Double-stranded nucleic acid compounds that inhibit ZPI

Abstract

The present application provides novel nucleic acid compounds suitable for therapeutic use. In addition, the present application provides methods of making these compounds, and methods of using these compounds in the treatment of various diseases and conditions.

Description

Technical Field

[0001] The present application provides novel nucleic acid compounds suitable for therapeutic use. In addition, the present application provides methods for preparing these compounds, and methods for using these compounds to treat various diseases and conditions. Background Art

[0002] Nucleic acid compounds have important therapeutic applications in medicine. Nucleic acids can be used to silence genes that cause specific diseases. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencers are promising alternatives to traditional small organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotides / oligonucleosides that prevent protein formation through gene silencing.

[0003] Over the past two decades, a large number of modified siRNA compounds have been developed for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutics for the treatment of various diseases, including central nervous system diseases, inflammatory diseases, metabolic disorders, tumors, infectious diseases, and ocular diseases.

[0004] The present application relates to nucleic acid compounds for treating and / or preventing diseases. Summary of the invention

[0005] According to the first aspect of the present application, a nucleic acid for inhibiting ZPI expression is provided, which comprises a duplex region, wherein the duplex region comprises a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) is at least partially complementary to a portion of RNA transcribed from the ZPI gene, and (ii) comprises at least 17 consecutive nucleosides that differ by 0 or 1 nucleoside from any one of the first strand sequences listed in Table 2.

[0006] According to the second aspect of the present application, a nucleic acid for inhibiting ZPI expression is provided, which comprises a duplex region, wherein the duplex region comprises a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) is at least partially complementary to a portion of RNA transcribed from a ZPI gene, and (ii) comprises at least 17 consecutive nucleosides that differ by 0 or 1 nucleoside from any one of the first strand modified sequences listed in Table 3.

[0007] The nucleic acid as described herein, wherein the first strand comprises nucleotides 2-18 of any one of the sequences of the first and second aspects of the present application.

[0008] According to the nucleic acid of the first aspect of the present application, the second strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differs by 0 or 1 nucleotide from any second strand sequence listed in Table 2, and wherein the second strand has a region that is at least 85% complementary to the first strand over the 17 consecutive nucleotides.

[0009] According to the nucleic acid of the first aspect of the present application, the second strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differs by 0 or 1 nucleotide from any second strand sequence listed in Table 2, and the duplex region comprises at least 14, 15, 16 or 17 complementary base pairs.

[0010] According to the nucleic acid of the above-mentioned second aspect of the present application, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs by 0 or 1 nucleoside from any second strand modified sequence listed in Table 4, and wherein the second strand has a region that is at least 85% complementary to the first strand in 17 consecutive nucleosides.

[0011] According to the nucleic acid of the above-mentioned second aspect of the present application, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs by 0 or 1 nucleoside from any second strand modified sequence listed in Table 4, and the duplex region comprises at least 14, 15, 16 or 17 complementary base pairs.

[0012] According to the nucleic acid of the first aspect of the present application, the first strand comprises any one of the first strand sequences listed in Table 2.

[0013] According to the nucleic acid of the second aspect of the present application, the first strand comprises any one of the first strand modified sequences listed in Table 3.

[0014] According to the nucleic acid of the first aspect of the present application, the second strand comprises any second strand sequence listed in Table 2.

[0015] According to the nucleic acid of the second aspect of the present application, the second strand comprises any one of the second strand modified sequences listed in Table 4.

[0016] The nucleic acid according to the first aspect of the present application, wherein the first strand comprises any one of the following sequences: the first strand comprises any one of the following sequences: SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:238, SEQ ID NO:239.

[0017] The nucleic acid according to the second aspect of the present application, wherein the first strand comprises any one of the following sequences: SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:372, SEQ ID NO:378, SEQ ID NO:379, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:498, SEQ ID NO:518, SEQ ID NO:538, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550 NO:551, SEQ ID NO:552, SEQ ID NO:558, SEQ ID NO:559.

[0018] The nucleic acid according to the first aspect of the present application, wherein the second chain comprises any one of the following sequences: SESEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:346, SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:358, SEQ ID NO:359.

[0019] The nucleic acid according to the second aspect of the present application, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 566, SEQ ID NO: 567, SEQ ID NO: 568, SEQ ID NO: 569, SEQ ID NO: 571, SEQ ID NO: 572, SEQ ID NO: 578, SEQ ID NO: 579, SEQ ID NO: 584, SEQ ID NO: 585, SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 698, SEQ ID NO: 718, SEQ ID NO: 738, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: NO:751, SEQ ID NO:752, SEQ ID NO:758, SEQ ID NO:759.

[0020] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0021]

[0022]

[0023] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0024]

[0025]

[0026] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0027] Unmodified first strand Unmodified second strand SEQ ID NO:128 SEQ ID NO:248 SEQ ID NO:144 SEQ ID NO:264 SEQ ID NO:148 SEQ ID NO:268 SEQ ID NO:149 SEQ ID NO:269 SEQ ID NO:138 SEQ ID NO:258

[0028] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0029] Modified first strand Modified second strand SEQ ID NO:368 SEQ ID NO:568 SEQ ID NO:384 SEQ ID NO:584 SEQ ID NO:388 SEQ ID NO:588 SEQ ID NO:389 SEQ ID NO:589 SEQ ID NO:538 SEQ ID NO:738

[0030] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0031] Unmodified first strand Unmodified second strand SEQ ID NO:148 SEQ ID NO:268 SEQ ID NO:145 SEQ ID NO:265 SEQ ID NO:144 SEQ ID NO:264 SEQ ID NO:165 SEQ ID NO:285 SEQ ID NO:202 SEQ ID NO:322

[0032] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of, or consist essentially of a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide:

[0033] Modified first strand Modified second strand SEQ ID NO:388 SEQ ID NO:588 SEQ ID NO:385 SEQ ID NO:585 SEQ ID NO:384 SEQ ID NO:584 SEQ ID NO:405 SEQ ID NO:605 SEQ ID NO:442 SEQ ID NO:642

[0034] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0035] Modified first strand Modified second strand SEQ ID NO:762 SEQ ID NO:772 SEQ ID NO:763 SEQ ID NO:773 SEQ ID NO:764 SEQ ID NO:774 SEQ ID NO:765 SEQ ID NO:775 SEQ ID NO:766 SEQ ID NO:776

[0036]

[0037] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0038] Modified first strand Modified second strand SEQ ID NO:385 SEQ ID NO:585 SEQ ID NO:388 SEQ ID NO:588 SEQ ID NO:764 SEQ ID NO:774 SEQ ID NO:765 SEQ ID NO:775 SEQ ID NO:766 SEQ ID NO:776 SEQ ID NO:767 SEQ ID NO:777

[0039] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0040] Unmodified first strand Unmodified second strand SEQ ID NO:145 SEQ ID NO:265 SEQ ID NO:148 SEQ ID NO:268

[0041] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0042]

[0043]

[0044] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0045] Modified first strand Modified second strand SEQ ID NO:388 SEQ ID NO:588 SEQ ID NO:766 SEQ ID NO:776 SEQ ID NO:767 SEQ ID NO:777 SEQ ID NO:810 SEQ ID NO:820 SEQ ID NO:811 SEQ ID NO:820 SEQ ID NO:812 SEQ ID NO:820 SEQ ID NO:813 SEQ ID NO:820 SEQ ID NO:814 SEQ ID NO:820 SEQ ID NO:815 SEQ ID NO:820 SEQ ID NO:816 SEQ ID NO:820 SEQ ID NO:817 SEQ ID NO:820 SEQ ID NO:818 SEQ ID NO:820

[0046] A nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise a nucleotide sequence that differs from any one of the following first and second sequences by 0 or 1 nucleotide, or consists of or consists essentially of the nucleotide sequence:

[0047] Modified first strand Modified second strand SEQ ID NO:764 SEQ ID NO:774 SEQ ID NO:766 SEQ ID NO:776

[0048] A conjugate for inhibiting the expression of a ZPI target gene in a cell, the conjugate comprising a nucleic acid disclosed herein and one or more ligand moieties.

[0049] A pharmaceutical composition comprising the nucleic acid disclosed herein and a pharmaceutically acceptable excipient or carrier.

[0050] A nucleic acid or pharmaceutical composition for use in therapy.

[0051] A nucleic acid or pharmaceutical composition for preventing or treating a disease associated with a hemostatic disorder, such as a disease associated with a hemostatic disorder, such as hemophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : The linker and ligand parts of the constructs suitable for use in the present application, including the tether la. Figure 1 Linkers are described as being conjugated to oligonucleotides, but it should be understood that the present application also encompasses conjugates of the same linkers to the oligonucleosides disclosed herein.

[0053] It should also be understood that although Figure 1 Describes the Figure 1 The product molecule of the linker and ligand moiety specifically depicted in the embodiment of the present invention is as described herein, but the product may further comprise a linker and a ligand moiety substantially as described herein. Figure 1 molecule linked to an oligonucleoside moiety as depicted in or consisting essentially of Figure 1 As shown in , the F substituent on the cyclooctyl ring is replaced by a substituent generated by hydrolytic displacement (such as an OH substituent). In this way, (a) the tethered la construct can be essentially composed of a structure having Figure 1 The tethered 1a construct can be composed essentially of a molecule having a linker and a ligand portion as shown, wherein the cyclooctyl ring has an F substituent; or (b) the tethered 1a construct can be composed essentially of a molecule having a Figure 1 The molecular composition of the linker and ligand parts shown, but wherein Figure 1 The F substituent on the cyclooctyl ring shown is substituted with a substituent generated by hydrolytic displacement (e.g., an OH substituent), or (c) the tethered la construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0054] Figure 2 : The linker and ligand parts of the constructs suitable for use in the present application, including the tether lb. Figure 2 Linkers are described as being conjugated to oligonucleotides, but it should be understood that the present application also encompasses conjugates of the same linkers to the oligonucleosides disclosed herein.

[0055] against Figure 1 The comments and Figure 1 The possible replacement of the F substituent on the cyclooctyl ring shown as being replaced by a substituent generated by hydrolytic displacement (e.g., an OH substituent) is also applicable to the tethered lb construct. In this manner, (a) the tethered lb construct can be essentially composed of a tethered 1b having Figure 2 The tethered lb construct can be composed essentially of a molecule having a linker and a ligand portion as shown, wherein the cyclooctyl ring has an F substituent; or (b) the tethered lb construct can be composed essentially of a molecule having a Figure 2 The molecular composition of the linker and ligand parts shown, but wherein Figure 2 The F substituent on the cyclooctyl ring shown is substituted with a substituent generated by hydrolytic displacement (eg, an OH substituent), or (c) the tethered 1b construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0056] Figure 3 : The linker and ligand parts suitable for the constructs of the present application, including tether 2a. Although Figure 3 Linkers are described as being conjugated to oligonucleotides, but it should be understood that the present application also encompasses conjugates of the same linkers to the oligonucleosides disclosed herein.

[0057] Figure 4 : The linker and ligand parts suitable for the constructs of the present application, including tether 2b. Although Figure 4 Linkers are described as being conjugated to oligonucleotides, but it should be understood that the present application also encompasses conjugates of the same linkers to the oligonucleosides disclosed herein.

[0058] Figure 5 : The formula described in items 1-101 disclosed herein.

[0059] Figure 6 : The formula described in items 1-56 disclosed herein.

[0060] Figure 7a and 7b : A reverse abasic construct that can be used with the nucleic acid sequence of the present application described herein. Figure 7a , the GalNAc linker is attached to the 5' region of the sense strand being used ( Figure 7a Not shown in ). Figure 7b , the GalNAc linker is attached to the 3' region of the sense strand being used ( Figure 7b not shown).

[0061] Figure 7a The iaia shown in the 3' terminal region of the sense strand indicates: (i) two abasic nucleosides are provided as the penultimate and terminal nucleosides of the 3' terminal region of the sense strand, (ii) wherein a 3'-3' reverse bond is provided between the penultimate nucleoside of the sense strand (i.e., position 21 of the sense strand, wherein position 1 is the terminal 5' nucleoside of the sense strand) and the adjacent penultimate abasic residue, and (iii) when reading toward the 3' terminal region comprising the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 5'-3'.

[0062] Figure 7bThe iaia shown in the 5' region of the sense strand indicates: (i) two abasic nucleosides are provided as the penultimate and terminal nucleosides of the 5' region of the sense strand, (ii) wherein a 5'-5' reverse bond is provided between the penultimate nucleoside of the sense strand (i.e., position 1 of the sense strand, excluding the iaia motif in the 5' region of the sense strand in the nucleoside position numbering on the sense strand) and the adjacent penultimate abasic residue, and (iii) when reading toward the 5' region including the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'-5'.

[0063] Figure 8a and 8b : Duplex construct according to Table 5.

[0064] Fig. 9 : Results of a dose response experiment for inhibition of ZPI mRNA expression in human Huh7 cells. Each point represents the mean relative expression of ZPI mRNA compared to untreated wells after treatment with the siRNA construct at the concentration indicated on the x-axis. Error bars represent the standard deviation of the mean. The dashed curve represents the 95% confidence interval. The dashed line and shaded area represent the mean relative expression + / - standard deviation compared to untreated wells on the same plate.

[0065] Fig.10 : Changes in liver ZPI mRNA expression over time following subcutaneous delivery of GalNAc-conjugated siRNA in C57BL / 6 mice. ETXM1180, 1188 and 1192 were all murinized for this assay as described in Example 11. Data are mean + / - standard deviation, n=3 mice per time point.

[0066] Fig.11 : Changes in liver ZPI mRNA expression over time following subcutaneous delivery of GalNAc-conjugated siRNA in C57BL / 6 mice. ETXM1181, 1189 and 1193 were all murinized for this assay as described in Example 11. Data are mean + / - standard deviation, n=3 mice per time point.

[0067] Figure 12: 3 days after injury ( Fig.10 A) and 10 days ( Fig.10 B) Visual bleeding scores of mice from three different treatment groups (wild-type control group, Haem A mice receiving vehicle (0.9% saline), and Haem A mice receiving the GalNAc-siRNA construct ETXM1184). Definitions of bleeding scores are provided below.

[0068] Figure 13: (A) Comparison of knee diameters at day 3 and day 10 after injury in three different treatment groups of mice (wild type control group, Haem A mice receiving vehicle (0.9% saline), and Haem A mice receiving GalNAc-siRNA construct ETXM1184). (B) Comparison of skinned knee diameters at day 10 after injury in the same three treatment groups of mice.

[0069] Figure 14: Comparison of the severity of (A) bone marrow hyperplasia, (B) osteoarthritis, (C) chondrocyte degeneration / necrosis, (D) hemorrhage, (E) hemosiderin deposition, (F) hematoma, (G) osteoclastic bone resorption, (H) osteolysis, (I) periostitis, (J) subchondral bone sclerosis, (K) tendinosis, (L) tendinitis, and (M) tenosynovitis in three different treatment groups (wild-type control group, Haem A mice receiving vehicle (0.9% saline), and Haem A mice receiving the GalNAc-siRNA construct ETXM1184) (Figure 12M).

[0070] Fig.15 : Joint protection: Dose-response effects documented across multiple endpoints. Prophylactic administration of ETXM1184 demonstrated dose-dependent protection in key tissue readouts 10 days post-injury. ETXM1184 demonstrated efficacy in the same range as clinical comparators: FVIII replacement therapy as a gold standard for acute treatment (Advate) and a siRNA-based prophylactic rebalancing agent in late-stage clinical development that demonstrated good bleeding protection (fitusiran). *Grade: 0 = normal; 1 = mild; 2 = moderate; 3 = marked; 4 = severe. [1] Glasson et al., Osteoarthritis Cartilage. 2010 Oct;18 Suppl 3:S17-23. doi:10.1016 / j.joca.2010.05.025. PMID:20864019.

[0071] Fig.16: The composite histopathological score quantified: tendinitis, tendinosis, tenosynovitis, periostitis, osteolysis, osteoclastic bone resorption, hemorrhage, hematoma, hemosiderin deposition, chondrocyte necrosis, cartilage OARSI grade, subchondral bone sclerosis, and myeloproliferation. ETX-148 showed a significant dose-response effect (Bayesian linear model fit to the composite score). Compared with the control group, the median composite score was reduced: -1.25 in the ETXM1184 10 mg / kg group (significance level equivalent to p < 0.01); -0.91 in the ETXM1184 3 mg / kg group (significance level equivalent to p < 0.05). The comparator fitusiran showed a median reduction of -1.04 in the 3 mg / kg group (significance level equivalent to p < 0.05).

[0072] Fig.17 : Prophylactic administration of ETXM1184 improves hematoma joint pathology in mice with hemophilia A. Administration of 3 mg / kg ETXM1184 improves hematoma knee joint pathology, reduces inflammation, and decreases bleeding area.

[0073] Fig.18 : Prophylactic administration of ETXM1184 reduces post-injury bleeding (in vivo visual bleeding score (VBS)) in hemophilia A mice. Bleeding events occurred in the knee joints of hemophilia A mice 8 days after siRNA administration. Bleeding was monitored for 10 days after injury and terminal histological analysis was performed. Prophylactic administration of a single dose of 10 mg / kg ETXM1184 effectively reduced the visual bleeding score (VBS), which was comparable to a factor VIII replacement (Advate) 10 days after injury.

[0074] Fig.19 : Prophylactic administration of ETXM1184 reduces bleeding after knee injury in hemophilia A mice (measurement of the diameter of the living injured knee compared to the diameter of the uninjured knee). Bleeding events occurred in the knee joints of hemophilia A mice 8 days after siRNA administration. Bleeding was monitored for 10 days after injury and terminal histological analysis was performed. Prophylactic administration of a single dose of 10 mg / kg ETXM1184 effectively reduced blood accumulation in the knee joint, with an effect comparable to that of a factor VIII replacement (Advate) 10 days after injury.

[0075] Fig. 20 : Prophylactic administration of ETXM1184 reduces hemarthrosis in a mouse model of hemophilia A (terminal measurements taken 18 days after siRNA administration and 10 days after injury). Prophylactic administration of a single dose of 10 mg / kg ETXM1184 effectively reduced joint bleeding and features of hemophilic arthropathy, comparable to factor VIII replacement (Advate) 10 days after injury.

[0076] Fig.21:ETXM1184 (ETXS1036 and ETXS1035), ETXM1199 (ETXS2398 and ETXS2397), ETXM1200 (ETXS2400 and ETXS2397), ETXM1201 (ETXS2402 and ETXS2397), ETXM1202 (ETXS2404 and ETCS2397), ETXM Inhibition of ZPI expression by ETXM1203 (ETXS2406 and ETXS2397), ETXM1204 (ETXS2408 and ETXS2397), ETXM1205 (ETXS2410 and ETXS2397), ETXM1206 (ETXS2412 and ETXS2397), and ETXM1207 (ETXS2414 and ETXS2397).

[0077] definition

[0078] "First strand" is also referred to herein as antisense strand or guide strand, and is used interchangeably herein to refer to a nucleic acid strand, such as a strand of an siRNA, such as a dsiRNA, that includes a region that is substantially complementary to a target sequence (e.g., mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence). When the complementary region is not completely complementary to the target sequence, the mismatch is typically in the interior or terminal region of the molecule. In some embodiments, the double-stranded nucleic acid (e.g., siRNA agent) of the present application includes a nucleoside mismatch in the antisense strand.

[0079] "Second strand" (also referred to herein as the sense strand or passenger strand, and used interchangeably herein) refers to the strand of a nucleic acid (eg, siRNA) that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0080] In the context of molecules comprising a nucleic acid provided with a ligand moiety, and optionally also provided with a linker moiety, the nucleic acid of the present application may be referred to as an oligonucleoside or an oligonucleoside moiety.

[0081] Oligonucleotide is a short nucleic acid polymer. Although oligonucleotide contains phosphodiester bond between its nucleoside components (base sugaring), the application is not limited to the oligonucleotide always connected by this phosphodiester bond between adjacent nucleosides, and other nucleoside oligomers connected by bonds other than phosphodiester bond are also considered. For example, the bond between nucleosides can be a thiophosphate bond. Therefore, the term "oligonucleoside" used herein covers oligonucleotide and other nucleoside oligomers. According to the application, preferably oligonucleoside, it is a nucleic acid having at least a portion of oligonucleotide. According to the application, it is also preferred to have one or more or most of phosphodiester backbone bonds between nucleosides. According to the application, it is also preferred to have one or more or most of phosphodiester backbone bonds between nucleosides, and it is also preferred to have one or more thiophosphate backbone bonds between nucleosides (usually in the terminal region of the first chain and / or the second chain) between nucleosides.

[0082] It is preferred herein that nucleic acid according to the present application is a double-stranded oligonucleoside comprising one or more thiophosphate backbone bonds between nucleosides.Therefore, in all cases where the present application mentions oligonucleotides, specifically in the chemical structures disclosed herein, oligonucleotides can be oligonucleosides defined herein equally.

[0083] In some embodiments, the double-stranded nucleic acids (eg, siRNA agents) of the present application include nucleoside mismatches in the sense strand. In some embodiments, the nucleoside mismatches are located within, for example, 5, 4, 3, 2, or 1 nucleosides from the 3' end of the nucleic acid (eg, siRNA).

[0084] In another embodiment, the nucleoside mismatch is, for example, in the 3' terminal nucleoside of the nucleic acid (eg, siRNA).

[0085] "Target sequence" (also referred to as target RNA or target mRNA) refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, including mRNA that is a product of RNA processing of a primary transcription product.

[0086] The target sequence can be about 10-35 nucleosides in length, for example, about 15-30 nucleosides in length. For example, the target sequence can be about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 21-22 nucleosides. Ranges and lengths between the above ranges and lengths are also considered to be part of this application.

[0087] The term "ribonucleoside" or "nucleoside" may also refer to modified nucleosides, as described in further detail below.

[0088] The nucleic acid may be DNA or RNA, and may contain modified nucleosides. RNA is the preferred nucleic acid.

[0089] The terms "iRNA", "siRNA", "RNAi agent", and "iRNA agent", "RNA interfering agent" are used interchangeably herein and refer to an agent containing RNA that mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. siRNA directs sequence-specific degradation of mRNA through RNA interference (RNAi).

[0090] Double-stranded RNA is referred to herein as a "double-stranded siRNA (dsiRNA) agent," "double-stranded siRNA (dsiRNA) molecule," "double-stranded RNA (dsRNA) agent," "double-stranded RNA (dsRNA) molecule," "dsiRNA agent," "dsiRNA molecule," or "dsiRNA," which refers to a ribonucleic acid molecule complex having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, with "sense" and "antisense" orientations relative to the target RNA.

