linked to the 5’ end of the first sense strand, linked to the 3’ end of the first sense strand, linked to the 5’ end of the second sense strand, or linked to the 3’ end of the second sense strand; preferably, the delivery system is linked to the 3’ end of the second sense strand.

12. The compound according to claim 10 or 11, wherein each of the delivery systems is independently a ligand, preferably the ligand alters the distribution, targeting or longevity, more preferably the ligand provides enhanced affinity to a target, such as a molecule, a cell or cell type, a compartment, a receptor, such as a cellular or organ compartment, a tissue, an organ or a body region, more preferably the ligand enables delivery of the compound to a target tissue and produces an RNA interference effect.

13. The compound according to any one of claims 10-12, wherein each of the ligands is independently selected from a GalNAc ligand, a lipophilic ligand, or other ligand targeting a receptor to facilitate endocytosis of the compound, such as a TfR targeting ligand, a LDL-R targeting ligand or an integrin targeting ligand; optionally each of the ligands is independently NAG37 or L96; preferably, the ligand is L96.

14. The compound according to any one of claims 10-13, wherein the second sense strand comprises or has the following sequence (5’-3’): a*augguaaUfgGfaCfagaguuau[L96].

15. The compound of any one of the preceding claims, selected from Z1-1, or a pharmaceutically acceptable salt thereof.

16. A pharmaceutical composition comprising a compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

17. A method of inhibiting expression of a target gene in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 16.

18. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 16, optionally the disease or disorder is related to a first target RNA or a first target gene and / or a second target RNA or a second target gene.

19. The method of claim 18, the disease or disorder comprises, but is not limited to, atherosclerotic cardiovascular disease (e.g., coronary artery disease (CAD), myocardial infarction (MI), ischemic stroke, peripheral arterial disease (PAD)), calcific aortic valve stenosis (CAVS), familial hypercholesterolemia (FH), diabetes with CVD, chronic kidney disease (CKD) with cardiovascular disease, etc.

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