[0091] The majority of nucleosides in each strand of a nucleic acid (e.g., a dsiRNA molecule) are preferably ribonucleosides, but in this case, each strand or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides or modified nucleosides. In addition, the "siRNA" used in this specification may include chemically modified ribonucleosides.

[0092] The term "modified nucleoside" refers to a nucleoside independently having a modified sugar moiety, a modified internucleoside bond, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleoside" encompasses substitutions, additions, or removals of, for example, functional groups or atoms to internucleoside bonds, sugar moieties, or nucleobases. For purposes of this specification and claims, any such modifications (such as used in siRNA-type molecules) are encompassed within "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent."

[0093] The two strands forming the duplex structure may be different parts of one larger molecule, or they may be separate molecules, such as RNA molecules.

[0094] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the nucleic acid duplex structure of the present application. The nucleic acid according to the present application may include an overhang of at least one nucleoside; or, the overhang may include at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides or more. The nucleoside overhang may include or consist of nucleosides / nucleoside analogs (including deoxynucleosides). The overhang may be located on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleosides of the overhang may be on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand.

[0095] In certain embodiments, the antisense strand has a 1-10 nucleoside overhang at the 3' or 5' end, eg, 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhang.

[0096] "Flat" or "blunt end" means that there is no unpaired nucleoside at the end of the double-stranded nucleic acid, that is, there is no nucleoside overhang. The nucleic acid of the present application includes a nucleic acid that does not have a nucleoside overhang at one end or at either end.

[0097] Unless otherwise indicated, the term "complementary" when used to describe the first nucleoside sequence relative to the second nucleoside sequence, refers to the ability of the oligonucleoside comprising the first nucleoside sequence to hybridize with the oligonucleoside comprising the second nucleoside sequence and form a duplex structure under certain conditions, as understood by those skilled in the art. Such conditions can be, for example, stringent conditions, wherein stringent conditions can include: 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).

[0098] As described herein, the complementary sequence in nucleic acid (e.g., dsiRNA) includes base pairing of an oligonucleoside comprising a first nucleoside sequence and an oligonucleoside comprising a second nucleoside sequence over the entire length of one or two nucleoside sequences. Such sequences may be referred to herein as "completely complementary" to each other. However, when the first sequence is referred to herein as "substantially complementary" or "partially complementary" relative to the second sequence, the two sequences may be completely complementary, or they may form one or more mismatched base pairs, such as 2,4 or 5 mismatched base pairs, but preferably not more than 5, while remaining on the ability to hybridize under the conditions most relevant to its final application (e.g., inhibiting gene expression by RISC pathway). In terms of the determination of complementarity, overhangs should not be considered as mismatches. For example, a nucleic acid (e.g., dsiRNA) comprising an oligonucleoside having a length of 17 nucleosides and another oligonucleoside having a length of 19 nucleosides, wherein a longer oligonucleoside comprises a sequence of 17 nucleosides that are completely complementary to a shorter oligonucleoside, and may still be referred to as "completely complementary".

[0099] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, or may be formed entirely by non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, as long as the above-mentioned requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0100] The terms "complementary," "fully complementary," and "substantially / partially complementary" herein may be used with respect to base matching between the sense and antisense strands of a nucleic acid (e.g., a dsiRNA), or between the antisense strand of a double-stranded nucleic acid (e.g., an siRNA agent) and a target sequence.

[0101] In the present application, the second strand of the nucleic acid according to the present application, in particular the dsiRNA for inhibiting ZPI, is at least partially complementary to the first strand of the nucleic acid. In certain embodiments, the first strand and the second strand of the nucleic acid of the present application are partially complementary if they form a duplex region of at least 17 base pairs in length and contain no more than 1, 2, 3, 4 or 5 mismatched base pairs.

[0102] In certain embodiments, if the first and second strands of the nucleic acid of the present application form a duplex region of 19 base pairs in length and comprising no more than 1, 2, 3, 4 or 5 mismatched base pairs, they are partially complementary. In certain embodiments, if the first and second strands of the nucleic acid of the present application form a duplex region of 21 base pairs in length and comprising no more than 1, 2, 3, 4 or 5 mismatched base pairs, they are partially complementary.

[0103] Alternatively, the first and second strands of the nucleic acid of the present application are partially complementary if they form a double-stranded region of at least 17 bases in length, wherein at least 14, 15, 16 or 17 of the base pairs are complementary base pairs, in particular Watson-Crick base pairs.

[0104] In certain embodiments, if the first and second strands of the nucleic acid of the present application form a duplex region of 19 base pairs in length, they are partially complementary, wherein at least 14, 15, 16, 17, 18 or all 19 base pairs are complementary base pairs, particularly Watson-Crick base pairs. In certain embodiments, if the first and second strands of the nucleic acid of the present application form a duplex region of 21 base pairs in length, they are partially complementary, wherein at least 16, 17, 18, 19, 20 or all 21 base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0105] As used herein, a nucleic acid that is "substantially complementary" or "partially complementary" to at least a portion of a messenger RNA (mRNA) refers to a nucleic acid that is substantially or partially complementary to a continuous portion of a target mRNA (e.g., an mRNA encoding a gene). In certain embodiments, the continuous portion of the mRNA is a sequence listed in Table 1, i.e., any one of SEQ ID NOs: 2-121. For example, if a nucleic acid sequence is substantially or partially complementary to an uninterrupted portion of an mRNA encoding a target gene, then the nucleic acid is complementary to at least a portion of the mRNA of the target gene.

[0106] Thus, in some preferred embodiments, the antisense oligonucleotides disclosed herein are fully complementary to the target gene sequence.

[0107] In other embodiments, the antisense oligonucleosides disclosed herein are substantially or partially complementary to a target RNA sequence and comprise a contiguous nucleoside sequence that is at least about 80% complementary to an equivalent region of a target RNA sequence over its entire length, e.g., at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0108] In certain embodiments, the first strand (antisense strand) of the nucleic acid according to the present application is partially or fully complementary to a consecutive portion of RNA transcribed from the ZPI gene. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a consecutive portion of at least 17 nucleosides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a consecutive portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of the ZPI mRNA. The first strand of the nucleic acid described in the present application is partially or fully complementary to a consecutive portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 2-121).

[0109] In certain embodiments, if the first strand (antisense) of the nucleic acid of the present application comprises a continuous nucleoside sequence of at least 17 nucleosides, it is partially complementary to a continuous portion of a ZPI mRNA, wherein at least 14, 15, 16 or 17 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of a ZPI mRNA. In certain embodiments, the first strand of the nucleic acid of the present application comprises a continuous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 2-121). In certain embodiments, the first strand of the nucleic acid of the present application comprises a continuous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18 or all 19 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 2-121). In certain embodiments, the first strand of the nucleic acid of the present application comprises a continuous nucleoside sequence of 23 nucleosides, wherein at least 18, 19, 20, 21, 22 or all 23 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 2-101).

[0110] In some embodiments, the nucleic acid (e.g., siRNA) of the present application includes a sense strand that is substantially or partially complementary to an antisense oligonucleoside, and the antisense oligonucleoside is complementary to the target gene sequence and includes a continuous nucleoside sequence. The nucleoside sequence of the sense strand is usually at least about 80% complementary to the equivalent region of the nucleoside sequence of the antisense strand over its entire length, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0111] In some embodiments, the nucleic acids (e.g., siRNA) of the present application include an antisense strand that is substantially or partially complementary to a target sequence, and comprises a continuous nucleoside sequence that is at least 80% complementary to the target sequence over its entire length, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0112] As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey and a chimpanzee) or a non-primate or a bird, which expresses a target gene endogenously or heterologously when the target gene sequence has sufficient complementarity with a nucleic acid (such as an siRNA agent) to promote target knockdown. In certain preferred embodiments, the subject is a human.

[0113] The term "treating" or "treatment" refers to a beneficial or desired result, including but not limited to alleviating or ameliorating one or more symptoms associated with gene expression. "Treatment" may also mean prolonging survival compared to expected survival without treatment. Treatment may include preventing the development of complications, such as reducing liver damage in individuals with liver infection.

[0114] As used herein, "therapeutically effective amount" is intended to include an amount of a nucleic acid (e.g., siRNA) that, when administered to a patient for treating a subject suffering from a disease, is sufficient to effect treatment of the disease (e.g., by alleviating, ameliorating, or maintaining an existing disease, or one or more symptoms of the disease or its associated complications).

[0115] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, or dosage forms that are suitable for contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0116] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material, used to carry or transport the target compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated.

[0117] When values ​​or ranges of values ​​are recited for parameters, it is intended that values ​​or ranges intermediate to the recited values ​​also be part of the present application.

[0118] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0119] As used herein, the term "including" is intended to be used interchangeably with the phrase "including, but not limited to,"

[0120] The term "or" as used herein means "and / or" and can be used interchangeably with the term unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" should be understood as "sense strand or antisense strand or sense strand and antisense strand."

[0121] As used herein, the term "about" refers to within the typical tolerance range of the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When "about" appears before a series of numbers or ranges, it should be understood that "about" can modify each number in the series or range.

[0122] The term "at least" before a number or a series of numbers should be understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that can be logically included, which can be clearly seen from the context. For example, the number of nucleosides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides in a 21 nucleoside nucleic acid molecule" means that 18, 19, 20 or 21 nucleosides have the characteristics shown. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0123] As used herein, "not greater than" or "less than" should be understood to refer to the value adjacent to the phrase and the logically lower value or integer, such as zero, as logically derived from the context. For example, a duplex having an overhang of "not greater than 2 nucleosides" has 2, 1, or 0 nucleoside overhangs. When "not greater than" appears before a series of numbers or ranges, it should be understood that "not greater than" can modify each number in the series or range.

[0124] The terminal region of a chain is the last five nucleosides from the 5' or 3' end.

[0125] Those skilled in the art can combine various embodiments of the present application as needed.

[0126] Abasic nucleosides

[0127] In certain embodiments, there is 1, such as 2, such as 3, such as 4 or more abasic nucleosides in the nucleic acids of the present application. Abasic nucleosides are modified nucleosides because they lack the base that usually appears at position 1 of the sugar moiety. Typically, there is a hydrogen at position 1 of the sugar moiety of the abasic nucleosides present in the nucleic acids of the present application.

[0128] The abasic nucleoside is located in the terminal region of the second strand, preferably within the terminal 5 nucleosides of the end of the strand. The terminal region may be the terminal 5 nucleosides, which include the abasic nucleoside.

[0129] The second chain may include the following features, as preferred features (combinations are specifically contemplated unless mutually exclusive):

[0130] There are 2 or more abasic nucleosides in the terminal region of the second strand; and / or

[0131] The 5' or 3' terminal region of the second strand has 2 or more abasic nucleosides; and / or

[0132] The 5' or 3' terminal region of the second strand has 2 or more abasic nucleosides, wherein the abasic nucleosides are in an overhang as described herein; and / or

[0133] There are 2 or more consecutive abasic nucleosides in the terminal region of the second strand, wherein preferably one such abasic nucleoside is the terminal nucleoside; and / or

[0134] There are 2 or more consecutive abasic nucleosides in the 5' or 3' terminal region of the second strand, wherein preferably one such abasic nucleoside is the terminal nucleoside in the 5' or 3' terminal region of the second strand; and / or

[0135] A reverse internucleoside bond connects at least one abasic nucleoside to an adjacent basic nucleoside in a terminal region of the second strand; and / or

[0136] A reverse internucleoside bond connects at least one abasic nucleoside to an adjacent basic nucleoside in the 5' or 3' terminal region of the second strand; and / or

[0137] The abasic nucleoside that is the penultimate nucleoside is linked to a nucleoside that is not the terminal nucleoside (referred to herein as the penultimate nucleoside) via a reverse bond; and / or

[0138] When the strand is read toward the end containing the terminal nucleoside, the abasic nucleoside acts as the 2 terminal nucleosides connected by a 5'-3' bond;

[0139] When the strand is read toward the end containing the terminal nucleoside, the abasic nucleoside acts as the 2 terminal nucleosides connected by a 3'-5' bond;

[0140] an abasic nucleoside as the terminal 2 positions, wherein the second to last nucleoside is linked to the third to last nucleoside by a reverse bond, and wherein the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond;

[0141] an abasic nucleoside as the terminal 2 positions, wherein the second to last nucleoside is linked to the third to last nucleoside via a reverse bond, and wherein

[0142] (1) the inverted bond is a 5-5' inverted bond and when reading toward the end containing the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 3'5'; or

[0143] (2) The reverse bond is a 3-3' reverse bond, and when reading toward the end including the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 5'3'.

[0144] Preferably, the second strand has an abasic nucleoside at its terminus.

[0145] Preferably, the terminal region of the second strand has 2 or at least 2 abasic nucleosides, preferably at the terminal and penultimate positions.

[0146] Preferably, 2 or more abasic nucleosides are consecutive, for example, all abasic nucleosides may be consecutive. For example, the terminal 1 or terminal 2 or terminal 3 or terminal 4 nucleosides may be abasic nucleosides.

[0147] Abasic nucleosides can also be linked to adjacent nucleosides via 5'-3' phosphodiester bonds or inverted bonds, unless there is only 1 abasic nucleoside at the end, in which case it will have an inverted bond to the adjacent nucleoside.

[0148] Inverted linkages (also referred to as inverted linkages, as is common in the art) include 5'-5', 3'3', 3'-2', or 2'-3' phosphodiester bonds between adjacent sugar moieties of a nucleoside.

[0149] A non-terminal abasic nucleoside will have 2 phosphodiester bonds, one to each adjacent nucleoside, and these bonds may be inverted, or may be 5'-3 phosphodiester bonds, or may be one of each.

[0150] A preferred embodiment comprises 2 abasic nucleosides at the terminal and penultimate positions of the second strand, and wherein the inverted internucleoside linkage is located between the penultimate (abasic) nucleoside and the penultimate nucleoside.

[0151] Preferably, there are 2 abasic nucleosides at the terminal and penultimate positions of the second strand, and the penultimate nucleoside is linked to the penultimate nucleoside by an inverted internucleoside bond and to the terminal nucleoside by a 5'-3' or 3'-5' phosphodiester bond (reading toward the terminal end of the molecule).

[0152] Preferably, the nucleic acid according to the present application comprises one or more abasic nucleosides, optionally wherein the one or more abasic nucleosides are located in the terminal region of the second strand, and / or wherein at least one abasic nucleoside is linked to an adjacent basic nucleoside via an inverted internucleoside bond.

[0153] Typically, the second strand comprises 2 consecutive abasic nucleosides in its 5' terminal region, wherein one such abasic nucleoside is the terminal nucleoside of the 5' terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside of the 5' terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent first basic nucleoside in the adjacent 5' proximal terminal region by an inverted internucleoside bond; (b) the inverted bond is a 5-5' inverted bond; and (c) when reading toward the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 3'5'. More typically, (i) the first and second strands are each 23 nucleosides in length; (ii) two phosphorothioate internucleoside bonds are located between three consecutive positions of the 5' proximal region of the second strand, wherein the first phosphorothioate internucleoside bond is located between the adjacent first base nucleoside and the adjacent second base nucleoside of (a) in the 5' proximal region of the second strand, and the second phosphorothioate internucleoside bond is located between the adjacent th di-base nucleoside and the adjacent th tri-base nucleoside of the 5' proximal region of the second strand; (iii) two wherein each of the terminal nucleosides of the 5' and 3' terminal regions of the first strand is linked to the corresponding 5' and 3' adjacent penultimate nucleosides, respectively, by a phosphorothioate internucleoside bond, and each of the 5' and 3' penultimate nucleosides is linked to the corresponding 5' and 3' adjacent penultimate nucleosides by a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is conjugated directly or indirectly to one or more ligand moieties of the 3' terminal region of the second strand.

[0154] Alternatively, the second strand comprises 2 consecutive abasic nucleosides, preferably located at the overhang of the 3' terminal region of the second strand, wherein one such abasic nucleoside is the terminal nucleoside of the 3' terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside of the 3' terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent first basic nucleoside of the adjacent 3' proximal terminal region by a reverse internucleoside bond; (b) the reverse bond is a 3-3' reverse bond; and (c) when reading toward the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 5'-3'. More typically, (i) the first strand and the second strand are each 23 nucleosides in length; (ii) two phosphorothioate internucleoside bonds are located between three consecutive positions of the 3' proximal region of the second strand, wherein the first phosphorothioate internucleoside bond is located between the adjacent first base nucleoside and the adjacent second base nucleoside of (a) in the 3' proximal region of the second strand, and the second phosphorothioate internucleoside bond is located between the adjacent second base nucleoside and the adjacent third base nucleoside of the 3' proximal region of the second strand; (iii) two The phosphorothioate internucleoside bonds are located between three consecutive positions of the 5' and 3' terminal regions of the first strand, respectively, whereby each terminal nucleoside of the 5' and 3' terminal regions of the first strand is linked to the corresponding 5' and 3' adjacent penultimate nucleosides, respectively, by a phosphorothioate internucleoside bond, and each first 5' and 3' penultimate nucleoside is linked to the corresponding 5' and 3' adjacent penultimate nucleoside by a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties of the 5' terminal region of the second strand.

[0155] Examples of structures are as follows (wherein the specific RNA nucleoside shown is not limiting and can be any RNA nucleoside):

[0156] A3'-3' reverse bond (also shows the 5'-3' orientation of the last phosphodiester bond between two abasic molecules when reading toward the end of the molecule)

[0157]

[0158] B illustrates the 5'-5' reverse bond (also shows the 3'-5' orientation of the last phosphodiester bond between two abasic molecules when reading toward the end of the molecule)

[0159]

[0160] The one or more abasic nucleosides present in the nucleic acid provide in the presence of one or more reverse nucleoside bonds, i.e. 5'-5' or 3'-3' reverse internucleoside bonds. The reverse bond occurs because the direction of the adjacent nucleoside sugar changes, so that the sugar has a 3'-5' direction, rather than a traditional 5'-3' direction (with reference to the numbering of ring atoms on the nucleoside sugar). The one or more abasic nucleosides present in the nucleic acid of the present application preferably include this type of reverse nucleoside sugar.

[0161] In the case where the terminal nucleoside has the reverse direction, this will result in the " inversion " end configuration of the whole nucleic acid. Although some structures drawn and quoted herein use conventional 5'-3' direction to represent (with reference to the ring atom numbering on the nucleoside sugar), it is to be understood that the existence of the terminal nucleoside with a change in direction and a near-end 3'-3' reverse bond will result in the nucleic acid with an overall 5'-5' end structure (i.e. conventional 3' terminal nucleoside becomes 5' terminal nucleoside). Or it should be understood that the existence of the terminal nucleoside with a change in direction and a near-end 5'-5' reverse bond will result in the nucleic acid with an overall 3'-3' end structure.

[0162] Near-end 3'-3' or 5'-5' reverse key as described herein can comprise directly adjacent / connected to the reverse key of the terminal nucleoside (such as the single terminal nucleoside with reverse direction) with reverse direction.Or near-end 3'-3' or 5'-5' reverse key as described herein can comprise adjacent 2 or more nucleoside with reverse direction, such as 2 or more than 2 terminal region nucleoside with reverse direction, such as the reverse key of terminal and penultimate nucleoside.In this way, reverse key can be connected to the penultimate nucleoside with reverse direction.Although skilled technicians (addressee) will understand that reverse direction as described above can cause nucleic acid molecules to have overall 3'-3' or 5'-5' end structure as described herein, it should also be understood that, due to the presence of one or more additional reverse connections and / or nucleoside with reverse direction, therefore overall nucleic acid can have the 3'-5' end structure corresponding to the 5' / 3' end of conventional positioning.

[0163] In one aspect, the nucleic acid can have a 3'-3' inverted linkage and the terminal sugar moiety can comprise a 5' OH, rather than a 5' phosphate group, at the 5' position of the terminal sugar.

[0164] Thus, it will be clearly understood by those skilled in the art that 5'-5', 3'-3' and 3'-5' (reading in this terminal direction) terminal variants of the more conventional 5'-3' structure drawn herein (numbering with reference to the ring atoms on the terminal nucleoside sugar) are included within the scope of the present disclosure wherein one or more inverted bonds are present.

[0165] In the case where, for example, an inverted internucleoside bond and / or one or more nucleosides have an inverted orientation to produce an inverted terminus, and the relative position of the bond (e.g., relative position to a linker) or the position of an internal feature (e.g., a modified nucleoside) is defined relative to the 5' or 3' end of the nucleic acid, then the 5' or 3' end is the conventional 5' or 3' end that would be present if there were no inverted bond, and wherein the conventional 5' or 3' end is determined by considering the directionality of the majority of internal nucleoside bonds within the nucleic acid and / or the orientation of the nucleosides. From these internal bonds and / or nucleoside orientations, it can be determined which ends of the nucleic acid would constitute the conventional 5' and 3' ends of the molecule (with reference to the numbering of the ring atoms on the terminal nucleoside sugar) in the absence of the inverted bond.

[0166] For example, in the structure shown below, there are abasic residues in the first 2 positions of the 5' end. When the terminal nucleoside has an inverted orientation, the 5' end shown in the figure below (which is a conventional 5' end) may actually contain a 3'OH according to the inverted nucleoside in the terminal position. Nevertheless, when reading along the standard 5'[PO4] to 3'[OH] direction of nucleic acid molecules (with reference to the numbering of the ring atoms on the nucleoside sugars), most molecules will contain a conventional internucleoside bond from the 3'OH of the sugar to the 5' phosphate of the next sugar, which can be used to determine the conventional 5' and 3' ends that would be found in the absence of the inverted end configuration.

[0167] 5'AA-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me 3'

[0168] In some embodiments, the second strand (sense strand) of the nucleic acid according to the present application comprises 2 consecutive abasic nucleosides in the 5' terminal region, as shown in the following 5' terminal motif:

[0169]

[0170] in:

[0171] B represents nucleoside base,

[0172] T represents H, OH or 2' ribose modification,

[0173] Z represents the remaining nucleosides of the second strand.

[0174] In some embodiments, the second strand (sense strand) of the nucleic acid according to the present application comprises 2 consecutive abasic nucleosides in the 5' terminal region, as shown in the following 5' terminal motif:

[0175]

[0176] in:

[0177] B represents nucleoside base,

[0178] T represents H, OH or 2'ribose modification (preferably 2'ribose modification, more preferably 2'Me or 2'F ribose modification),

[0179] V represents O or S (preferably O),

[0180] R stands for H or C 1-4 Alkyl (preferably H),

[0181] Z represents the remaining nucleosides of the second strand,

[0182] More preferably, the following 5' terminal motif is present:

[0183]

[0184] in:

[0185] B represents nucleoside base,

[0186] T represents a 2' ribose modification (preferably a 2'Me or 2'F ribose modification),

[0187] Z represents the remaining nucleosides of the second strand.

[0188] The reverse bond is preferably located at the end of the nucleic acid (eg, RNA) that is distal to the ligand portion of the molecule (eg, the GalNAc-containing portion).

[0189] The GalNAc-siRNA construct having 5'-GalNAc on the sense strand may have an inverted bond at the other end of the sense strand.

[0190] The GalNAc-siRNA construct having 3'-GalNAc on the sense strand may have an inverted bond at the other end of the sense strand.

[0191] In a preferred embodiment, the second strand (sense strand) of the nucleic acid according to the present application comprises 2 consecutive abasic nucleosides in the 5' terminal region, as shown in the following 5' terminal motif,

[0192]

[0193] in:

[0194] B represents nucleoside base,

[0195] T represents H, OH or 2'ribose modification (preferably 2'ribose modification, more preferably 2'Me or 2'F ribose modification),

[0196] V represents O or S (preferably O),

[0197] R stands for H or C 1-4 Alkyl (preferably H),

[0198] Z comprises 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, more preferably 19 consecutive nucleosides,

[0199] More preferred is the following 5' terminal motif:

[0200]

[0201] in:

[0202] B represents nucleoside base,

[0203] T represents a 2' ribose modification (preferably a 2'Me or 2'F ribose modification),

[0204] Z comprises 19 consecutive nucleosides.

[0205] Nucleic acid length

[0206] In one aspect, i) the length of the first strand of the nucleic acid is 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the length of the second strand of the nucleic acid is 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0207] Typically, the duplex region of the nucleic acid is 17 to 30 nucleotides in length, more preferably 19 or 21 nucleotides in length. Similarly, the complementary region between the first strand and a portion of the RNA transcribed from the ZPI gene is 17 to 30 nucleotides in length.

[0208] Nucleic acid modification

[0209] In certain embodiments, the nucleic acids of the present application, eg, RNA, eg, dsiRNA, do not comprise further modifications, eg, chemical modifications or conjugations known in the art and described herein.

[0210] In other preferred embodiments, the nucleic acids of the present application, such as RNA, such as dsiRNA, are further chemically modified to enhance stability or other beneficial properties.

[0211] In certain embodiments of the present application, substantially all nucleosides are modified.

[0212] The nucleic acids described in this application can be synthesized or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference.

[0213] Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse bond) or 3'-terminal modifications (conjugation, DNA nucleosides within RNA or RNA nucleosides within DNA, reverse bond, etc.); base modifications, such as replacement with stabilizing bases, destabilizing bases, or bases that pair with an expanded library of paired substances, conjugated bases; sugar modifications (e.g., at the 2'-position or the 4'-position) or sugar replacement; or backbone modifications, including modification or replacement of phosphodiester bonds.

[0214] In the embodiments described herein, the specific examples of useful nucleic acids (such as siRNA compounds) include but are not limited to RNA containing modified backbones or no natural internucleoside bonds. Nucleic acids (such as RNA) with modified backbones include but are not limited to nucleic acids without phosphorus atoms in the backbone. For the purpose of this specification, and as sometimes cited in the art, modified nucleic acids (such as RNA) that do not contain phosphorus atoms in their internucleoside backbones can also be considered as oligonucleosides. In some embodiments, modified nucleic acids (such as siRNA) have phosphorus atoms in their internucleoside backbones.

[0215] Modified nucleic acid (e.g., RNA) backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates with normal 3'-5' bonds, 2'-5' connection analogs of these, and those with reversed polarity, wherein adjacent nucleoside units are connected to 5'-3' or 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0216] Modified nucleic acids (e.g., RNA) may also contain one or more substituted sugar moieties. Nucleic acids (e.g., siRNAs, e.g., dsiRNAs) described herein may include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted. 2'O-methyl and 2'-F are preferred modifications.

[0217] In certain preferred embodiments, the nucleic acid comprises at least one modified nucleoside.

[0218] The nucleic acid of the present application may comprise one or more modified nucleosides on the first strand and / or the second strand.

[0219] In some embodiments, substantially all nucleosides of the sense strand and all nucleosides of the antisense strand comprise modifications.

[0220] In some embodiments, all nucleosides of the sense strand and substantially all nucleosides of the antisense strand comprise modifications.

[0221] In some embodiments, all nucleosides of the sense strand and all nucleosides of the antisense strand comprise a modification.

[0222] In one embodiment, at least one modified nucleoside is selected from the group consisting of deoxynucleosides, 3'-terminal deoxythymidine (dT) nucleosides, 2'-O-methyl modified nucleosides (also referred to herein as 2'-Me, wherein Me is methoxy), 2'-fluoro modified nucleosides, 2'-deoxy modified nucleosides, locked nucleosides, unlocked nucleosides, conformationally restricted nucleosides, constrained ethyl nucleosides, abasic nucleosides, 2'-amino modified nucleosides, 2'-O-allyl modified nucleosides, 2'- In another embodiment, the modified nucleoside comprises a 2'-O-alkyl-modified nucleoside, a 2'-hydroxy-modified nucleoside, a 2'-methoxyethyl-modified nucleoside, a 2'-O-alkyl-modified nucleoside, a morpholine nucleoside, a phosphoramidate, a non-natural base comprising a nucleoside, a tetrahydropyran-modified nucleoside, a 1,5-anhydrohexitol-modified nucleoside, a cyclohexenyl-modified nucleoside, a nucleoside comprising a thiophosphate group, a nucleoside comprising a methylphosphonate group, a nucleoside comprising a 5'-phosphate, and a nucleoside comprising a 5'-phosphate analog. In another embodiment, the modified nucleoside comprises a short sequence of 3'-terminal deoxy-thymidine (dT).

[0223] The modification on the nucleoside can be preferably the following group: including but not limited to LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl and combinations thereof. In another embodiment, the modification on the nucleoside is 2-O-methyl ("2'-Me") or 2'-fluoro modification.

[0224] A preferred modification is a modification at the 2'-OH group of the ribose, optionally selected from a 2'-Me or 2'-F modification.

[0225] Preferred nucleic acids contain one or more modified nucleosides on the first strand and / or the second strand to form modified nucleosides as shown below:

[0226] A nucleic acid wherein the modification is at the 2'-OH group of the ribose sugar, optionally selected from a 2'-Me or 2'-F modification.

[0227] A nucleic acid, wherein the first strand comprises a 2'-F modification at any one of position 2, position 6, position 14, or any combination thereof, counting from position 1 of the first strand.

[0228] A nucleic acid, wherein the second strand comprises a 2'-F modification at any one of position 7, position 9, position 11, or any combination thereof, counting from position 1 of the second strand.

[0229] A nucleic acid wherein the first strand and the second strand each comprise a 2'-Me modification and a 2'-F modification.

[0230] A nucleic acid comprising at least one thermal destabilizing modification, counting from position 1 of the first strand, suitably at one or more of positions 1 to 9 of the first strand, and / or at one or more positions aligned with positions 1 to 9 of the first strand at position 7 of the first strand on the second strand, wherein the destabilizing modification is selected from: modified non-locked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid, more preferably (S)-glycol nucleic acid.

[0231] A nucleic acid comprising at least one thermal destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand.

[0232] A nucleic acid which is a siRNA oligonucleoside, wherein the siRNA oligonucleoside comprises 3 or more 2'-F modifications at positions 6 to 12 of the second strand, counting from position 1 of the second strand, such as 4, 5, 6 or 7 2'-F modifications at positions 6 to 12 of the second strand.

[0233] A nucleic acid which is a siRNA oligonucleoside, wherein the second strand comprises at least 3, such as 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand, counting from position 1 of the second strand.

[0234] A nucleic acid which is a siRNA oligonucleotide, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region, or at least within 1 or 2 nucleosides of the terminal nucleoside of the 3' terminal region.

[0235] A nucleic acid which is a siRNA oligonucleotide, wherein the first strand comprises seven consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside in the 3' terminal region.

[0236] A nucleic acid which is a siRNA oligonucleoside, wherein each of the first and second strands comprises an alternating modification pattern, preferably a completely alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified by (i) a 2'Me modification on odd-numbered nucleosides, counting from position 1 of the first strand, and (ii) a 2'F modification on even-numbered nucleosides, counting from position 1 of the first strand, and the nucleosides of the second strand are modified by (i) a 2'F modification on odd-numbered nucleosides, counting from position 1 of the second strand, and (ii) a 2'Me modification on even-numbered nucleosides, counting from position 1 of the second strand. Typically, this completely alternating modification pattern is present in a blunt-ended oligonucleoside, wherein the length of the first and second strands is 19 nucleosides each.

[0237] Position 1 of the first or second strand is the nucleoside closest to the end of the nucleic acid (ignoring any abasic nucleosides) and is linked to the adjacent nucleoside (at position 2) by a 3' to 5' internal bond, referenced to the bonds between the backbone sugar moieties, and read in a direction away from the end of the molecule.

[0238] It can be seen that "position 1 of the sense strand" is the 5'-most nucleoside of the conventional 5' end of the sense strand (excluding abasic nucleosides). Typically, the nucleoside at this position 1 of the sense strand will be equivalent to the 5' nucleoside of the selected target nucleic acid sequence, and more generally, the sense strand will have nucleosides that are equivalent to the nucleosides of the target nucleic acid sequence starting from this position 1 of the sense strand, while also allowing for acceptable mismatches between sequences.

[0239] As used herein, "position 1 of the antisense strand" is the 5'most nucleoside of the conventional 5' end of the antisense strand (excluding abasic nucleosides). As mentioned above, there is a complementary region between the sense strand and the antisense strand, so that the antisense strand also has a region complementary to the target nucleic acid sequence.

[0240] In certain embodiments, the nucleic acid (e.g., siRNA) agent further comprises at least one phosphorothioate or methylphosphonate internucleoside bond. For example, the phosphorothioate or methylphosphonate internucleoside bond can be located at the 3'-end or terminal region of one strand (i.e., sense strand or antisense strand); or at the ends of two strands (i.e., sense strand and antisense strand).

[0241] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkage is located at the 5' terminus or terminal region of one strand (ie, the sense strand or the antisense strand); or at the termini of both strands (ie, the sense strand and the antisense strand).

[0242] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkages are located at the 5'- and 3'-termini or terminal regions of one strand (ie, the sense strand or the antisense strand); or at the ends of both strands (ie, the sense strand and the antisense strand).

[0243] Any nucleic acid can comprise one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside linkages at the chain ends.

[0244] At least one oligoribonucleoside chain preferably comprises at least two consecutive phosphorothioate modifications in the last three nucleosides of the oligonucleoside.

[0245] Therefore, the present application also relates to: a nucleic acid disclosed herein, comprising phosphorothioate internucleoside bonds between at least two or three consecutive positions, respectively, such as in the 5' and / or 3' terminal region and / or the proximal terminal region of the second chain, whereby the proximal terminal region is preferably adjacent to the terminal region where the one or more abasic nucleosides of the second chain are located.

[0246] The nucleic acids disclosed herein comprise phosphorothioate internucleoside bonds between at least two or three consecutive positions of the 5' and / or 3' terminal region of the first strand, whereby preferably the terminal positions of the 5' and / or 3' terminal region of the first strand are linked to their adjacent positions via phosphorothioate internucleoside bonds.

[0247] The nucleic acid strand may be an RNA comprising phosphorothioate internucleoside bonds between three nucleosides adjacent to two terminal abasic nucleosides.

[0248] The preferred nucleic acid is a double-stranded RNA comprising two adjacent abasic nucleosides at the 5' end of the second strand and a ligand portion comprising one or more GalNAc ligand portions at the opposite 3' end of the second strand. Further preferably, the same nucleic acid may also comprise phosphorothioate bonds between nucleotides at positions 3-4 and 4-5 of the second strand, read from position 1 of the second strand. Further preferably, the same nucleic acid may also comprise 2'F modifications at positions 7, 9, 11 of the second strand.

[0249] Preferred modifications are as follows.

[0250] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of:

[0251] Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0252] Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0253] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0254] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0255] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me.

[0256] A nucleic acid, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following:

[0257] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0258] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0259] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0260] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0261] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0262] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or

[0263] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0264] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0265] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0266] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0267] where (s) is a phosphorothioate internucleoside bond.

[0268] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0269] ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0270] ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0271] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0272] ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0273] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0274] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, or

[0275] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0276] Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0277] Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0278] Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0279] Among them, the ia representative of the opposite directionless red base nucleus is present, and the ia-ia representative is the opposite directionless red base nucleus present at the second 3' end, and the opposite directionless red base nucleus is present at the two nucleus protruding end.

[0280] One kind of nucleic acid, including the second modified nucleic acid mentioned below, any one modification model (5'-3'):

[0281] ia–ia-Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0282] ia–ia-Me(s)Me(s)Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0283] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0284] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0285] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0286] Me–Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia, or

[0287] Me–Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0288] Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0289] Me–Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0290] Me–Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0291] in:

[0292] (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is at the 3' end of the second chain, the inverted abasic nucleoside is at a 2-nucleoside overhang.

[0293] A nucleic acid wherein the modified nucleosides include any one of the following modification patterns:

[0294] Modification mode 1: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0295] Or modification mode 2: second strand (5'-3'): Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0296] Or modification pattern 3: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0297] Or modification pattern 4: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0298] Or modification pattern 5: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0299] Or modification pattern 6: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me.

[0300] A nucleic acid, wherein the modified nucleosides include any one of the following modification patterns:

[0301] Modification pattern 1: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0302] Or modification pattern 2: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0303] Or modification pattern 3: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0304] Or modification pattern 4: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0305] Or modification pattern 5: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0306] Or modification pattern 6: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0307] Where (s) is a phosphorothioate internucleoside bond.

[0308] A nucleic acid, wherein the modified nucleoside includes any one of the following modification patterns:

[0309] Modification pattern 1: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0310] Or modification pattern 2: Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0311] Or modification pattern 3: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0312] Or modification pattern 4: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0313] Or modification pattern 5: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0314] Or modification pattern 6: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0315] Wherein (s) is a phosphorothioate internucleoside bond.

[0316] A nucleic acid, wherein the modified nucleosides include any one of the following modification patterns:

[0317] Modification pattern 1: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0318] Or modification pattern 2: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0319] Or modification pattern 3: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0320] Or modification pattern 4: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0321] Or modification mode 5: second link (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, first link (5'-3'): Me-F-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-F-Me-Me-Me-Me-Me–Me

[0322] Or modification mode 6: second link (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, first link (5'-3'): Me-F-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-F-Me-Me-Me-Me-Me–Me,

[0323] Among them, the representative of IA is unopposed.

[0324] One kind of nucleic acid, among which modified nucleic acid, including any one of the following modifications:

[0325] Modification mode 1: Second link (5'-3'): Me-Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, First link (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-Me-Me

[0326] Or modification mode 2: second link (5'-3'): Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first link (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-Me

[0327] Or modification mode 3: second link (5'-3'): Me-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first link (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-Me

[0328] Or modification mode 4: second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, first strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0329] Or modification pattern 5: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me–Me

[0330] Or modification pattern 6: second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me,

[0331] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is at the 3' end of the second strand, the inverted abasic nucleoside is at a 2-nucleoside overhang.

[0332] A nucleic acid wherein the modified nucleosides include any one of the following modification patterns:

[0333] Modification pattern 1: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me , first strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0334] Or modification pattern 2: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0335] Or modification pattern 3: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0336] Or modification pattern 4: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0337] Or modification pattern 5: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0338] Or modification pattern 6: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me;

[0339] Wherein:

[0340] (s) This is the base of the base, the representative of the base.

[0341] One type of nucleic acid, among which the modified nucleic acids are inclusive, and any one of the following modified models:

[0342] Modification pattern 1: Second pin (5'-3'): Me–Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0343] Modification model 2: Second pin (5'-3'): Me–Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0344] Modification model 3: Second pin (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–i a, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0345] Modification model 4: Second pin (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0346] Or modification pattern 5: second strand (5'-3'): Me–Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–i a, first strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0347] Or modification pattern 6: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–i a, first strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0348] wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is at the 3' end of the second strand, the inverted abasic nucleoside is at a 2-nucleoside overhang.

[0349] Particularly preferred are nucleic acids wherein the modified nucleosides comprise the following modification pattern:

[0350] Modification pattern 4: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0351] Wherein: (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside.

[0352] A nucleic acid wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

[0353] A nucleic acid wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

[0354] A nucleic acid wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of 3 2'F modifications.

[0355] A nucleic acid wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of 5 2'F modifications.

[0356] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0357] Me–F–Me–X 2 –Me–F–(Me) 7 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0358] Where X 2 , X 3 and X 4 Selected from 2'Me and 2'F sugar modifications, provided that for X 2 , X 3 and X 4 , at least one is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications.

[0359] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0360] Me–F–Me–X 2 –Me–F–(Me) 7 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0361] Where X 2 is 2'F sugar modification, X 3 and X 4 It is a 2'Me sugar modification.

[0362] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0363] Me–F–Me–X 2 –Me–F–(Me) 7 –(F–Me) 2 –X 3 –Me–X 4 –(Me)3

[0364] Where X 3 is 2'F sugar modification, X 2 and X 4 It is a 2'Me sugar modification.

[0365] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0366] Me–F–Me–X 2 –Me–F–(Me) 7 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0367] Where X 4 is 2'F sugar modification, X 2 and X 3 It is a 2'Me sugar modification.

[0368] A nucleic acid wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of seven 2'F modifications.

[0369] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0370] Me–F–Me–X 2 –Me–F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0371] Where X 2 , X 3 and X 4 Selected from 2'Me and 2'F sugar modifications, provided that for X 2 , X 3 and X 4 , at least one is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications.

[0372] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0373] Me–F–Me–X 2 –Me–F–Me–(F) 2 –(Me)4 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0374] Where X 2 is 2'F sugar modification, X 3 and X 4 It is a 2'Me sugar modification.

[0375] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0376] Me–F–Me–X 2 –Me–F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0377] Where X 3 is 2'F sugar modification, X 2 and X 4 It is a 2'Me sugar modification.

[0378] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0379] Me–F–Me–X 2 –Me–F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –X 3 –Me–X 4 –(Me) 3

[0380] Where X 4 is 2'F sugar modification, X 2 and X 3 It is a 2'Me sugar modification.

[0381] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0382] Me–F–(Me) 3 –X 1 –(Me) 7 –F–Me–F–(Me) 7

[0383] Where X 1 It is a thermal destabilizing modification.

[0384] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0385] Me–F–(Me) 3 –X 1 –Me–(F) 2 –(Me) 4 –F–Me–F–(Me) 7

[0386] Where X 1 It is a thermal destabilizing modification.

[0387] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0388] (Me) 8 –(F) 3 –(Me) 10 .

[0389] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0390] (Me) 8 –(F) 3 –(Me) 10 ,

[0391] wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

[0392] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0393] (Me) 8 –(F) 3 –(Me) 10 ,

[0394] wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of three, five or seven 2'F modifications.

[0395] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0396] (Me)8 –(F) 3 –(Me) 10 ,

[0397] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0398] Me–F–(Me) 3 –X 1 –(Me) 7 –F–Me–F–(Me) 7 , where X 1 It is a thermal destabilizing modification.

[0399] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0400] (Me) 8 –(F) 3 –(Me) 10 ,

[0401] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0402] (Me–F) 3 –(Me) 7 –F–Me–F–(Me) 7 .

[0403] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0404] (Me) 8 –(F) 3 –(Me) 10 ,

[0405] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0406] Me–F–(Me) 3 –F–(Me) 7 –(F–Me) 2 –F–(Me) 5 .

[0407] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0408] (Me) 8 –(F) 3 –(Me) 10 ,

[0409] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0410] Me–F–(Me) 3 –F–(Me) 7 –F–Me–F–(Me) 3 –F–(Me) 3 .

[0411] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0412] (Me) 8 –(F) 3 –(Me) 10 ,

[0413] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0414] Me–F–(Me) 3 –X 1 –Me–(F) 2 –(Me) 4 –F–Me–F–(Me) 7 ,

[0415] Where X 1 It is a thermal destabilizing modification.

[0416] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0417] (Me)8 –(F) 3 –(Me) 10 ,

[0418] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0419] (Me–F) 3 –Me–(F) 2 –(Me) 4 –(F–Me) 2 –(Me) 6 .

[0420] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0421] (Me) 8 –(F) 3 –(Me) 10 ,

[0422] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0423] Me–F–(Me) 3 –F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –F–(Me) 5 .

[0424] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0425] (Me) 8 –(F) 3 –(Me) 10 ,

[0426] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0427] Me–F–(Me) 3 –F–Me–(F) 2 –(Me) 4 –(F–Me)2 –(Me) 2 –F–(Me) 3 .

[0428] A nucleic acid wherein the second strand comprises the following 2' sugar and abasic modification pattern (5'-3'):

[0429] ia-ia-(Me) 8 –(F) 3 –(Me) 10

[0430] Where ia represents the inverted abasic nucleoside.

[0431] A nucleic acid wherein the second strand comprises the following 2' sugar and abasic modification pattern (5'-3'):

[0432] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

[0433] A nucleic acid wherein the second strand comprises the following 2' sugar and abasic modification pattern (5'-3'):

[0434] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of three, five or seven 2'F modifications.

[0435] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0436] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0437] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7, where X 1 It is a thermal destabilizing modification.

[0438] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0439] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0440] (Me–F) 3 –(Me) 7 –F–Me–F–(Me) 7 .

[0441] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0442] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0443] Me–F–(Me) 3 –F–(Me) 7 –(F–Me) 2 –F–(Me) 5 .

[0444] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0445] ia-ia-(Me) 8 –(F)3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0446] Me–F–(Me) 3 –F–(Me) 7 –F–Me–F–(Me) 3 –F–(Me) 3 .

[0447] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0448] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0449] Me–F–(Me) 3 –X 1 –Me–(F) 2 –(Me) 4 –F–Me–F–(Me) 7 ,

[0450] Where X 1 It is a thermal destabilizing modification.

[0451] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0452] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0453] (Me–F) 3 –Me–(F) 2 –(Me) 4–(F–Me) 2 –(Me) 6 .

[0454] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0455] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0456] Me–F–(Me) 3 –F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –F–(Me) 5 .

[0457] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0458] ia-ia-(Me) 8 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein the nucleosides of the first strand comprise the following 2' sugar modification pattern (5'-3'):

[0459] Me–F–(Me) 3 –F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –(Me) 2 –F–(Me) 3 .

[0460] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0461] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me)10 ,

[0462] Where ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond.

[0463] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0464] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond; and

[0465] wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

[0466] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0467] ia-ia-Me(s)Me(s)(Me)e-(F)s-(Me)io, wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond; and

[0468] wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of three, five or seven 2'F modifications.

[0469] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0470] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, (s) represents a phosphorothioate bond, and

[0471] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0472] Me(s)F(s)(Me) 3 –X 1 –(Me) 7 –F–Me–F–(Me)5 (s)Me(s)Me, where X 1 It is a thermal destabilizing modification.

[0473] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0474] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond, and

[0475] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0476] Me(s)F(s)Me–F–Me–F–(Me) 7 –F–Me–F–(Me) 5 (s)Me (s)Me.

[0477] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0478] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, (s) represents a phosphorothioate bond, and

[0479] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0480] Me(s)F(s)(Me) 3 –F–(Me) 7 –(F–Me) 2 –F–(Me) 3 (s)Me (s)Me.

[0481] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0482] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, (s) represents a phosphorothioate bond, and

[0483] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0484] Me(s)F(s)(Me) 3 –F–(Me) 7 –F–Me–F–(Me) 3 –F–Me(s)Me(s)Me.

[0485] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0486] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, (s) represents a phosphorothioate bond, and

[0487] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0488] Me(s)F(s)(Me) 3 –X 1 –Me–(F) 2 –(Me) 4 –F–Me–F–(Me) 5 (s)Me(s)Me, where X 1 It is a thermal destabilizing modification.

[0489] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0490] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, (s) represents a phosphorothioate bond, and

[0491] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0492] Me(s)F(s)Me–F–Me–F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –(Me) 4 (s)Me (s)Me.

[0493] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0494] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, (s) represents a phosphorothioate bond, and

[0495] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0496] Me(s)F(s)(Me) 3 –F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –F–(Me) 3 (s)Me (s)Me.

[0497] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0498] ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond, and

[0499] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0500] Me(s)F(s)(Me) 3 –F–Me–(F) 2 –(Me) 4 –(F–Me) 2 –(Me) 2 –F–Me(s)Me(s)Me.

[0501] Preferred modifications are as follows:

[0502] Modification mode 1:

[0503] Second strand (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0504] First strand (5'-3'): Me–F–Me–Me–Me–X 1 –Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me–Me-Me, where X 1 It is a thermal destabilization modification;

[0505] Or modifier mode 2:

[0506] Second strand (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0507] First strand (5'-3'): Me–F–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me;

[0508] Modification model 3:

[0509] Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0510] 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me–Me–Me ；

[0511] Modification model 4:

[0512] Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0513] 1st pin (5'-3'): Me–F–Me–Me-Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–F–Me–Me-Me ；

[0514] Modification model 5:

[0515] Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0516] 1st pin (5'-3'): Me–F–Me–Me-Me–X 1 –Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, part X 1 This is heat removal fixation;

[0517] Modification model 6:

[0518] Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0519] 1st pin (5'-3'): Me–F–Me–F–Me-F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me；

[0520] Modification model 7:

[0521] Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0522] 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me–Me–Me ；

[0523] Modification model 8:

[0524] Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0525] 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–F–Me–Me–Me,

[0526] Among them, the representative of IA is unopposed.

[0527] Step by step graceful practice:

[0528] Ceremony model 1:

[0529] Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0530] 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–X 1 –Me–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me–Me(s)Me(s)Me, where X1 is the heat-stabilizing modifier;

[0531] A modification model 2:

[0532] Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0533] 1st pin (5'-3'): Me(s)F(s)Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me;

[0534] Modification model 3:

[0535] Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0536] 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–F–Me–Me–Me(s)Me(s)Me;

[0537] Modification model 4:

[0538] Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0539] 1st pin (5'-3'): Me(s)F(s)Me–Me-Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–F–Me(s)Me(s)Me;

[0540] Modification model 5:

[0541] Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0542] 1st pin (5'-3'): Me(s)F(s)Me–Me-Me–X 1 –Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, in which X1 is heat removal fixation;

[0543] Modification model 6:

[0544] Second strand (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0545] First strand (5'-3'): Me(s)F(s)Me–F–Me-F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me;

[0546] Or with modifier mode 7:

[0547] Second strand (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0548] First strand (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me(s)Me(s)Me;

[0549] Or in modifier mode 8:

[0550] Second strand (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me,

[0551] First strand (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–F–Me(s)Me(s)Me;

[0552] where (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside.

[0553] Conjugation

[0554] Another modification of the nucleic acid (RNA (e.g., siRNA) of the present application involves linking the nucleic acid (e.g., siRNA) to one or more ligand moieties, for example, to enhance the activity of the nucleic acid (e.g., siRNA), such as entry into cells, cellular distribution or cellular uptake.

[0555] In some embodiments, the ligand moiety can be attached to a nucleic acid, such as a siRNA oligonucleotide, via a cleavable or non-cleavable linker.The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, for example, covalently connects two parts of a compound.

[0556] The ligand can be attached to the 3' or 5' end of the sense strand.

[0557] The ligand is preferably conjugated to the 3' end of the sense strand of a nucleic acid (eg, siRNA agent).

[0558] Therefore, the present application relates in another aspect to a conjugate for inhibiting the expression of a target gene in a cell, the conjugate comprising a nucleic acid portion and one or more ligand portions, the nucleic acid portion comprising a nucleic acid as disclosed herein.

[0559] In one aspect, the second strand of the nucleic acid is conjugated directly or indirectly (eg, via a linker) to one or more ligand moieties, wherein the ligand moiety is typically at the terminal region of the second strand, preferably at its 3' terminal region.

[0560] In certain embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative attached to a nucleic acid (eg, a dsiRNA) via a linker.

[0561] The present application therefore relates to a conjugate, wherein the ligand part comprises:

[0562] i) one or more GalNAc ligands; and / or

[0563] ii) one or more GalNAc ligand derivatives; and / or

[0564] iii) one or more GalNAc ligands conjugated to the nucleic acid via a linker.

[0565] The GalNAc ligand may be directly or indirectly conjugated to the 5' or 3' terminal region of the second nucleic acid strand, preferably conjugated to the 3' terminal region thereof.

[0566] GalNAc ligands are well known in the art and are described in EP3775207A1 and the like.

[0567] In some embodiments, the GalNAc ligand is contained in Figures 1 to 4 or Figure 5In any one of the linkers shown in (Formula XI), wherein "oligonucleotide" can be any nucleic acid disclosed herein. Therefore, "oligonucleotide" can contain other bonds besides phosphodiester bonds, such as one or more phosphorothioate bonds. Preferably, the nucleic acid according to the present application is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand via a phosphodiester bond, more preferably to the 3' terminal region of the second strand.

[0568] In some embodiments, the GalNAc ligand is contained in Figure 3 In the connector shown, "oligonucleotide" can be any nucleic acid disclosed herein. Therefore, "oligonucleotide" can contain other bonds except phosphodiester bonds, such as one or more phosphorothioate bonds. Preferably, the nucleic acid according to the present application is a double-stranded oligonucleoside as defined herein, and the connector is conjugated to the second strand via a phosphodiester bond, more preferably to the 3' terminal region of the second strand.

[0569] In some embodiments, the GalNAc ligand is contained in Figure 5 In the connector shown in (Formula XI), "oligonucleotide" can be any nucleic acid disclosed herein. Therefore, "oligonucleotide" can contain other bonds besides phosphodiester bonds, such as one or more phosphorothioate bonds. Preferably, the nucleic acid according to the present application is a double-stranded oligonucleoside as defined herein, and the connector is conjugated to the second strand via a phosphodiester bond, more preferably to the 3' terminal region of the second strand.

[0570] In some embodiments, the GalNAc ligand is contained in Figures 1 to 4 or Figure 5 In any one of the linkers shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified or unmodified second chain, and the second chain comprises SEQ ID NO: 265 or SEQ ID NO: 268 or consists of it, preferably wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 265 or SEQ ID NO: 268) via a phosphodiester bond.

[0571] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified or unmodified second chain, and the second chain comprises SEQ ID NO: 265 or SEQ ID NO: 268 or consists of it, preferably wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 265 or SEQ ID NO: 268) via a phosphodiester bond.

[0572] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified or unmodified second chain, and the second chain comprises SEQ ID NO: 265 or SEQ ID NO: 268 or consists of it, preferably wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 265 or SEQ ID NO: 268) via a phosphodiester bond.

[0573] In some embodiments, the GalNAc ligand is contained in Figures 1 to 4 or Figure 5 In any one of the linkers shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified second chain, and the second chain comprises SEQ ID NO: 774 or SEQ ID NO: 776 or consists of it, preferably wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 774 or SEQ ID NO: 776) via a phosphodiester bond.

[0574] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified second chain, wherein the second chain comprises SEQ ID NO: 774 or SEQ ID NO: 776 or consists of it, preferably wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 774 or SEQ ID NO: 776) via a phosphodiester bond.

[0575] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified second chain, and the second chain comprises SEQ ID NO: 774 or SEQ ID NO: 776 or consists of it, preferably wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 774 or SEQ ID NO: 776) via a phosphodiester bond.

[0576] In some embodiments, the GalNAc ligand is contained in Figures 1 to 4 or Figure 5In any one of the linkers shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first chain and a modified second chain, the first chain comprises SEQ ID NO: 764 or consists of it, and the second chain comprises SEQ ID NO: 774 or consists of it, preferably, wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQID NO: 774) via a phosphodiester bond.

[0577] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first chain and a modified second chain, the first chain comprises SEQ ID NO: 764 or consists of it, and the second chain comprises SEQ ID NO: 774 or consists of it, preferably, wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 774) via a phosphodiester bond.

[0578] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first chain and a modified second chain, the first chain comprises SEQ ID NO: 764 or consists of it, and the second chain comprises SEQ ID NO: 774 or consists of it, preferably, wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 774) via a phosphodiester bond.

[0579] In some embodiments, the GalNAc ligand is contained in Figures 1 to 4 or Figure 5 In any one of the linkers shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first chain and a modified second chain, the first chain comprises SEQ ID NO: 766 or consists of it, and the second chain comprises SEQ ID NO: 776 or consists of it, preferably, wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQID NO: 776) via a phosphodiester bond.

[0580] In some embodiments, the GalNAc ligand is contained in Figure 3In the linker shown, "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first chain and a modified second chain, the first chain comprises SEQ ID NO: 766 or consists of it, and the second chain comprises SEQ ID NO: 776 or consists of it, preferably, wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 776) via a phosphodiester bond.

[0581] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first chain and a modified second chain, the first chain comprises SEQ ID NO: 766 or consists of it, and the second chain comprises SEQ ID NO: 776 or consists of it, preferably, wherein the linker is conjugated to the 3' terminal region of the second chain (i.e., to the 3' terminal region of SEQ ID NO: 776) via a phosphodiester bond.

[0582] In some embodiments, the GalNAc ligand is contained in Figure 3 In the connector shown, "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, wherein the first strand comprises SEQ ID NO: 764 or consists of it, and the second strand comprises SEQ ID NO: 774 or consists of it, wherein the second strand has the following structure:

[0583]

[0584] in:

[0585] T represents 2'Me ribose modification,

[0586] B represents the nucleobases of the first two base nucleosides in the 5' terminal region of SEQ ID NO: 774, and

[0587] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO:774.

[0588] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, wherein the first strand comprises SEQ ID NO: 764 or consists thereof, and the second strand comprises SEQ ID NO: 774 or consists thereof, wherein the second strand has the following structure:

[0589]

[0590] in:

[0591] T represents 2'Me ribose modification,

[0592] B represents the nucleobases of the first two base nucleosides in the 5' terminal region of SEQ ID NO: 774, and

[0593] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO:774.

[0594] In some embodiments, the GalNAc ligand is contained in Figure 3 In the connector shown, "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, wherein the first strand comprises SEQ ID NO: 766 or consists of it, and the second strand comprises SEQ ID NO: 776 or consists of it, wherein the second strand has the following structure:

[0595]

[0596] in:

[0597] T represents 2'Me ribose modification,

[0598] B represents the nucleoside bases of the first two base nucleosides in the 5' terminal region of SEQ ID NO: 776, and

[0599] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO:776.

[0600] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein "oligonucleotide" represents the nucleic acid according to the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, wherein the first strand comprises SEQ ID NO: 766 or consists thereof, and the second strand comprises SEQ ID NO: 776 or consists thereof, wherein the second strand has the following structure:

[0601]

[0602] in:

[0603] T represents 2'Me ribose modification,

[0604] B represents the nucleoside bases of the first two base nucleosides in the 5' terminal region of SEQ ID NO: 776, and

[0605] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO:776.

[0606] Vectors and cells

[0607] In one aspect, the application provides cells containing a nucleic acid, such as an inhibitory RNA [RNAi] described herein.

[0608] In one aspect, the present application provides a cell comprising a vector described herein.

[0609] Pharmaceutically acceptable compositions

[0610] In one aspect, the present application provides a pharmaceutical composition for inhibiting the expression of a target gene, the composition comprising the nucleic acid disclosed herein.

[0611] A pharmaceutically acceptable composition may comprise an excipient and / or a carrier.

[0612] Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) 3) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) fillers such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL and LDL; and (22) other nontoxic compatible substances used in pharmaceutical preparations.

[0613] Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0614] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and not having adverse reactions with nucleic acids can also be used to prepare the compositions of the present application. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, etc.

[0615] Preparations for topical administration of nucleic acid may include aseptic and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as ethanol or nucleic acid solutions in liquid or solid oil bases. The solution may also contain buffer, diluent and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and not having adverse reactions with nucleic acid may be used.

[0616] In one embodiment, nucleic acid or composition is applied in non-buffered solution. In certain embodiments, non-buffered solution is saline or water. In other embodiments, nucleic acid (e.g., siRNA agent) is applied in buffered solution. In such embodiments, buffered solution can include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. For example, buffered solution can be phosphate buffered saline (PBS).

[0617] dose

[0618] The pharmaceutical composition of the application can be used with a dosage sufficient to inhibit gene expression. Typically, the suitable dosage of the nucleic acid (e.g., siRNA) of the application is about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, typically about 1 to 50 mg per kilogram of body weight per day. Typically, the suitable dosage of the nucleic acid (e.g., siRNA) of the application is about 0.1 mg / kg to about 5.0 mg / kg, for example, about 0.3 mg / kg and about 3.0 mg / kg.

[0619] Repeated dosage regimens can include administering a therapeutic amount of nucleic acid, such as siRNA, regularly (e.g., every other day or once a year). In certain embodiments, nucleic acid (e.g., siRNA) is administered about once a month to about once a quarter (i.e., about once every three months).

[0620] In various embodiments, nucleic acid (e.g., siRNA agent) is administered with a dosage of about 0.01mg / kg to about 10mg / kg or about 0.5mg / kg to about 50mg / kg. In some embodiments, nucleic acid (e.g., siRNA agent) is administered with a dosage of about 10mg / kg to about 30mg / kg. In certain embodiments, nucleic acid (e.g., siRNA agent) is administered with a dosage selected from about 0.5mg / kg, 1mg / kg, 1.5mg / kg, 3mg / kg, 5mg / kg, 10mg / kg and 30mg / kg. In certain embodiments, nucleic acid (e.g., siRNA agent) is administered with a dosage of about 0.1mg / kg to about 5.0mg / kg approximately once a week, once a month, once every two months or once a quarter (i.e., once every three months). In certain embodiments, nucleic acid (e.g., siRNA agent) is administered to a subject once a week. In certain embodiments, nucleic acid (e.g., siRNA agent) is administered to a subject once a month. In certain embodiments, nucleic acid (e.g., siRNA agent) is administered once a quarter (i.e., every three months).

[0621] After the initial treatment regimen, treatment may be given less frequently, for example, once a week or every two weeks for three months, followed by repeated monthly administration for six months or a year, or longer.

[0622] The pharmaceutical composition can be applied once daily, or applied with two, three or more sub-doses at appropriate intervals in a day, or even using continuous infusion or delivering by controlled release formulations. In this case, the nucleic acid (e.g., siRNA) contained in each sub-dose must be correspondingly smaller, to reach a daily total dose. The dosage unit can also be compounded to deliver within a few days, for example, using conventional sustained-release formulations, which can provide the sustained release of nucleic acid (e.g., siRNA) within a few days. Sustained-release formulations are well-known in the art, and are particularly suitable for delivering medicaments at specific locations, such as can be used together with the medicament of the present application. In this embodiment, the dosage unit contains a daily dose of corresponding multiples.

[0623] In other embodiments, the single dose of the pharmaceutical composition can last for a long time, so that the subsequent dose is used at an interval of no more than 3,4 or 5 days, or is used at an interval of no more than 1,2,3 or 4 weeks. In some embodiments of the application, the pharmaceutical composition of the application of a single dose is used once a week. In other embodiments of the application, the pharmaceutical composition of the application of a single dose is used once every two months. In certain embodiments, siRNA is used about once a month to about once a quarter (that is, about once every three months), or even once every 6 months or 12 months.

[0624] Effective dosages and in vivo half-lives of individual nucleic acids (eg, siRNAs) encompassed by the present application can be estimated using conventional methods or based on in vivo testing using appropriate animal models (as is known in the art).

[0625] The pharmaceutical composition of the present application can be applied in a variety of ways, depending on whether local treatment or systemic treatment is needed and depending on the area to be treated. Application can be local (for example, by transdermal patch), pulmonary (for example, by inhalation or blowing into powder or aerosol, including by a nebulizer), intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example, by implantation device; or intracranial, for example, by intraparenchymal, intrathecal or intraventricular administration. In certain preferred embodiments, the composition is applied by intravenous infusion or injection. In certain embodiments, the composition is applied by subcutaneous injection.

[0626] In one embodiment, the nucleic acid (eg, agent) is administered to the subject subcutaneously.

[0627] Nucleic acids (eg, siRNA) can be delivered in a manner that targets specific tissues (eg, particularly hepatocytes).

[0628] Methods for inhibiting ZPI gene expression

[0629] The present application also provides a method for inhibiting the expression of a ZPI gene in a cell. The method comprises contacting a cell with a nucleic acid of the present application (e.g., an siRNA agent, such as a double-stranded siRNA agent), the amount of which is effective in inhibiting the expression of the ZPI gene in the cell, thereby inhibiting the expression of the ZPI gene in the cell. It should be noted that the nucleic acid "for inhibiting the expression of ZPI" is a nucleic acid capable of inhibiting the expression of ZPI, preferably as described below.

[0630] The contact of cells with nucleic acids (e.g., siRNA, such as double-stranded siRNA agents) can be carried out in vitro or in vivo. Cells are contacted with nucleic acids in vivo, for example, including contacting cells or a group of cells in a subject (e.g., a human subject) with nucleic acids (e.g., siRNA). In vitro and in vivo methods of contacting cells can also be combined. As described above, contacting cells can be direct or indirect. In addition, contacting cells can be achieved by targeting ligand moieties, including any ligand moieties described herein or known in the art. In a preferred embodiment, the targeting ligand moiety is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand moiety that guides the siRNA agent to the target site.

[0631] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0632] In some embodiments of the methods of the present application, expression of the ZPI gene is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or to below the level of detection as determined, preferably when assayed by qPCR as described herein and / or when siRNA is introduced into target cells by transfection. In certain embodiments, the method includes clinically relevant inhibition of ZPI target gene expression, e.g., as demonstrated by clinically relevant results following treatment of a subject with an agent to reduce gene expression.

[0633] In some embodiments, when transfected into cells, the nucleic acids of the present application inhibit the expression of the ZPI gene with an IC50 value of less than 2500 pM, 2400 pM, 2300 pM, 2200 pM, 2100 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0634] In a preferred embodiment, when the nucleic acid of the present application is transfected into a cell, its IC50 value for inhibiting the expression of the ZPI gene is less than 2500 pM. In a more preferred embodiment, when the nucleic acid of the present application is transfected into a cell, its IC50 value for inhibiting the expression of the ZPI gene is less than 1000 pM. In a more preferred embodiment, when the nucleic acid of the present application is transfected into a cell, its IC50 value for inhibiting the expression of the ZPI gene is less than 500 pM. In a most preferred embodiment, when the nucleic acid of the present application is transfected into a cell, its IC50 value for inhibiting the expression of the ZPI gene is less than 100 pM.

[0635] Inhibition of ZPI gene expression can be quantified by:

[0636] Huh7 cells (a human hepatocyte-derived cell line obtained from JCRB Cell Bank) were cultured at 37°C and 5% CO 2Atmosphere in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS. Then, siRNA duplexes or negative control siRNAs targeting ZPI mRNA (siRNA control; positive strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) can be used to transfect cells, using 10x3 times serial dilution, and the final duplex concentration range is 20nM to 1pM. Transfection can be performed by adding 9.7μL Opti-MEM (ThermoFisher) and 0.3μL Lipofectamine RNAiMAX (ThermoFisher) to each siRNA duplex of 10μL. The mixture can be incubated at room temperature for 15 minutes and then added to 100μL of complete growth medium containing 20,000 Huh7 cells. Before purifying total RNA using the RNeasy 96 kit (Qiagen), cells were incubated at 37°C / 5% CO 2 Each duplex can be tested in a single experiment by transfection in duplicate wells.

[0637] cDNA synthesis can be performed using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using specific primers for human ZPI (Hs01547819_ml) and human GAPDH (Hs02786624_gl) using the FastStart Universal Probe Master kit (Roche).

[0638] qPCR can be performed in duplicate on cDNA from each well and the average cycle threshold (Ct) calculated. Relative ZPI expression can be calculated from the average Ct values ​​using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells. Maximum percent inhibition of ZPI expression and IC50 values ​​can be calculated using GraphPad Prism 9 using a four-parameter (variable slope) model.

[0639] Alternatively or additionally, inhibition of ZPI gene expression may be characterized by a decrease in the mean relative expression of the ZPI gene.

[0640] In some embodiments, when cells are transfected with 0.1 nM of a nucleic acid of the present application, the average relative expression of ZPI is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0641] In some embodiments, when cells are transfected with 5 nM of a nucleic acid of the present application, the average relative expression of ZPI is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0642] The average relative expression of ZPI genes can be quantified by:

[0643] Huh7 cells (a human hepatocyte-derived cell line obtained from JCRB Cell Bank) were cultured at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS under an environment of 40 °C. Cells can be transfected with siRNA duplexes targeting ZPI mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) at final duplex concentrations of 5 nM and 0.1 nM. Transfection can be performed by adding 9.7 μL Opti-MEM (ThermoFisher) and 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture can be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells can be grown at 37 °C / 5% CO 2 After incubation for 24 hours at 4 °C, total RNA was purified using the RNeasy 96 kit (Qiagen). Each duplex was tested by transfection in duplicate wells in two independent experiments.

[0644] cDNA synthesis can be performed using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using specific primers for human ZPI (Hs01547819_ml) and human GAPDH (Hs02786624_gl) using the FastStart Universal Probe Master kit (Roche).

[0645] qPCR can be performed in duplicate on cDNA from each well and the average Ct calculated. Relative ZPI expression can be calculated from the average Ct values ​​using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells.

[0646] Inhibition of ZPI gene expression can be demonstrated by a decrease in the amount of mRNA for the target ZPI gene compared to a suitable control.

[0647] In other embodiments, inhibition of ZPI gene expression can be assessed by a decrease in a parameter associated with gene expression function, such as protein expression or signaling pathways.

[0648] Methods for treating or preventing diseases associated with ZPI gene expression

[0649] The present application also provides a method for reducing or inhibiting the expression of the ZPI gene in a cell using a nucleic acid (e.g., siRNA) of the present application or a composition containing the nucleic acid (e.g., siRNA) of the present application. The method includes contacting the cell with the nucleic acid (e.g., dsiRNA) of the present application, and maintaining the cell for a sufficient time to obtain degradation of the ZPI mRNA transcript, thereby inhibiting the expression of the ZPI gene in the cell. The reduction of gene expression can be assessed by any method known in the art.

[0650] In the methods of the present application, the cells may be contacted in vitro or in vivo, ie, the cells may be within the body of a subject.

[0651] Cells suitable for treatment using the methods of the present application can be any cells that express a target gene associated with a disease associated with a hemostatic disorder (eg, a disease associated with a hemostatic disorder, such as hemophilia).

[0652] The in vivo method of the present application may include administering to a subject a composition containing a nucleic acid (eg, siRNA) of the present application, wherein the nucleic acid (eg, siRNA) includes a nucleotide sequence complementary to at least a portion of an RNA transcript of a ZPI gene of a mammal to be treated.

[0653] The present application further provides a method for treating a subject in need thereof. The method of treatment of the present application comprises administering a nucleic acid (such as siRNA) of the present application, such as a nucleic acid (such as siRNA) targeting ZPI or a pharmaceutical composition comprising a nucleic acid targeting ZPI, to a subject (e.g., a subject benefiting from reducing or inhibiting ZPI gene expression) in a therapeutically effective amount. The disease to be treated is related to a hemostatic disorder, such as a disease associated with a hemostatic disorder, such as hemophilia.

[0654] Hemophilia (haemophilia or hemophilia) is a genetic disorder caused primarily by inheritance that impairs the body's ability to produce blood clots, a process necessary to stop bleeding. This causes subjects to bleed longer after an injury, bruise easily, and have an increased risk of bleeding into the joints or brain. Subjects with mild forms of the disease may only experience symptoms after an accident or during surgery. Bleeding into the joints (also called hemarthrosis) can lead to permanent injury, while bleeding into the brain can cause long-term headaches, seizures, or a decreased level of consciousness.

[0655] There are two main types of hemophilia: hemophilia A, which occurs due to low levels of clotting factor VIII, and hemophilia B, which occurs due to low levels of clotting factor IX. Both types are usually inherited from parents through the X chromosome that carries a non-functional gene. Rarely, a new mutation may occur during early development or later in life due to the formation of antibodies against clotting factors. Other types include hemophilia C, which occurs due to low levels of factor XI, von Willebrand disease, which occurs due to low levels of a substance called von Willebrand factor, and parahemophilia, which occurs due to low levels of factor V. Hemophilia A, B, and C prevent the intrinsic pathway from functioning properly; this clotting pathway is necessary when the lining of the blood vessels is damaged. Acquired hemophilia has been linked to cancer, autoimmune diseases, and pregnancy. Diagnosis is by testing the blood's ability to clot and the levels of clotting factors.

[0656] In certain embodiments, the nucleic acids of the present application are suitable for treatment, or for use in the treatment of hemophilia A, B, and / or C. In certain embodiments, the nucleic acids of the present application are suitable for treatment, or for use in the treatment of hemophilia A and / or B. In certain embodiments, the nucleic acids of the present application are suitable for treatment, or for use in the treatment of acquired hemophilia. In certain embodiments, the nucleic acids of the present application are suitable for treatment, or for use in the treatment of von Willebrand disease. In certain embodiments, the nucleic acids of the present application are suitable for treatment, or for use in the treatment of parahemophilia.

[0657] Without wishing to be bound by theory, treatment with the nucleic acid of the present application may result in an increase in the level of coagulation factors, thereby reducing or preventing bleeding, as shown in Figure 12. Therefore, in a preferred embodiment, treatment with the nucleic acid of the present application reduces or prevents bleeding episodes in subjects with hemophilia. In another preferred embodiment, treatment with the nucleic acid of the present application reduces or prevents joint bleeding in subjects with hemophilia. In certain embodiments, treatment with the nucleic acid of the present application reduces or prevents muscle or brain bleeding in subjects with hemophilia.

[0658] Alternatively or additionally, treating a subject, preferably a subject suffering from a hemostatic disorder such as hemophilia, with a nucleic acid of the present application may result in one or more of the following outcomes:

[0659] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in decreased bone marrow proliferation. Fig.14A As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced bone marrow proliferation in the mice.

[0660] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in osteoarthritis. Fig. 14B As shown, treatment of Haem A mice with the nucleic acid of the present application significantly alleviated osteoarthritis in the mice.

[0661] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in chondrocyte degeneration / necrosis. Fig. 14C As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced chondrocyte degeneration / necrosis in the mice.

[0662] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in reduced bleeding. Fig.14D As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced bleeding in the mice.

[0663] In certain embodiments, treatment of a subject, preferably a subject suffering from a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in hemosiderin deposition. Fig.14E As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced the deposition of hemosiderin in the mice.

[0664] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduced incidence of hematomas. Fig.14FAs shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced the hematoma of the mice.

[0665] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a decrease in osteoclastic bone resorption. Figure 14G As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced osteoclastic bone resorption in the mice.

[0666] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a decrease in osteolysis. Fig.14H As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced osteolysis in the mice.

[0667] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in periostitis. Fig.14I As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced periostitis in the mice.

[0668] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in subchondral bone sclerosis. Fig.14J As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced subchondral bone sclerosis in the mice.

[0669] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in tendon degeneration. Figure 14K As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced tendon degeneration in said mice.

[0670] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in tendinitis. Figure 14L As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced tendinitis in the mice.

[0671] In certain embodiments, treatment of a subject, preferably a subject with a hemostatic disorder such as hemophilia, with a nucleic acid of the present application results in a reduction in tenosynovitis. Figure 14M As shown, treatment of Haem A mice with the nucleic acid of the present application significantly reduced tenosynovitis in the mice.

[0672] Therefore, in a specific embodiment, the application relates to nucleic acids suitable for or for treating hemophilia, wherein the treatment of hemophilia is characterized by reducing bleeding and one or more of the following: reducing bone marrow hyperplasia, reducing osteoarthritis, reducing chondrocyte degeneration / necrosis, reducing bleeding, reducing hemosiderin deposition, reducing hematomas, reducing osteoclastic bone resorption, reducing osteolysis, reducing periostitis, reducing subchondral bone sclerosis, reducing tendon degeneration, reducing tendinitis and / or reducing tenosynovitis. The nucleic acid (e.g., siRNA) of the present application can be administered as "free" nucleic acid or "free" siRNA, administered without a pharmaceutical composition. The exposed nucleic acid can be in a suitable buffer solution. The buffer solution can include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH value and osmotic pressure of the buffer solution can be adjusted to make it suitable for administration to a subject.

[0673] Alternatively, the nucleic acids (eg, siRNA) of the present application can be administered as a pharmaceutical composition (eg, dsiRNA liposome formulation).

[0674] In one embodiment, the method comprises administering a composition as described herein such that expression of the ZPI gene is reduced, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, or about 36 hours. In one embodiment, expression of the ZPI target gene is reduced for a longer period of time, such as at least about two, three, four days or longer, such as about one, two, three, or four weeks or longer, such as about one, two, or three months.

[0675] A therapeutic amount of a nucleic acid (eg, siRNA), such as about 0.01 mg / kg to about 200 mg / kg, can be administered to a subject to treat a disease associated with a hemostatic disorder, such as a disease associated with a hemostatic disorder, such as hemophilia.

[0676] Nucleic acids (e.g., siRNA) can be administered by intravenous infusion regularly over a period of time. In certain embodiments, after the initial treatment regimen, the treatment can be administered less frequently. The administration of siRNA can reduce, for example, the gene product level of the ZPI target gene in the patient's cells or tissues by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or below the detection level of the assay method used. In certain embodiments, the administration results in clinical stabilization or preferably a clinically relevant reduction in at least one sign or symptom of a ZPI gene-related disorder.

[0677] Or nucleic acid (e.g., siRNA) can be administered subcutaneously, i.e., administered by subcutaneous injection. One or more injections can be used to deliver a desired daily dose of nucleic acid (e.g., siRNA) to a subject. Injections can be repeated over a period of time. Administration can be repeated regularly. In certain embodiments, after the initial treatment regimen, treatment can be administered at a lower frequency. Repeated dose regimens can include regularly administering a therapeutic amount of nucleic acid, such as every other day or once a year. In certain embodiments, nucleic acid is administered approximately once a month to approximately once a quarter (i.e., approximately once every three months).

[0678] In one aspect, the present application can be applied to the compounds, methods, compositions or uses of the following Sentences 1-101, wherein reference to any formula in Sentences 1-101 refers only to those formulas defined in Sentences 1-101. Figure 5 Specifically, the oligonucleoside moiety represented by Z in any of the following may comprise a nucleic acid for inhibiting ZPI expression as defined in any of the claims below.

[0679] 1. A compound comprising the following structure:

[0680]

[0681] in:

[0682] R 1 is independently selected at each occurrence from hydrogen, methyl, and ethyl;

[0683] R 2 Selected from hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0684] X 1 and X 2 is independently selected at each occurrence from methylene, oxygen, and sulfur;

[0685] m is an integer from 1 to 6;

[0686] n is an integer from 1 to 10;

[0687] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0688] (i) q and r cannot be 0 at the same time; and

[0689] (ii) s, t and v cannot be 0 at the same time;

[0690] Z is an oligonucleoside moiety.

[0691] 2. The compound according to item 1, wherein R 1At each occurrence it is hydrogen.

[0692] 3. The compound according to item 1, wherein R 1 It's methyl.

[0693] 4. The compound according to item 1, wherein R 1 It is ethyl.

[0694] 5. A compound according to any one of items 1 to 4, wherein R 2 It's a hydroxyl group.

[0695] 6. A compound according to any one of items 1 to 4, wherein R 2 It's a halogen.

[0696] 7. The compound according to item 6, wherein R 2 It's fluorine.

[0697] 8. The compound according to item 6, wherein R 2 It's chlorine.

[0698] 9. The compound according to item 6, wherein R 2 It's bromine.

[0699] 10. The compound according to item 6, wherein R 2 It's iodine.

[0700] 11. The compound according to item 6, wherein R 2 It's nitro.

[0701] 12. A compound according to any one of items 1 to 11, wherein X 1 It is a methylene group.

[0702] 13. A compound according to any one of items 1 to 11, wherein X 1 It's oxygen.

[0703] 14. A compound according to any one of items 1 to 11, wherein X 1 It's sulfur.

[0704] 15. A compound according to any one of items 1 to 14, wherein X 2 It is a methylene group.

[0705] 16. A compound according to any one of items 1 to 15, wherein X 2 It's oxygen.

[0706] 17. A compound according to any one of items 1 to 16, wherein X 2 It's sulfur.

[0707] 18. The compound according to any one of items 1 to 17, wherein m=3.

[0708] 19. The compound according to any one of items 1 to 18, wherein n=6.

[0709] 20. The compound according to items 13 and 15, wherein X 1 is oxygen, and X 2 is a methylene group, and preferably wherein:

[0710] q=l,

[0711] r = 2,

[0712] s=1,

[0713] t=1,

[0714] v=1.

[0715] 21. The compound according to items 12 and 15, wherein X 1 and X 2 are all methylene, and preferably among them:

[0716] q=l,

[0717] r = 3,

[0718] s=1,

[0719] t=1,

[0720] v=1.

[0721] 22. A compound according to any one of items 1 to 21, wherein Z is:

[0722]

[0723] in:

[0724] Z 1 , Z 2 , Z 3 , Z 4 is independently oxygen or sulfur at each occurrence; and P and Z 2 Between P and Z 3 One of the bonds between is a single bond and the other is a double bond.

[0725] 23. A compound according to item 22, wherein the oligonucleoside is an RNA compound capable of regulating, preferably inhibiting, the expression of a target gene.

[0726] 24. A compound according to item 23, wherein the RNA compound comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end.

[0727] 25. A compound according to item 24, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[0728] 26. A compound according to item 24, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[0729] 27. A compound of formula (II):

[0730]

[0731] 28. A compound of formula (III)

[0732]

[0733] 29. A compound according to item 27 or 28, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[0734] 30. A composition comprising a compound of formula (II) as defined in item 27, and a compound of formula (III) as defined in item 28, optionally subject to item 29.

[0735] 31. The composition according to item 30, wherein the content of the compound of formula (III) as defined in item 28 is 10 to 15% by weight of the composition.

[0736] 32. A compound of formula (IV):

[0737]

[0738] 33. A compound of formula (V)

[0739]

[0740] 34. A compound according to item 32 or 33, wherein the oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is connected to an adjacent phosphate group at the 3' end of its second strand.

[0741] 35. A composition comprising a compound of formula (IV) as defined in item 32, and a compound of formula (V) as defined in item 33, optionally subject to item 34.

[0742] 36. The composition according to item 35, wherein the content of the compound of formula (V) defined in item 33 is 10 to 15% by weight of the composition.

[0743] 37. A compound as defined in any one of items 1 to 29 or 32 to 34, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[0744] 38. A compound according to item 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0745] 39. A compound according to any one of items 1 to 29, or 32 to 34, or 37 to 38, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0746] 40. A compound according to claim 39, wherein the one or more degradation protection parts are not at the end of the oligonucleoside chain carrying the ligand part, and / or wherein the one or more degradation protection parts are selected from thiophosphate internucleoside bonds, dithiophosphate internucleoside bonds and reverse abasic nucleosides, wherein the reverse abasic nucleosides are at the distal end of the chain carrying the ligand part.

[0747] 41. A compound according to any one of items 1 to 29, or 32 to 34, or 37 to 40, wherein the ligand portion represented by formula (I) in item 1 comprises one or more ligands.

[0748] 42. A compound according to item 41, wherein the ligand portion represented by formula (I) in item 1 comprises one or more carbohydrate ligands.

[0749] 43. A compound according to item 42, wherein the one or more carbohydrates may be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide.

[0750] 44. A compound according to item 43, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0751] 45. A compound according to item 44, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[0752] 46. ​​A compound according to item 45, which comprises two or three N-acetylgalactosamine moieties.

[0753] 47. A compound according to any one of items 41 to 46, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[0754] 48. A compound according to item 47, wherein the one or more ligands are connected in a biantennary or triantennary branching configuration.

[0755] 49. The compound according to items 46 to 48, wherein the moiety represented by formula (I) in item 1 is:

[0756]

[0757] is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0758]

[0759] in:

[0760] A I is hydrogen, or a suitable hydroxy protecting group;

[0761] a is an integer of 2 or 3; and

[0762] b is an integer from 2 to 5; or

[0763]

[0764] in:

[0765] A I is hydrogen, or a suitable hydroxy protecting group;

[0766] a is an integer of 2 or 3; and

[0767] c and d are independently an integer from 1 to 6; or

[0768]

[0769] in:

[0770] A I is hydrogen, or a suitable hydroxy protecting group;

[0771] a is an integer of 2 or 3; and

[0772] e is an integer from 2 to 10.

[0773] 50. The compound according to items 46 to 48, wherein the moiety represented by formula (I) in item 1 is:

[0774]

[0775] Formula (VII):

[0776]

[0777] in:

[0778] A I It is hydrogen;

[0779] a is an integer of 2 or 3.

[0780] 51. A compound according to item 49 or 50, wherein a=2.

[0781] 52. A compound according to item 49 or 50, wherein a=3.

[0782] 53. A compound according to item 49, wherein b=3.

[0783] 54. A compound of formula (VIII):

[0784]

[0785] 55. A compound of formula (IX):

[0786]

[0787] 56. A compound according to item 54 or 55, wherein the oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and 3' end, and wherein the RNA duplex is connected to an adjacent phosphate group at the 5' end of its second strand.

[0788] 57. A composition comprising a compound of formula (VIII) as defined in item 54, and a compound of formula (IX) as defined in item 55, optionally subject to item 56.

[0789] 58. The composition according to item 57, wherein the content of the compound of formula (IX) as defined in item 55 is 10 to 15% by weight of the composition.

[0790] 59. A compound of formula (X):

[0791]

[0792] 60. A compound of formula (XI):

[0793]

[0794] 61. A compound according to item 59 or 60, wherein the oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is connected to an adjacent phosphate group at the 3' end of its second strand.

[0795] 62. A composition comprising a compound of formula (X) as defined in item 59, and a compound of formula (XI) as defined in item 60, optionally subject to item 61.

[0796] 63. The composition according to item 62, wherein the content of the compound of formula (XI) defined in item 60 is from 10 to 15% by weight of the composition.

[0797] 64. A compound as defined in any one of items 54 to 63, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex further comprising one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[0798] 65. A compound according to item 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0799] 66. A compound according to any one of items 54 to 65, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0800] 67. A compound according to claim 66, wherein the one or more degradation protection parts are not at the end of the oligonucleoside chain carrying the ligand part, and / or wherein the one or more degradation protection parts are selected from thiophosphate internucleoside bonds, dithiophosphate internucleoside bonds and reverse abasic nucleosides, wherein the reverse abasic nucleosides are at the distal end of the chain carrying the ligand part, as shown in any one of formulas (VIII), (IX), (X) or (XI) in claims 54, 55, 59 or 60.

[0801] 68. A method for preparing a compound of any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67 and / or a composition of any one of items 30, 31, 35, 36, 57, 58, 62, 63, which comprises reacting compounds of formula (XII) and (XIII):

[0802]

[0803] In this article:

[0804] R 1 is independently selected at each occurrence from hydrogen, methyl, and ethyl;

[0805] R 2 Selected from hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0806] X 1 and X 2 is independently selected at each occurrence from methylene, oxygen, and sulfur;

[0807] m is an integer from 1 to 6;

[0808] n is an integer from 1 to 10;

[0809] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0810] (i) q and r cannot be 0 at the same time; and

[0811] (ii) s, t and v cannot be 0 at the same time;

[0812] Z is an oligonucleoside moiety;

[0813] And where appropriate, deprotection of the ligand and / or annealing of the second strand of the oligonucleoside moiety is performed.

[0814] 69. A process according to item 68, wherein the compound of formula (XII) is prepared by reacting compounds of formula (XIV) and (XV):

[0815]

[0816] R 1 is independently selected at each occurrence from hydrogen, methyl, and ethyl;

[0817] R 2 Selected from hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0818] X 1 and X 2 is independently selected at each occurrence from methylene, oxygen, and sulfur;

[0819] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0820] (i) q and r cannot be 0 at the same time; and

[0821] (ii) s, t and v cannot be 0 at the same time;

[0822] Z is an oligonucleoside moiety.

[0823] 70. A method according to claim 68 for preparing a compound of any one of claims 20, 25, 27, 29, 54, 56, and / or a composition of any one of claims 30, 31, 57, 58, wherein:

[0824] The compound of formula (XII) is formula (XIIa):

[0825]

[0826] The compound of formula (XIII) is formula (XIIIa):

[0827]

[0828] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is linked to adjacent phosphate groups at the 5' end of the second strand thereof.

[0829] 71. A method according to claim 68 for preparing a compound of any one of claims 20, 25, 28, 29, 55, 56, and / or a composition of any one of claims 30, 31, 57, 58, wherein: the compound of formula (XII) is formula (XIIb):

[0830]

[0831] The compound of formula (XIII) is formula (XIIIa):

[0832]

[0833] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is linked to adjacent phosphate groups at the 5' end of the second strand thereof.

[0834] 72. A method according to claim 68 for preparing a compound of any one of claims 21, 26, 32, 34, 59, 61, and / or a composition of any one of claims 35, 36, 62, 63, wherein:

[0835] The compound of formula (XII) is formula (XIIc):

[0836]

[0837] The compound of formula (XIII) is formula (XIIIa):

[0838]

[0839] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is linked to adjacent phosphate groups at the 3' end of the second strand thereof.

[0840] 73. A method according to claim 68 for preparing a compound of any one of claims 21, 26, 33, 34, 60, 61, and / or a composition of any one of claims 35, 36, 62, 63, wherein:

[0841] The compound of formula (XII) is formula (XIId):

[0842]

[0843] The compound of formula (XIII) is formula (XIIIa):

[0844]

[0845] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of the second strand thereof.

[0846] 74. A method according to any one of items 70 to 73, wherein:

[0847] The compound of formula (XIIIa) is formula (XIIIb):

[0848]

[0849] 75. The method according to item 69, subject to items 70 to 73, wherein: the compound of formula (XIV) is formula (XIVa) or formula (XIVb):

[0850]

[0851] And the compound of formula (XV) is formula (XVa) or formula (XIVb):

[0852]

[0853]

[0854] wherein the oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein (i) the RNA duplex is linked to adjacent phosphate groups in formula (XVa) at the 5' end of the second strand, or (ii) the RNA duplex is linked to adjacent phosphate groups in formula (XVb) at the 3' end of the second strand.

[0855] 76. A compound of formula (XII):

[0856]

[0857] in:

[0858] R 1 is independently selected at each occurrence from hydrogen, methyl, and ethyl;

[0859] R 2 Selected from hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0860] X 1 and X 2 is independently selected at each occurrence from methylene, oxygen, and sulfur;

[0861] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0862] (i) q and r cannot be 0 at the same time; and

[0863] (ii) s, t and v cannot be 0 at the same time;

[0864] Z is an oligonucleoside moiety.

[0865] 77. A compound of formula (XIIa)

[0866]

[0868] 78. A compound of formula (XIIb):

[0869]

[0870] 79. A compound of formula (XIIc):

[0871]

[0872] 80. A compound of formula (XIId):

[0873]

[0874] 81. A compound of formula (XIII):

[0875]

[0876] in:

[0877] R 1 is independently selected at each occurrence from hydrogen, methyl, and ethyl;

[0878] m is an integer from 1 to 6;

[0879] n is an integer of 1 to 10.

[0880] 82. A compound of formula (XIIIa):

[0881]

[0882] 83. A compound of formula (XIIIb):

[0883]

[0884] 84. A compound of formula (XIV):

[0885]

[0886] in:

[0887] R 1 is selected from the group consisting of hydrogen, methyl and ethyl;

[0888] R 2 Selected from hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0889] X 2 is selected from methylene, oxygen and sulfur;

[0890] s, t, and v are independently integers from 0 to 4, provided that s, t, and v cannot be 0 at the same time.

[0891] 85. A compound of formula (XIVa):

[0892]

[0893] 86. A compound of formula (XIVb):

[0894]

[0895] 87. A compound of formula (XV):

[0896]

[0897] in:

[0898] R 1 is independently selected at each occurrence from hydrogen, methyl, and ethyl;

[0899] X 1 is selected from methylene, oxygen and sulfur;

[0900] q and r are independently integers from 0 to 4, provided that q and r cannot be 0 at the same time;

[0901] Z is an oligonucleoside moiety.

[0902] 88. A compound of formula (XVa)

[0903]

[0904] 89. A compound of formula (XVb):

[0905]

[0906] 90. Use of a compound of any one of items 76, 81 to 84, 87 in the preparation of a compound of any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67 and / or a composition of any one of items 30, 31, 35, 36, 57, 58, 62 and 63.

[0907] 91. Use of a compound of item 85 in the preparation of a compound of any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition of any one of items 30, 31, 35, 36, 57, 58, 62 and 63, wherein R 2 =F.

[0908] 92. Use of a compound of item 86 in the preparation of a compound of any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition of any one of items 30, 31, 35, 36, 57, 58, 62 and 63, wherein R 2 =OH.

[0909] 93. Use of the compound of item 77 in the preparation of the compound of any one of items 20, 25, 27, 29, 54, 56 and / or the composition of any one of items 30, 31, 57, 58.

[0910] 94. Use of the compound of item 78 in the preparation of any compound of items 20, 25, 28, 29, 55, 56 and / or any composition of items 30, 31, 57, 58.

[0911] 95. Use of the compound of item 79 in the preparation of any compound of items 21, 26, 32, 34, 59, 61 and / or any composition of items 35, 36, 62, 63.

[0912] 96. Use of the compound of item 80 in the preparation of any compound of items 21, 26, 33, 34, 60, 61 and / or any composition of items 35, 36, 62, 63.

[0913] 97. Use of the compound of item 88 in the preparation of the compound of any one of items 20, 25, 27 to 29, 54 to 56 and / or the composition of any one of items 30, 31, 57, 58.

[0914] 98. Use of the compound of item 89 in the preparation of the compound of any one of items 21, 26, 32 to 34, 59 to 61 and / or the composition of any one of items 35, 36, 62, 63.

[0915] 99. A compound or composition obtained or obtainable by the method of any one of items 68 to 75.

[0916] 100. A pharmaceutical composition comprising a compound of any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition of any one of items 30, 31, 35, 36, 57, 58, 62 and 63, and a pharmaceutically acceptable carrier, diluent or excipient.

[0917] 101. A compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, for use in therapy.

[0918] In another aspect, the present application may be applied to the compounds, methods, compositions or uses of the following clauses 1-56, wherein reference to any formula in a clause refers only to those formulas defined in clauses 1-56. Figure 6 Specifically, the oligonucleoside portion represented by Z in any of the following clauses may comprise a nucleic acid for inhibiting ZPI expression as defined in any of the following claims.

[0919] 1. A compound comprising the following structure:

[0920]

[0921] wherein: r and s are independently integers selected from 1 to 16; and

[0922] Z is an oligonucleoside moiety.

[0923] 2. The compound according to item 1, wherein s is an integer selected from 4 to 12.

[0924] 3. A compound according to clause 2, wherein s is 6.

[0925] 4. The compound according to any one of clauses 1 to 3, wherein r is an integer selected from 4 to 14.

[0926] 5. A compound according to clause 4, wherein r is 6.

[0927] 6. A compound according to clause 4, wherein r is 12.

[0928] 7. The compound according to clause 5, which is subject to clause 3.

[0929] 8. The compound according to clause 6, which is subject to clause 3.

[0930] 9. A compound according to any one of clauses 1 to 8, wherein Z is:

[0931]

[0932] in:

[0933] Z 1 , Z 2 , Z 3 , Z 4 is independently at each occurrence oxygen or sulfur; and

[0934] P and Z 2 Between P and Z 3 One of the bonds between is a single bond and the other is a double bond.

[0935] 10. The compound according to any one of clauses 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0936] 11. A compound according to item 10, wherein the RNA compound comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end.

[0937] 12. The compound according to clause 11, preferably also subject to clauses 3 and 6, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[0938] 13. The compound according to clause 11, preferably also subject to clauses 3 and 5, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[0939] 14. A compound of formula (II), preferably belonging to clause 12:

[0940]

[0941] 15. A compound of formula (III), preferably belonging to clause 13:

[0942]

[0943] 16. A compound as defined in any one of clauses 1 to 15, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex further comprising one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[0944] 17. A compound according to clause 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0945] 18. A compound according to any one of clauses 1 to 17, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0946] 19. A compound according to clause 18, wherein the one or more degradation protection parts are not at the end of the oligonucleoside chain carrying the linker / ligand part, and / or wherein the one or more degradation protection parts are selected from thiophosphate internucleoside bonds, dithiophosphate internucleoside bonds and reverse abasic nucleosides, wherein the reverse abasic nucleosides are at the distal end of the chain that is the same as the end carrying the linker / ligand part.

[0947] 20. The compound according to any one of clauses 1 to 19, wherein the ligand portion represented by formula (I) in clause 1 comprises one or more ligands.

[0948] 21. A compound according to item 20, wherein the ligand portion represented by formula (I) in item 1 comprises one or more carbohydrate ligands.

[0949] 22. A compound according to clause 21, wherein the one or more carbohydrates may be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide.

[0950] 23. A compound according to clause 22, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties and / or one or more mannose moieties.

[0951] 24. A compound according to clause 23, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[0952] 25. A compound according to clause 24, comprising two or three N-acetylgalactosamine moieties.

[0953] 26. The compound according to any of the preceding clauses, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[0954] 27. A compound according to clause 26, wherein the one or more ligands are connected in a biantennary or triantennary branching configuration.

[0955] 28. The compound according to clauses 20 to 27, wherein the moiety represented by formula (I) in clause 1 is:

[0956]

[0957] Any one of formula (IV), (V) or (VI), preferably formula (IV):

[0958]

[0959] in:

[0960] A I is hydrogen, or a suitable hydroxy protecting group;

[0961] a is an integer of 2 or 3;

[0962] b is an integer from 2 to 5; or

[0963]

[0964] in:

[0965] A I is hydrogen, or a suitable hydroxy protecting group;

[0966] a is an integer of 2 or 3; and

[0967] c and d are independently an integer from 1 to 6; or

[0968]

[0969] in:

[0970] A I is hydrogen, or a suitable hydroxy protecting group;

[0971] a is an integer of 2 or 3; and

[0972] e is an integer from 2 to 10.

[0973] 29. A compound according to any one of clauses 1 to 28, wherein the moiety represented by formula (I) in clause 1 is:

[0974]

[0975] Formula (VII):

[0976]

[0977]

[0978] in:

[0979] A I It is hydrogen;

[0980] a is an integer of 2 or 3.

[0981] 30. The compound according to clause 28 or 29, wherein a=2.

[0982] 31. A compound according to clause 28 or 29, wherein a=3.

[0983] 32. A compound according to clause 28, wherein b=3.

[0984] 33. A compound of formula (VIII)

[0985]

[0986] 34. A compound of formula (IX):

[0987]

[0988] 35. A compound according to clause 33 or 34, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex further comprising one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[0989] 36. A compound according to clause 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0990] 37. A compound according to any one of clauses 33 to 36, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0991] 38. A compound according to clause 37, wherein the one or more degradation protection parts are not at the end of the oligonucleoside chain carrying the linker / ligand part, and / or wherein the one or more degradation protection parts are selected from thiophosphate nucleoside bonds, dithiophosphate nucleoside bonds and reverse abasic nucleosides, wherein the reverse abasic nucleosides are at the far end of the chain that is the same as the end carrying the linker / ligand part.

[0992] 39. A compound according to item 33, wherein the oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is connected to an adjacent phosphate group at the 5' end of its second strand.

[0993] 40. A compound according to item 34, wherein the oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[0994] 41. A method for preparing a compound of any one of clauses 1 to 40, comprising reacting compounds of formula (X) and (XI):

[0995]

[0996] in:

[0997] r and s are independently integers selected from 1 to 16; and

[0998] Z is an oligonucleoside moiety; and

[0999] Where appropriate, deprotection of the ligand and / or annealing of the second strand of the oligonucleotide is performed.

[1000] 42. A method according to clause 41 to prepare a compound according to any one of clauses 6, 8 to 14, 16 to 33 and 35 to 40, wherein:

[1001] The compound of formula (X) is of formula (Xa):

[1002]

[1003]

[1004] The compound of formula (XI) is of formula (XIa):

[1005]

[1006] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is linked to adjacent phosphate groups at the 5' end of the second strand thereof.

[1007] 43. A method according to clause 41 to prepare a compound according to any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40, wherein:

[1008] The compound of formula (X) is of formula (Xb):

[1009]

[1010] The compound of formula (XI) is of formula (XIa):

[1011]

[1012] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is linked to adjacent phosphate groups at the 3' end of the second strand thereof.

[1013] 44. A method according to clause 42 or 43, wherein: the compound of formula (XIa) is formula (XIb):

[1014]

[1015] 45. A compound of formula (X):

[1016]

[1017] wherein: r is independently an integer selected from 1 to 16; and Z is an oligonucleoside moiety.

[1018] 46. ​​A compound of formula (Xa):

[1019]

[1020] 47. A compound of formula (Xb):

[1021]

[1022] 48. A compound of formula (XI):

[1023]

[1024]

[1025] in:

[1026] s is independently an integer selected from 1 to 16; and

[1027] Z is an oligonucleoside moiety.

[1028] 49. A compound of formula (XIa):

[1029]

[1030] 50. A compound of formula (XIb):

[1031]

[1032] 51. Use of a compound of any one of clauses 45 and 48 to 50 in the preparation of a compound of any one of clauses 1 to 40.

[1033] 52. Use of a compound of clause 46 in the preparation of a compound of any one of clauses 6, 8 to 14, 16 to 33 and 35 to 40.

[1034] 53. Use of a compound of clause 47 in the preparation of a compound of any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40.

[1035] 54. A compound or composition obtained or obtainable by the method of any one of clauses 41 to 44.

[1036] 55. A pharmaceutical composition comprising a compound of any one of clauses 1 to 40 and a pharmaceutically acceptable carrier, diluent or excipient.

[1037] 56. A compound according to any one of clauses 1 to 40 for use in therapy. Example

[1038] The application will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the application. It should be understood that the embodiments and embodiments described herein are only for illustrative purposes, and those skilled in the art can make various modifications or changes accordingly, and these modifications or changes should all be included in the spirit and scope of the application and the scope of the additional clauses.

[1039] Example 1: Synthesis of Tether 1

[1040] General experimental conditions:

[1041] Thin layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. The fluorescence was determined under UV light (254 nm) or according to Stahl (from Sigma-Aldrich) using 5% H 2 O in methanol (MeOH). 2 SO 4 Compounds were visualized by spraying with or ninhydrin reagent followed by heating.Flash chromatography was performed with a Biotage Isolera One flash chromatography instrument equipped with a bivariate UV wavelength detector (200-400 nm) using Biotage Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1042] All moisture-sensitive reactions were performed under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercial reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK scientific.

[1043] HPLC / ESI-MS was performed on a Dionex UltiMate 3000RS UHPLC system and a Thermo Scientific MSQPlus mass spectrometer using a Waters Acquity UPLC Protein BEH C4 column (300A, 1.7 pm, 2.1 x 100 mm) at 60°C. The solvent system consisted of solvent A with water containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient of B was increased from 5% to 100% in 15 minutes with a flow rate of 0.4 mL / min. Detector and conditions: Corona supercharged aerosol detector (from esa). Nebulizer temperature: 25°C. N 2 Pressure: 35.1psi. Filter: Corona.

[1044] 1 H and 13 C NMR spectra were acquired at room temperature on a VaR1an spectrometer at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR) records. Chemical shifts are in ppm, referenced to the residual solvent peak (CDCl 3 – 1 H NMR: δ at 7.26 ppm and 13 CNMR: δ at 77.2 ppm; DMSO-d 6 –1H NMR: δ at 2.50 ppm and13 C NMR: δ at 39.5 ppm). Coupling constants are in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).

[1045] Synthesis route of conjugated structural unit three GalNAc_tether 1:

[1046]

[1047] Preparation of compound 2: Under argon, D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 hours. The reaction mixture was diluted with DCM (50 mL) and quenched with cold saturated NaHCO 3 The organic layer was separated and washed with aqueous solution (100 mL) and water (100 mL). 2 SO 4 Drying and concentration gave the title compound as a yellow oil which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM, 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1048]

[1049] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) were dissolved in anhydrous DCM (40 mL) under argon and molecular sieves were added. (5 g) was added to the solution. The mixture was stirred at room temperature for 1 hour. TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieve was filtered, the filtrate was diluted with DCM (100 mL) and the mixture was washed with cold saturated NaHCO 3 The organic layer was separated and washed with aqueous solution (100 mL) and water (100 mL). 2 SO 4 Dry and remove the solvent under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM, 10CV) to give the title compound as a light yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: C 20 H 32 N4 O 11 The calculated value is 504.21. The found value is 505.4. 1 H NMR (500 MHz, CDCl 3 )δ6.21-6.14(m,1H),5.30(dd,J=3.4,1.1Hz,1H),5.04(dd,J=11.2,3.4Hz,1H),4.76(d,J=8.6Hz,1H),4.23-4.08 (m,3H),3.91-3.80(m,3H),3.74-3.59(m,9H),3.49-3.41(m,2H),2.14(s,3H),2.02(s,3H),1.97(d,J=4.2Hz,6H). 13 C NMR (125 MHz, CDCl 3 )δ170.6(C),170.5(C),170.4(C),170.3(C),102.1(CH),71.6(CH),70.8(CH),70.6(CH),70.5(CH),70.3(CH 2 ),69.7(CH 2 ),68.5(CH 2 ),66.6(CH 2 ),61.5(CH 2 ),23.1(CH 3 ),20.7(3xCH 3 ).

[1050]

[1051] Preparation of compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1:1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed with vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through diatomaceous earth and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to obtain the title compound as a colorless oil (0.95 g, quantitative yield, rf=0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: C 20 H 34 N 2 O 11 The calculated value is 478.2. The measured value is 479.4.

[1052]

[1053] Preparation of compound 7: Tris{[2-(tert-butyloxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a DCM / water mixture (40 mL 1:1 v / v) and Na 2 CO 3 (0.18 g, 1.7 mmol, 0.25 eq.). Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture and stirred at room temperature for 24 hours. The reaction mixture was washed with CH 2 Cl 2 (100 mL) and washed with water (100 mL). The organic layer was separated and washed with Na 2 SO 4 The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane, 12 CV) to afford the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: C 33 H 53 NO 11 The calculated value is 639.3. The measured value is 640.9. 1 H NMR (500 MHz, DMSO-d 6 )δ7.38-7.26(m,5H),4.97(s,2H),3.54(t,6H),3.50(s,6H),2.38(t,6H),1.39(s,27H). 13 C NMR (125 MHz, DMSO-d 6 )δ170.3(3xC),154.5(C),137.1(C),128.2(2xCH),127.7(CH),127.6(2xCH),79.7(3xC),68.4(3xCH 2 ),66.8(3xCH 2 ),64.9(C),58.7(CH 2 ),35.8(3xCH 2 ),27.7(9xCH 3 ).

[1054]

[1055] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH 2 Cl 2(1 mL), trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) 3 times and dried under high vacuum to give the compound as a TFA salt (0.183 g, 98%). The compound was used without further purification. MS: C 21 H 29 NO 11 The calculated value is 471.6. The measured value is 472.4.

[1056]

[1057] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH 2 5 (3.56 g, 7.44 mmol, 5.0 eq.) was dissolved in N, N-dimethylformamide (DMF) (25 mL). Then N, N, N', N'-tetramethyl-O-(1H-benzotriazole-1-yl) uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N, N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution, and the reaction was stirred for 72 hours. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and heated with saturated NaHCO 3 The organic layer was washed with Na 2 SO 4 Dry, evaporate the solvent and purify the crude material by flash chromatography (gradient elution: 0-5% MeOH in DCM, 14CV). The product is obtained as a pale yellow oil (1.2 g, 43%, rf=0.20 (5% MeOH in DCM)). MS: C 81 H 125 N 7 O 41 The calculated value is 1852.9. The measured value is 1854.7. 1 H NMR (500 MHz, DMSO-d 6)δ7.90-7.80(m,10H),7.65-7.62(m,4H),7.47-7.43(m,3H),7.38-7.32(m, 8H),5.24-5.22(m,3H),5.02-4.97(m,4H),4.60-4.57(m,3H),4.07-3.90(m 10H),3.67-3.36(m,70H),3.23-3.07(m,25H),2.18(s,10H),2.00(s,13H),1.89(s,11H),1.80-1.78(m,17H). 13 C NMR (125 MHz, DMSO-d 6 )δ170.1(C),169.8(C),169.7(C),169.4(C),169.2(C),169.1(C),142.7(C),126.3(CH),123. 9(CH),118.7(CH),109.7(CH),100.8(CH),70.5(CH),69.8(CH),69.6(CH),69.5(CH),69.3(CH 2 ),69.0(CH 2 ),68.2(CH 2 ),67.2(CH 2 ),66.7(CH 2 ),61.4(CH 2 ),22.6(CH 2 ),22.4(3xCH 3 ),20.7(9xCH 3 ).

[1058]

[1059] Preparation of compound 10: The triantennary GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) was dissolved in MeOH (15 mL) and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed with vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. The completion of the reaction was tracked by mass spectrometry and the resulting mixture was filtered through a thin diatomaceous earth pad. The solvent was evaporated and the obtained residue was dried under high vacuum and used in the next step without further purification. A pale yellow oily product (0.24 g, quantitative yield) was obtained. MS: C 73 H 119 N 7 O 39 The calculated value is 1718.8. The measured value is 1719.3.

[1060]

[1061] Preparation of compound 11: Commercially available bis(N-hydroxysuccinimide suberate) (3.67 g, 9.9 mmol, 1.0 eq.) was dissolved in DMF (5 mL) and triethylamine (1.2 mL) was added. A solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 eq.) in DMF (5 mL) was added dropwise to the solution. The reaction was stirred at room temperature for 3 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated and purified by Na 2 SO 4 Dry and remove the solvent under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 16CV). The product was obtained as a white solid (1.54 g, 43%, rf=0.71 (5% MeOH in DCM)). MS: C 15 H 23 N 5 O 5 The calculated value is 353.4. The measured value is 354.3.

[1062]

[1063] Preparation of triGalNAc (12): triantennary GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) were dissolved in DCM (5 mL) under argon, and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and Na 2 SO 4 The solvent was evaporated and the resulting crude material was purified by flash chromatography (elution gradient: 0-10% MeOH in DCM, 20CV) to afford the title compound as a white fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: C 84 H 137 N 11 O 41 The calculated value is 1957.1. The measured value is 1959.6.

[1064] Conjugation of tether 1 to siRNA strand: Monofluorocyclooctyne (MFCO) conjugation at the 5' or 3' end

[1065] 5' end MFCO conjugation

[1066]

[1067] 3' end MFCO conjugation

[1068]

[1069] General conditions for MFCO conjugation: Amine-modified single strands were dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer pH 9.6 / dimethyl sulfoxide (DMSO) 4:6 (v / v), and one molar equivalent of a 35 mM solution of MFCO-C6-NHS ester in DMF (Berry & Associates, Cat. # LK 4300) was added to the solution. The reaction was carried out at room temperature and another molar equivalent of MFCO solution was added after 1 hour. The reaction was continued for one hour and monitored by LC / MS. At least a two-molar excess of MFCO NHS ester reagent relative to the amino-modified oligonucleotide was required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius, and then incubated at 4 °C for 1 hr. Purification was performed by retaining phase (RP HPLC) on a Pure instrument (GE Healthcare).

[1070] Purification was performed using a Waters Xbridge C18 Prep 19x50mm column. Buffer A was 100mM TEAAc pH 7 and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10mL / min and a temperature of 60°C were used. UV traces were recorded at 280nm. A gradient of 0-100% B in 60 column volumes was used.

[1071] Fractions containing the full-length conjugated oligonucleotide were pooled, precipitated in a freezer with 3M NaOAc, pH 5.2, and 85% ethanol, and the collected precipitate was dissolved in water. The sample was desalted by size exclusion chromatography and concentrated using a vacuum concentrator to give an isolated yield of 40–80% of the conjugated oligonucleotide.

[1072] 5'-GalNAc-T1 conjugate

[1073]

[1074] 3'-GalNAc-T1 conjugate

[1075]

[1076] General procedure for tri-GalNAc conjugation: MFCO-modified single strand was dissolved in water at 2000 OD / mL, and one equivalent of compound 12 (10 mM) in DMF was added to the solution. The reaction was carried out at room temperature, and 0.7 molar equivalent of compound 12 solution was added after 3 hours. The reaction was allowed to proceed overnight, and the completion of the reaction was monitored by LCMS. The conjugate was diluted 15-fold in water, filtered through a 1.2 μm filter from Sartorius, and then Purification was performed by RPHPLC on a Pure instrument (GE Healthcare).

[1077] Purification was performed using a Waters Xbridge C18 Prep 19x50mm column. Buffer A was 100 mM triethylammonium acetate pH 7 and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60°C were used. UV traces were recorded at 280 nm. A gradient of 0-100% B in 60 column volumes was used.

[1078] The fractions containing the full-length conjugated oligonucleotides were pooled, precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a refrigerator, and the collected precipitate was dissolved in water to obtain an oligonucleotide solution of about 1000 OD / mL. O-acetate was removed by adding 20% ​​ammonia. Quantitative removal of these protecting groups was verified by LC-MS.

[1079] Sephadex G25 Fine resin (GE Healthcare) was used in The conjugate was desalted by size exclusion chromatography on a Pure (GE Healthcare) instrument to give isolated yields of 50-70% of the conjugated oligonucleotide.

[1080] The following scheme further outlines the synthetic route:

[1081] Solution 1:

[1082]

[1083] Option 2:

[1084] Option 3:

[1085]

[1086] Solution 4:

[1087] Solution 5:

[1088]

[1089] Example 2: Double Annealing

[1090] To generate the desired siRNA duplex, the two complementary strands were annealed by combining equimolar aqueous solutions of the two strands. The mixture was placed in a 70°C water bath for 5 minutes and then cooled to ambient temperature within 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[1091] Superdex on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system TM The duplexes were analyzed by analytical SEC HPLC on a 75Increase 5 / 150GL column 5x153-158mm (Cytiva). The mobile phase consisted of 1x PBS containing 10% acetonitrile. An isocratic gradient was run over 10 minutes at room temperature at a flow rate of 1.5mL / min. UV traces were recorded at 260 and 280nm. Water (LC-MS grade) was purchased from Sigma-Aldrich and phosphate buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[1092] Example 3: Synthesis of Tether 2

[1093] General experimental conditions:

[1094] Thin layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. The fluorescence was determined under UV light (254 nm) or according to Stahl (from Sigma-Aldrich) using 5% H 2 O in methanol (MeOH). 2 SO 4 Compounds were visualized by spraying with ninhydrin reagent followed by heating. Flash chromatography was performed with a Biotage Isolera One flash chromatography instrument equipped with a bivariate UV wavelength detector (200-400 nm) using Biotage Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1095] All moisture-sensitive reactions were performed under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercial reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK scientific.

[1096] HPLC / ESI-MS was performed on a Dionex UltiMate 3000RS UHPLC system and a Thermo Scientific MSQPlus mass spectrometer using a Waters Acquity UPLC Protein BEH C4 column (300A, 1.7 pm, 2.1 x 100 mm) at 60°C. The solvent system consisted of solvent A with water containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient of B was increased from 5% to 100% in 15 minutes with a flow rate of 0.4 mL / min. Detector and conditions: Corona supercharged aerosol detector (from esa). Nebulizer temperature: 25°C. N 2 Pressure: 35.1psi. Filter: Corona.

[1097] 1 H and 13 C NMR spectra were acquired at room temperature on a VaR1an spectrometer at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR) records. Chemical shifts are in ppm, referenced to the residual solvent peak (CDCl 3 – 1 H NMR: δ at 7.26 ppm and 13 CNMR: δ at 77.2 ppm; DMSO-d 6 –1H NMR: δ at 2.50 ppm and 13 C NMR: δ at 39.5 ppm). Coupling constants are in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).

[1098] Synthesis route of conjugated structural unit three GalNAc_tether 2:

[1099]

[1100] Preparation of compound 2: Under argon, D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 hours. The reaction mixture was diluted with DCM (50 mL) and quenched with cold saturated NaHCO 3 The organic layer was separated and washed with aqueous solution (100 mL) and water (100 mL). 2 SO 4Drying and concentration gave the title compound as a yellow oil which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM, 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1101]

[1102] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) were dissolved in anhydrous DCM (40 mL) under argon and molecular sieves were added. (5 g) was added to the solution. The mixture was stirred at room temperature for 1 hour. TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieve was filtered, the filtrate was diluted with DCM (100 mL) and the mixture was washed with cold saturated NaHCO 3 The organic layer was separated and washed with aqueous solution (100 mL) and water (100 mL). 2 SO 4 Dry and remove the solvent under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM, 10CV) to give the title compound as a light yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: C 20 H 32 N 4 O 11 The calculated value is 504.21. The measured value is 505.4. 1 H NMR (500 MHz, CDCl 3 )δ6.21-6.14(m,1H),5.30(dd,J=3.4,1.1Hz,1H),5.04(dd,J=11.2,3.4Hz,1H),4.76(d,J=8.6Hz,1H),4.23-4.08 (m,3H),3.91-3.80(m,3H),3.74-3.59(m,9H),3.49-3.41(m,2H),2.14(s,3H),2.02(s,3H),1.97(d,J=4.2Hz,6H). 13 C NMR (125 MHz, CDCl 3 )δ170.6(C),170.5(C),170.4(C),170.3(C),102.1(CH),71.6(CH),70.8(CH),70.6(CH),70.5(CH),70.3(CH2 ),69.7(CH 2 ),68.5(CH 2 ),66.6(CH 2 ),61.5(CH 2 ),23.1(CH 3 ),20.7(3xCH 3 ).

[1103]

[1104] Preparation of compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1:1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed with vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through diatomaceous earth and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to obtain the title compound as a colorless oil (0.95 g, quantitative yield, rf=0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: C 20 H 34 N 2 O 11 The calculated value is 478.2. The measured value is 479.4.

[1105]

[1106] Preparation of compound 7: Tris{[2-(tert-butyloxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a DCM / water mixture (40 mL 1:1 v / v) and Na 2 CO 3 (0.18 g, 1.7 mmol, 0.25 eq.). Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture and stirred at room temperature for 24 hours. The reaction mixture was washed with CH 2 Cl 2 (100 mL) and washed with water (100 mL). The organic layer was separated and washed with Na 2 SO 4 The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane, 12 CV) to afford the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: C 33 H53 NO 11 The calculated value is 639.3. The measured value is 640.9. 1 H NMR (500 MHz, DMSO-d 6 )δ7.38-7.26(m,5H),4.97(s,2H),3.54(t,6H),3.50(s,6H),2.38(t,6H),1.39(s,27H). 13 C NMR (125 MHz, DMSO-d 6 )δ170.3(3xC),154.5(C),137.1(C),128.2(2xCH),127.7(CH),127.6(2xCH),79.7(3xC),68.4(3xCH 2 ),66.8(3xCH 2 ),64.9(C),58.7(CH 2 ),35.8(3xCH 2 ),27.7(9xCH 3 ).

[1107]

[1108] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH 2 Cl 2 (1 mL), trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) 3 times and dried under high vacuum to give the compound as a TFA salt (0.183 g, 98%). The compound was used without further purification. MS: C 21 H 29 NO 11 The calculated value is 471.6. The measured value is 472.4.

[1109]

[1110] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH 25 (3.56 g, 7.44 mmol, 5.0 eq.) was dissolved in N,N-dimethylformamide (DMF) (25 mL). Then N,N,N′,N′-tetramethyl-O-(1H-benzotriazole-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution, and the reaction was stirred for 72 hours. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and heated with saturated NaHCO 3 The organic layer was washed with Na 2 SO 4 Dry, evaporate the solvent and purify the crude material by flash chromatography (gradient elution: 0-5% MeOH in DCM, 14CV). The product is obtained as a pale yellow oil (1.2 g, 43%, rf=0.20 (5% MeOH in DCM)). MS: C 81 H 125 N 7 O 41 The calculated value is 1852.9. The measured value is 1854.7. 1 H NMR (500 MHz, DMSO-d 6 )δ7.90-7.80(m,10H),7.65-7.62(m,4H),7.47-7.43(m,3H),7.38-7.32(m, 8H),5.24-5.22(m,3H),5.02-4.97(m,4H),4.60-4.57(m,3H),4.07-3.90(m 10H),3.67-3.36(m,70H),3.23-3.07(m,25H),2.18(s,10H),2.00(s,13H),1.89(s,11H),1.80-1.78(m,17H). 13 C NMR (125 MHz, DMSO-d 6 )δ170.1(C),169.8(C),169.7(C),169.4(C),169.2(C),169.1(C),142.7(C),126.3(CH),123. 9(CH),118.7(CH),109.7(CH),100.8(CH),70.5(CH),69.8(CH),69.6(CH),69.5(CH),69.3(CH 2 ),69.0(CH 2 ),68.2(CH2 ),67.2(CH 2 ),66.7(CH 2 ),61.4(CH 2 ),22.6(CH 2 ),22.4(3xCH 3 ),20.7(9xCH 3 ).

[1111]

[1112] Preparation of compound 10: The triantennary GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) was dissolved in MeOH (15 mL) and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed with vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. The completion of the reaction was tracked by mass spectrometry and the resulting mixture was filtered through a thin diatomaceous earth pad. The solvent was evaporated and the obtained residue was dried under high vacuum and used in the next step without further purification. A pale yellow oily product (0.24 g, quantitative yield) was obtained. MS: C 73 H 119 N 7 O 39 The calculated value is 1718.8. The measured value is 1719.3.

[1113]

[1114] Preparation of compound 14: triantennary GalNAc compound 10 (0.45 g, 0.26 mmol, 1.0 eq.), HBTU (0.19 g, 0.53 mmol, 2.0 eq.) and DIPEA (0.23 mL, 1.3 mmol, 5.0 eq.) were dissolved in DCM (10 mL) under argon. A solution of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) in DCM (5 mL) was added dropwise to the mixture. The reaction was stirred at room temperature overnight. The solvent was removed and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL), and washed with Na 2 SO 4 Dry. Evaporate the solvent and purify the crude material by flash chromatography (gradient elution: 0-5% MeOH in DCM, 20CV). Obtain the product as a white fluffy solid (0.25 g, 48%, rf=0.4 (10% MeOH in DCM)). MS: Calculated for C88H137N7O42 1965.1. Found 1965.6.

[1115]

[1116] Preparation of triGalNAc (15): The triantennary GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL) and Pd / C (40 mg) was added. The reaction mixture was degassed by vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. The completion of the reaction was monitored by mass spectrometry and the resulting mixture was filtered through a thin celite pad. The solvent was removed under reduced pressure and the resulting residue was dried under high vacuum overnight. The residue was used for conjugation with oligonucleosides without further purification (0.28 g, quantitative yield). MS: C 81 H 131 N 7 O 42 The calculated value is 1874.9. The measured value is 1875.3.

[1117] Conjugation of tether 2 to siRNA strand: Conjugation of tri-GalNAc tether 2 (GalNAc-T2) at the 5' or 3' end

[1118] 5'-GalNAc-T2 conjugate

[1119]

[1120] 3'-GalNAc-T2 conjugate

[1121]

[1122] Preparation of tri-GalNAc tether 2 NHS ester: To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 μmol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N′-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol) were added. The solution was stirred at room temperature for 18 hours and used for subsequent conjugation reactions without purification.

[1123] General procedure for conjugation of tri-GalNAc tether 2: Amine-modified single strands were dissolved in 50 mM carbonate / bicarbonate buffer pH 9.6 / DMSO 4:6 (v / v) at 700 OD / mL and one molar equivalent of tether 2 NHS ester (57 mM) in DMF was added to the solution. The reaction was carried out at room temperature and after 1 hour another molar equivalent of NHS ester solution was added. The reaction was continued for one hour and the progress of the reaction was monitored by LCMS. At least two molar equivalents excess of NHS ester reagent relative to the amino-modified oligonucleoside was required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius and then incubated at 4 °C for 1 hr. Purification was performed by retaining phase (RPHPLC) on a Pure instrument (GE Healthcare).

[1124] Purification was performed using a Waters Xbridge C18 Prep 19 x 50 mm column. Buffer A was 100 mM TEAA pH 7 and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60°C were used. UV traces were recorded at 280 nm. A gradient of 0-100% B in 60 column volumes was used.

[1125] Fractions containing full-length conjugated oligonucleotides were pooled, precipitated in a refrigerator with 3M NaOAc, pH 5.2, and 85% ethanol, and then dissolved in water at 1000 OD / mL. O-acetate removal was performed using 20% ​​aqueous ammonium hydroxide until complete (monitored by LC-MS).

[1126] Sephadex G25 Fine resin (GE Healthcare) was used in The conjugate was desalted by size exclusion chromatography on a Pure (GE Healthcare) instrument to give isolated yields of 60-80% of the conjugated oligonucleotide.

[1127] HPLC-MS analysis of the conjugate was performed using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics) using a 2.1x50mm Xbridge C18 column (Waters). Buffer A was 16.3mM triethylamine, 100mM HFIP in 1% MeOH aqueous solution, and buffer B contained 95% MeOH in buffer A. A flow rate of 250 μL / min and a temperature of 60°C were used. UV traces were recorded at 260 and 280nm. A gradient of 1-100% B in 31 minutes was used.

[1128] The following scheme further outlines the synthetic route:

[1129] Solution 6:

[1130]

[1131] Solution 7:

[1132] Solution 8:

[1133] Solution 9:

[1134]

[1135] Example 4: Double Annealing

[1136] To generate the desired siRNA duplex, the two complementary strands were annealed by combining equimolar aqueous solutions of the two strands. The mixture was placed in a 70°C water bath for 5 minutes and then cooled to ambient temperature within 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[1137] Superdex on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system TM The duplexes were analyzed by analytical SEC HPLC on a 75Increase 5 / 150GL column 5x153-158mm (Cytiva). The mobile phase consisted of 1x PBS containing 10% acetonitrile. An isocratic gradient was run over 10 minutes at room temperature at a flow rate of 1.5mL / min. UV traces were recorded at 260 and 280nm. Water (LC-MS grade) was purchased from Sigma-Aldrich and phosphate buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[1138] Example 5: Alternative synthetic route for conjugated building block three GalNAc_tether 2:

[1139]

[1140]

[1141] Conjugation of tether 2 to siRNA strand: Conjugation of tri-GalNAc tether 2 (GalNAc-T2) at the 5' or 3' end

[1142] Conjugation conditions

[1143]

[1144] Pre-activation: To a solution of compound 15 (16 μmol, 4 eq.) in DMF (160 μL) at 25°C, TFA-O-PFP (15 μL, 21 eq.) was added, followed by DIPEA (23 μL, 32 eq.). The tube was shaken at 25°C for 2 h. 2 O (10 μL) to terminate the reaction.

[1145] Conjugation: The resulting mixture was diluted with DMF (400 μl) and then oligoamine solution (4.0 μmol in 10x PBS, pH 7.4, 500 μL; final oligonucleotide concentration in organic and aqueous solution: 4 μmol / ml = 4 mM) was added. The tube was shaken at 25°C for 16 hours and the reaction was analyzed by LCMS. The resulting mixture was washed with 28% NH 4 OH (4.5 ml) and shaken at 25° C. for 2 hours. The mixture was analyzed by LCMS, concentrated, and purified by IP-RP HPLC to yield an oligonucleotide conjugated to tether 2GalNAc.

[1146] 5'-GalNAc-T2 conjugate

[1147]

[1148] 3'-GalNAc-T2 conjugate

[1149]

[1150] Example 6: Solid Phase Synthesis: Scale ≤ 1 μmol

[1151] The synthesis of siRNA sense and antisense strands was performed on a MerMadel 92X synthesizer using a commercially available solid support made of controlled pore glass with a universal linker (universal CPG, loading of 40 μmol / g; LGC Biosearch or Glen Research).

[1152] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1153] The 2'-O-methylphosphoramidites used were as follows: 5'-(4,4'-dimethoxytrityl)-N-benzoyladenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetylcytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyrylguanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1154] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyldeoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyldeoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryldeoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5'-dimethoxytrityldeoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1155] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite which was dissolved in DMF / MeCN (1:4, v / v). 2 0.02 M iodine in 1:1 v / v acetonitrile / pyridine was used as the oxidizing agent. 0.2 M PADS (TCI) in 1:1 v / v acetonitrile / pyridine was used for thiolation of phosphorothioate bonds. 0.25 M mM 5-ethylthiotetrazolyl (ETT) in acetonitrile was used as the activator solution.

[1156] The reverse abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[1157] In each cycle, DMT was removed using deblocking solution (3% TCA in DCM, DNAchem).

[1158] The conjugation time was 180 seconds. The oxidant contact time was set to 80 seconds and the thiolation time was 2*100 seconds.

[1159] At the end of the synthesis, NH 4 OH:EtOH solution 4:1 (v / v) was reacted at 45°C (TCI) for 20 hours to cleave the oligonucleotide from the solid support. The solid support was then removed by filtration and the residue was washed with H 2 O. Rinse the filter thoroughly and reduce the volume of the combined solution by evaporation under reduced pressure.

[1160] Oligonucleotides were treated to form sodium salts by ultracentrifugation using an Amicon Ultra-2 centrifugal filter unit, PBS buffer (10x, Teknova, pH 7.4, sterile), or by EtOH precipitation from 1 M sodium acetate.

[1161] Single-strand identity was assessed by MS ESI-PCR followed by annealing in water to form the final duplex siRNA, and duplex purity was assessed by size exclusion chromatography.

[1162] Example 7: Solid phase synthesis: scale ≥ 5 μmol

[1163] The synthesis of siRNA sense and antisense strands was performed on a MerMadel 92X synthesizer using a commercially available solid support made of controlled pore glass with a universal linker (universal CPG, loading of 40 μmol / g; LGC Biosearch or Glen Research) at a scale of 5 μmol. 12 μmol of the sense strand for 3' conjugation was synthesized on the solid support, with a loading of 86 μmol / g (LGC).

[1164] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1165] The 2'-O-methylphosphoramidites used were as follows: 5'-(4,4'-dimethoxytrityl)-N-benzoyladenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetylcytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyrylguanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1166] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyldeoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyldeoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryldeoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5'-dimethoxytrityldeoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1167] The reverse abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[1168] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite which was dissolved in DMF / MeCN (1:4, v / v). 2 0.02 M iodine in 1:1 v / v acetonitrile / pyridine was used as the oxidizing agent. 0.2 M PADS (TCI) in 1:1 v / v acetonitrile / pyridine was used for thiolation of phosphorothioate bonds. 0.25 M mM 5-ethylthiotetrazolyl (ETT) in acetonitrile was used as the activator solution.

[1169] In each cycle, DMT was removed using deblocking solution (3% TCA in DCM, DNAchem).

[1170] For the chain synthesized on universal CPG, conjugation was performed with 8 eq. of amide for 130 sec. The oxidation time was 47 sec and the thiolation time was 210 sec.

[1171] For the strand synthesized on the 3'-PT-amino modifier C6 CPG, conjugation was performed with 8 eq. of amide for 2*150 sec. The oxidation time was 47 sec and the thiolation time was 250 sec.

[1172] At the end of the synthesis, NH 4 OH:EtOH solution 4:1 (v / v) was reacted at 45°C (TCI) for 20 hours to cleave the oligonucleotide from the solid support. The solid support was then removed by filtration and the residue was washed with H 2 O. Rinse the filter thoroughly and reduce the volume of the combined solution by evaporation under reduced pressure.

[1173] Oligonucleotides were precipitated with EtOH from 1 M sodium acetate to form the sodium salt.

[1174] Single-stranded oligonucleotides were purified by IP-RP HPLC on an Xbridge BEH C18 5 μm, Purification on a 19x150 mm (Waters) column with increasing gradient of B in A. Mobile phase A: 240 mM HFIP, 7 mM TEA and 5% methanol in water; Mobile phase B: 240 mM HFIP, 7 mM TEA in methanol.

[1175] Single-chain purity and identity were assessed by UPLC / MS ESI-on a Xbridge BEH C18 2.5 μm, 3x50 mm (Waters) column with a gradient increase of B in A. Mobile phase A: 100 mM HFIP, 5 mM TEA in water; Mobile phase B: 20% Mobile phase A: 80% acetonitrile (v / v).

[1176] The sense strand was conjugated according to the protocol provided in Examples 1, 3 or 5.

[1177] The positive and antisense strands were then annealed in water to form the final duplex siRNA, and the duplex purity was assessed by size exclusion chromatography.

[1178] Example 8: Nucleic acid sequences

[1179] The siRNA oligonucleotides of the present application target ZPI. The complete DNA sequence of the ZPI target is as follows (SEQ ID NO: 1):

[1180]

[1181] Table 1 below provides oligonucleotide mRNA target sequences for ZPI, and the corresponding positions in transcript NM_016186.3. It should be understood that SEQ ID NOs: 2 to 121 refer to human (Homo sapiens) mRNA sequences.

[1182] Table 1

[1183]

[1184]

[1185]

[1186]

[1187] Table 2 provides the unmodified first strand (antisense strand) and corresponding unmodified second strand (sense strand) sequences of the siRNA oligonucleotides of the present application, as well as the corresponding positions in the overall gene sequence of SEQ ID NO: 1, as shown below.

[1188] Table 2

[1189]

[1190]

[1191]

[1192]

[1193]

[1194]

[1195]

[1196]

[1197] Table 3 provides the modified first (antisense) sequences of the siRNA oligonucleotides of the present application, and the corresponding unmodified first (antisense) sequences, as shown below.

[1198] Table 3

[1199]

[1200]

[1201]

[1202]

[1203]

[1204]

[1205]

[1206]

[1207]

[1208]

[1209]

[1210]

[1211]

[1212] Table 4 provides the modified second (sense) sequences of the siRNA oligonucleotides of the present application, and the corresponding unmodified second (sense) sequences, as shown below.

[1213] Table 4

[1214]

[1215]

[1216]

[1217]

[1218]

[1219]

[1220]

[1221]

[1222]

[1223]

[1224]

[1225]

[1226]

[1227] As shown in Table 4 above, some modified second strand sequences include a preferred 5'iaia motif. However, it should also be understood that the scope of these modified second strand sequences also includes Me / F modified second strands without a 5'iaia motif.

[1228] Table 5 identifies the duplexes, whose duplex IDs refer to the modified antisense and sense IDs in Tables 3 and 4 above.

[1229] Table 5

[1230]

[1231]

[1232]

[1233]

[1234]

[1235]

[1236]

[1237] For the duplexes in Table 5:

[1238] ETXM316-ETXM415, ETXM436-ETXM515 and ETXM1180-ETXM1216 have Figure 8a The duplex structure shown has a 2-nucleoside overhang at the 3′ end of the antisense strand;

[1239] ETXM416-ETXM435 has Figure 8b The duplex structure shown is a 19mer blunt-ended construct.

[1240] Definitions listed in the table above:

[1241] A—Adenosine

[1242] C—Cytidine

[1243] G—Guanosine

[1244] T—thymidine

[1245] m—2'-O-methyl

[1246] f—2' fluorine

[1247] S—phosphorothioate bond

[1248] o—Heat-destabilized nucleosides

[1249] ia—inverted abasic nucleoside

[1250] Example 9: Inhibition screening of ZPI expression in human Huh7 cells

[1251] Huh7 cells (a human hepatocyte-derived cell line obtained from JCRB Cell Bank) were cultured at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS under atmosphere. Cells were transfected with siRNA duplexes targeting ZPI mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) at final duplex concentrations of 5nM and 0.1nM. Transfection was performed by adding 9.7μL Opti-MEM (ThermoFisher) and 0.3μL Lipofectamine RNAiMAX (ThermoFisher) to 10μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes, and then 100μL of complete growth medium containing 20,000 Huh7 cells was added. The cells were incubated at 37°C / 5% CO 2 After incubation for 24 hours at 4 °C, total RNA was purified using the RNeasy 96 kit (Qiagen). Each duplex was tested by transfection in duplicate wells in two independent experiments.

[1252] cDNA synthesis was performed using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 using specific primers for human ZPI (Hs01547819_ml) and human GAPDH (Hs02786624_gl) using the FastStart Universal Probe Master kit (Roche).

[1253] qPCR was performed in duplicate on cDNA from each well, and the average Ct was calculated. Relative ZPI expression was calculated from the average Ct values ​​using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells. Based on the results of the initial screening, siRNA duplexes that showed good activity were selected for dose response tracking. The results are shown in Fig. 9 The sequences of RNAi molecules are shown in Table 5.

[1254] Example 10: Dose response for inhibition of ZPI expression in human Huh7 cells

[1255] Huh7 cells (a human hepatocyte-derived cell line obtained from JCRB Cell Bank) were cultured at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS in an atmosphere of 4% CO. Cells were transfected with siRNA duplexes targeting ZPI mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) using 10x3-fold serial dilutions with a final duplex concentration of 20nM to 1pM. Transfection was performed by adding 9.7μL Opti-MEM (ThermoFisher) and 0.3μL Lipofectamine RNAiMAX (ThermoFisher) to 10μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes and then 100μL of complete growth medium containing 20,000 Huh7 cells was added. The cells were incubated at 37°C / 5% CO 2 After incubation for 24 hours at 4 °C, total RNA was purified using the RNeasy 96 kit (Qiagen). In one experiment, each duplex was tested by transfection in duplicate wells.

[1256] cDNA synthesis was performed using the FastQuant RT kit (with gDNase) (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 using specific primers for human ZPI (Hs01547819_ml) and human GAPDH (Hs02786624_gl) using the FastStart Universal Probe Master Kit (Roche).

[1257] qPCR was performed in duplicate on cDNA from each well, and the average Ct was calculated. Relative ZPI expression was calculated from the average Ct values ​​using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells. The maximum percent inhibition and IC50 values ​​of ZPI expression were calculated using a four-parameter (variable slope) model using GraphPad Prism 9. The results are shown in Fig. 9 The sequences of the RNAi molecules are described in the relevant tables of this article.

[1258] Table 6 Relative mRNA expression

[1259]

[1260]

[1261]

[1262]

[1263]

[1264]

[1265]

[1266] Table 7 Dose response data table

[1267]

[1268]

[1269] The application is not limited to the scope of the specific disclosed embodiments, and these embodiments are provided for example to illustrate the various aspects of the application. According to the description and teaching of this article, various modifications to the composition and method will become apparent. Such changes can be practiced without departing from the true scope and spirit of the present disclosure, and such changes are intended to fall within the scope of the present disclosure.

[1270] When there is ambiguity between the sequence in this specification and the sequence in the attached sequence listing, the sequence provided herein is considered to be the correct sequence.

[1271] Example 11: Murineization of ZPI siRNA Sequences

[1272] Table 8 shows five siRNA sequences identified as inhibitors of ZPI expression in the Huh7 cell line transfection screen.

[1273] Table 8

[1274]

[1275]

[1276] ETXM338, ETXM359 and ETXM396 do not cross-react with the mouse ZPI sequence and will not be used in mouse PoC studies. These sequences were murinized so that they are homologous to the mouse ZPI sequence for use in mouse studies. The siRNA sequences were aligned to the mouse ZPI transcripts NM_144834.4 and NM_001301404.1 and nucleotides that did not match the mouse sequence were changed to match the mouse sequence.

[1277] ETXM338 had its sense strand position 18 changed to C, and its antisense strand position 4 changed to G, resulting in ETXM1064.

[1278] ETXM359 had its sense strand position 19 changed to G, its antisense strand position 3 changed to C, and its antisense strand position 23 changed to C, resulting in ETXM1072.

[1279] The sense strand position 2 of ETXM396 was changed to U, the sense strand position 5 was changed to C, the antisense strand position 17 was changed to G, and the antisense strand position 20 was changed to A to obtain ETXM1076.

[1280] The murinized sequences were checked by BLAST searches to ensure that they did not cross-react with other mouse transcripts.

[1281] The murinized siRNA sequences are shown in Table 9. The positions that were changed to the mouse sequence are underlined.

[1282] Table 9

[1283]

[1284] Example 12: In vivo efficacy data in a hemophilia mouse model

[1285] Hemarthrosis, defined as bleeding into a joint cavity, is a common feature of hemophilia. The long-term consequence of recurrent hemarthrosis is the development of permanent joint disease, known as hemophilic arthropathy. Approximately 50% of people with hemophilia develop severe arthropathy, resulting in chronic joint pain, reduced range of motion and function, and decreased quality of life. Hemophilic arthropathy is characterized by synovial hyperplasia, chronic inflammation, fibrosis, and hemosiderosis.

[1286] The hemarthrosis model used was to induce knee bleeds in haem A mice and appropriate background wild type (WT) strains and monitor the progression of joint bleeds for 10 days after injury. Two replicate studies were performed to increase the number of animals used for analysis.

[1287] The purpose of these replicate studies was to demonstrate that prophylactic administration of ETXM1184 can reduce hemarthrosis following joint bleeding injury in mice with hemophilia A. Fitusiran (siRNA targeting antithrombin (AT)) was used as a reference. Advate (recombinant FVIII) was used as a positive control.

[1288] For this purpose, a total of 20 Haem A mice (Bi, L., Lawler, A., Antonarakis, S. et al. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 10, 119–121 (1995). https: / / doi.org / 10.1038 / ng0595-119) and 10 WT mice were used in this study:

[1289]

[1290] Eight days before induction of knee bleeds, mice were injected subcutaneously (sc) with the GalNAc-siRNA construct ETXM1184, fitusiran or vehicle (0.9% saline) at a dose volume of 5 ml / kg. Advate was injected intravenously 15 minutes before joint bleed induction.

[1291] To induce knee bleeding, mice were weighed and anesthetized using isoflurane inhalation anesthetic. The hair on both legs was shaved to expose the knee joints. 10 ml / kg buprenorphine was injected subcutaneously into the mice for analgesia, and the diameter of both knees was measured with an electronic caliper. Subsequently, both knees were wiped with 70% ethanol.

[1292] Insert a 30G sterile hypodermic needle into the infrapatellar ligament of one knee. Randomize the injected knees to the left and right sides and record the side of the injection. Remove the mouse from anesthesia and place it in a warm cage to recover before returning it to its cage.

[1293] Mice were monitored regularly during the first 6 h and analgesia was given subcutaneously with buprenorphine at a dose of 10 ml / kg 6 h after injury. Visual bleeding scores (VBS) of the injured knee were assessed 72 h and 10 days after injury.

[1294] All mice were carefully examined daily for clinical signs of excessive blood loss. Mice showing clinical signs of excessive blood loss, piloerection, avoidance of cage mates, or grimacing were killed for welfare reasons.

[1295] Ten days after injury, the mice were discontinued from the study.

[1296] Under isoflurane anesthesia, citrated blood samples were collected by cardiac puncture, plasma was prepared, and aliquots were frozen on dry ice and then stored at -80°C. To this end, blood was collected in a 1:9 ratio into 3.8% sodium citrate and then centrifuged at 7000 x g for 10 minutes at 4°C. Specifically, the following steps were performed:

[1297] 1. Collect blood by cardiac puncture.

[1298] 2. Rinse the syringe and needle with sodium citrate solution (3.8%), leaving the solution in the center of the syringe (about 30 μl).

[1299] 3. After blood collection, drain the sample into a 1.5 ml microcentrifuge tube, making sure to add enough sodium citrate solution (3.8%) to achieve a 1:9 ratio of sodium citrate to blood. Add the sodium citrate solution to the side of the tube, not directly to the sample. Invert 4 to 6 times to mix. If the sample is not centrifuged immediately, place it in a refrigerator (if available) or on packed ice, and continue to invert the collection tube regularly.

[1300] 4. As quickly as possible, centrifuge the sample at 7000 x g for 10 minutes at 4°C.

[1301] 5. Remove all plasma from the sample and place into a new microcentrifuge tube.

[1302] 6. Aliquot the plasma into pre-labeled tubes (Thermo Scientific; 10775974) as follows:

[1303] o 30 μl for potential TGA assay

[1304] o 100 μl for potential APTT assay

[1305] oThe rest were all used for potential target protein abundance analysis.

[1306] 7. Immediately place all aliquots on wet / dry ice.

[1307] 8. Transport samples on wet / dry ice.

[1308] 9. Transfer the samples to a -20℃ / -80℃ freezer and store until use.

[1309] The liver was removed and up to 3 sections of each lobe were placed in RNA and stored at 4°C for 24 to 72 hours. The tissue was then blotted dry, weighed, and stored at -80°C. Specifically, the following steps were performed:

[1310] 1. Kill mice by cervical dislocation immediately after cardiac puncture.

[1311] 2. Make an incision in the abdominal wall and remove the liver as quickly as possible.

[1312] 3. Place the liver in a Petri dish on wet ice to minimize degradation of the sample.

[1313] 4. Cut 3 x -50 mg liver slices from each of the following lobes: lateral lobe, medial lobe, lateral lobe and caudate lobe. Immediately place these liver slices into pre-labeled tubes (1.5 ml microcentrifuge tubes) containing 500 μl RNA and place the collection tubes on wet ice.

[1314] a. Transport on wet ice and transfer to 4°C for storage.

[1315] b. After 24-72 hours, drain and weigh the liver sample. Record the weight on a terminal table.

[1316] c. Transfer to -80℃ for long-term storage.

[1317] 5. Collect excess liver and place into separate pre-labeled collection tubes (2 mL microcentrifuge tubes).

[1318] a. Freeze on dry ice for potential future analysis.

[1319] b. Transport samples on dry ice.

[1320] c. Transfer to -80℃ for long-term storage.

[1321] 6. Clean all dissection tools between animals to prevent any cross contamination.

[1322] The skin of the legs was removed and the knee joints were measured. The legs were then placed in 10% formalin followed by decalcification and slide preparation. Specifically, the following steps were performed:

[1323] 1. After removal of the liver, measure and record the diameter of the injured and uninjured knees.

[1324] 2. Remove the skin of both knees. Perform visual bleeding scoring and measure the knee joints.

[1325] 3. Dissect the leg from the top of the femur to the ankle joint and remove some excess muscle, taking care not to cause any damage to the knee and associated structures. Place the knee in a pre-labeled tube (7 mL cuvette) containing 10% neutral buffered formalin for histological analysis.

[1326] On days 3 and 10 after induction of knee bleeding, visual bleeding scores were significantly reduced in Haem A mice receiving the GalNAc-siRNA construct ETXM1184 compared to Haem A mice receiving vehicle (0.9% saline) (see Fig. 12A and B). In addition, the knee diameter of mice receiving the GalNAc-siRNA construct ETXM1184 recovered faster after induction of knee bleeding compared with mice receiving vehicle treatment ( Fig.13AThis observation was confirmed by comparing the diameters of injured and uninjured skinned knees ( Fig. 13B ).

[1327] Analysis of Haem A mice 10 days after injury further showed that mice that received the GalNAc-siRNA construct ETXM1184 had less severe bone marrow hyperplasia compared to Haem A mice that received vehicle (0.9% saline). Fig.14A ), less severe osteoarthritis ( Fig. 14B ), chondrocyte degeneration / necrosis is less severe ( Fig. 14C ), less severe bleeding ( Fig.14D ), hemosiderin deposition is less severe ( Fig.14E ), hematoma is less serious ( Fig.14F ), osteoclastic bone resorption is less severe ( Figure 14G ), less severe osteolysis ( Fig.14H ), periostitis is less severe ( Fig.14I ), subchondral bone sclerosis is less severe ( Fig.14J ), less severe tendinosis ( Figure 14K ), tendinitis is less severe ( Figure 14L ), and less severe tenosynovitis ( Figure 14M ).

[1328] Figure 15-20 Comparative data between ETXM1184 and fitusiran are provided.

[1329] Example 13: Dose response for inhibition of ZPI expression in human Huh7 cells

[1330] Huh7 cells (a human hepatocyte-derived cell line obtained from JCRB Cell Bank) were cultured at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS in an atmosphere of 4% CO. The cells were transfected with 0.1 nM and 1 nM of siRNA duplexes designed against the target or negative control siRNA. Transfection was performed by adding 9.7 μL Opti-MEM (ThermoFisher) and 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes, and then 100 μL of complete growth medium containing 20,000 Huh7 cells was added. The cells were incubated at 37°C / 5% CO 2 After incubation for 24 hours at 4 °C, total RNA was purified using the RNeasy 96 kit (Qiagen). Each duplex was tested by transfection in duplicate wells, and the experiment was repeated three times.

[1331] cDNA synthesis was performed using the FastKing RT kit (with gDNase) (Tiangen). Real-time quantitative PCR (qPCR) was performed using TaqMan Gene Expression Assays (ThermoFisher Scientific) on an ABI Prism 7900HT or ABI QuantStudio 7 using specific primers for human ZPI (Hs01547819_ml) and human GAPDH (Hs02786624_gl).

[1332] Duplicate qPCR was performed on cDNA from each well and the average Ct was calculated. Relative target expression was calculated from the average Ct values ​​using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells.

[1333] To inhibit ZPI, siRNA duplexes ETXM1184, ETXM1199, ETXM1200, ETXM1201, ETXM1202, ETXM1203, ETXM1204, ETXM1205, ETXM1206, and ETXM1207 ( Fig.21 ).

Claims

1. A nucleic acid for inhibiting ZPI expression, comprising a duplex region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) is at least partially complementary to a portion of RNA transcribed from said ZPI gene, and (ii) comprising at least 17 consecutive nucleotides that differ from any first strand sequence listed in Table 2 by 0 or 1 nucleotide.

2. A nucleic acid for inhibiting ZPI expression, comprising a duplex region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) is at least partially complementary to a portion of RNA transcribed from said ZPI gene, and (ii) comprising at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any of the first-strand modified sequences listed in Table 3.

3. The nucleic acid according to claim 1 or 2, wherein the first strand comprises nucleosides 2-18 of any sequence defined in claim 1 or 2, specifically wherein the first strand comprises nucleosides 2-18 of any sequence defined in Table 2 or Table 3.

4. The nucleic acid of claim 1, wherein the second strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differs by 0 or 1 nucleotide from any second strand sequence listed in Table 2, and wherein the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleotides.

5. nucleic acid according to claim 2, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ by 0 or 1 nucleoside from any second strand modified sequence listed in Table 4, and wherein the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

6. The nucleic acid of claim 1, wherein the first strand comprises any one of the first strand sequences listed in Table 2.

7. The nucleic acid of claim 2, wherein the first strand comprises any one of the first strand modified sequences listed in Table 3.

8. The nucleic acid of claim 4, wherein the second strand comprises any one of the second strand sequences listed in Table 2.

9. The nucleic acid of claim 5, wherein the second strand comprises any one of the second strand modified sequences listed in Table 4.

10. The nucleic acid of claim 6, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 144, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 238, SEQ ID NO:

239.

11. The nucleic acid of claim 7, wherein the first strand comprises any one of the following sequences: SEQ ID NO:385, SEQ ID NO:388, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:372, SEQ ID NO:378, SEQ ID NO:379, SEQ ID NO:384, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:498, SEQ ID NO:518, SEQ ID NO:538, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551 NO:551, SEQ ID NO:552, SEQ ID NO:558, SEQ ID NO:

559.

12. The nucleic acid of claim 8, wherein the second strand comprises any one of the following sequences: SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:264, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:346, SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:358, SEQ ID NO:

359.

13. The nucleic acid of claim 9, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 585, SEQ ID NO: 588, SEQ ID NO: 566, SEQ ID NO: 567, SEQ ID NO: 568, SEQ ID NO: 569, SEQ ID NO: 571, SEQ ID NO: 572, SEQ ID NO: 578, SEQ ID NO: 579, SEQ ID NO: 584, SEQ ID NO: 587, SEQ ID NO: 589, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 698, SEQ ID NO: 718, SEQ ID NO: 738, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: NO:751, SEQ ID NO:752, SEQ ID NO:758, SEQ ID NO:

759.

14. The nucleic acid according to claim 1 and 4, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 15. The nucleic acid according to claim 2 and 5, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 16. The nucleic acid according to claim 14, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 17. The nucleic acid according to claim 15, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 18. The nucleic acid according to claims 1 and 4, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 19. The nucleic acid according to claim 2 and 5, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 20. The nucleic acid according to claims 1 and 4, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 21. The nucleic acid according to claim 2 and 5, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 22. The nucleic acid according to claim 2 and 5, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 23. The nucleic acid according to claim 2 and 5, comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 24. The nucleic acid according to any one of the preceding claims, wherein the first strand has a length of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides.

25. The nucleic acid according to any one of the preceding claims, wherein the second strand has a length of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

26. The nucleic acid according to any one of the preceding claims, wherein the duplex region of the nucleic acid is 17 to 30 nucleosides in length, more preferably 19 or 21 nucleosides in length.

27. The nucleic acid of any one of the preceding claims, wherein the region of complementarity between the first strand and a portion of RNA transcribed from the ZPI gene is 17 to 30 nucleosides in length.

28. A nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises one or more single-stranded nucleoside overhangs, optionally wherein the overhang is on the first or second strand, preferably at the 3' end of the first or second strand, and / or wherein the overhang comprises 1 to 4 nucleosides, more preferably 2 nucleosides.

29. The nucleic acid of any one of the preceding claims, wherein the nucleic acid is a siRNA oligonucleoside.

30. The nucleic acid of any one of the preceding claims, wherein one or more nucleosides on the first strand and / or the second strand are modified to form modified nucleosides.

31. The nucleic acid of claim 30, wherein one or more nucleosides on the first strand and / or the second strand comprise a terminal modification, a base modification, a sugar modification, and / or a backbone modification.

32. The nucleic acid of claim 30, wherein one or more nucleosides on the first strand and / or the second strand comprise a sugar modification, wherein the modification is at the 2'-OH group of the ribose.

33. The nucleic acid of claim 32, wherein the sugar modification comprises a 2'-Me modification and / or a 2'-F modification.

34. The nucleic acid of claim 30, wherein the first strand comprises a 2'-F modification at any one of position 2, position 6, position 14, or any combination thereof, counting from position 1 of the first strand.

35. The nucleic acid of claim 30, wherein the second strand comprises a 2'-F modification at any one of position 7, position 9, position 11, or any combination thereof, counting from position 1 of the second strand.

36. The nucleic acid of claim 30, wherein the first strand and the second strand each comprise a 2'-Me modification and a 2'-F modification.

37. A nucleic acid according to claim 30, wherein the nucleic acid comprises at least one thermal destabilizing modification, the thermal destabilizing modification being suitably at one or more positions among positions 1 to 9 of the first strand, counting from position 1 of the first strand, and / or at one or more positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from: modified non-locked nucleic acids (UNA) and glycol nucleic acids (GNA), preferably glycol nucleic acids, more preferably (S)-glycol nucleic acids, wherein more preferably, the nucleic acid comprises at least one thermal destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand.

38. The nucleic acid of claim 30, which is a siRNA oligonucleoside, wherein the siRNA oligonucleoside comprises 3 or more 2'-F modifications at positions 6 to 12 of the second strand, counting from position 1 of the second strand, such as 4, 5, 6 or 7 2'-F modifications at positions 6 to 12 of the second strand.

39. The nucleic acid of claim 30, which is a siRNA oligonucleoside, wherein the second strand comprises at least 3, such as 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand, counting from position 1 of the second strand.

40. The nucleic acid according to claim 30, which is a siRNA oligonucleoside, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside in the 3' terminal region, or within at least 1 or 2 nucleosides starting from the terminal nucleoside in the 3' terminal region.

41. The nucleic acid according to claim 30, which is a siRNA oligonucleotide, wherein the first strand comprises seven consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside in the 3' terminal region.

42. The nucleic acid of claim 30, which is a siRNA oligonucleoside, wherein each of the first and second strands comprises an alternating modification pattern, preferably a completely alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified by (i) a 2'Me modification on odd-numbered nucleosides, counting from position 1 of the first strand, and (ii) a 2'F modification on even-numbered nucleosides, counting from position 1 of the first strand, and the nucleosides of the second strand are modified by (i) a 2'F modification on odd-numbered nucleosides, counting from position 1 of the second strand, and (ii) a 2'Me modification on even-numbered nucleosides, counting from position 1 of the second strand.

43. The nucleic acid of claim 30 which is a siRNA oligonucleoside, wherein the nucleosides of the first strand comprise a 2' sugar modification pattern, wherein the modifications are at least selected from: a 2'Me sugar modification and a 2'F sugar modification, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

44. The nucleic acid of claim 30, which is a siRNA oligonucleoside, wherein the nucleosides of the first strand comprise a 2' sugar modification pattern, wherein the modifications are at least selected from: a 2'Me sugar modification and a 2'F sugar modification, wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

45. A nucleic acid according to any one of the preceding claims, further comprising one or more abasic nucleosides, optionally wherein the one or more abasic nucleosides are located in the terminal region of the second strand, and / or wherein at least one abasic nucleoside is linked to an adjacent basic nucleoside by a reverse internucleoside bond.

46. ​​The nucleic acid of claim 45, wherein the second strand comprises 2 consecutive abasic nucleosides at the 5' terminal region of the second strand, wherein one such abasic nucleoside is the terminal nucleoside at the 5' terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside at the 5' terminal region of the second strand, in: (a) the penultimate abasic nucleoside is linked to the adjacent first abasic nucleoside in the adjacent 5' proximal region via an inverted internucleoside bond; and (b) the reverse bond is a 5-5' reverse bond; and (c) When reading toward the terminus comprising the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 3'-5'.

47. The nucleic acid of claim 45, wherein the second strand comprises 2 consecutive abasic nucleosides, preferably located at an overhang at the 3' terminal region of the second strand, wherein one such abasic nucleoside is the terminal nucleoside at the 3' terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside at the 3' terminal region of the second strand, in: (a) the penultimate abasic nucleoside is linked to the adjacent first abasic nucleoside in the adjacent 3' proximal region via an inverted internucleoside bond; and (b) the inverted bond is a 3-3' inverted bond; and (c) When reading toward the end including the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 5'-3'.

48. The nucleic acid according to claim 46, wherein (i) the first strand and the second strand are each 23 nucleotides in length; (ii) two phosphorothioate internucleoside bonds are respectively located between three consecutive positions in the 5' proximal region of the second strand, wherein the first phosphorothioate internucleoside bond is located between the adjacent first base nucleoside in (a) and the adjacent second base nucleoside in the 5' proximal region of the second strand, and the second phosphorothioate internucleoside bond is located between the adjacent second base nucleoside and the adjacent third base nucleoside in the 5' proximal region of the second strand; (iii) two phosphorothioate internucleoside bonds are located between three consecutive positions of the 5' and 3' terminal regions of the first strand, respectively, whereby each terminal nucleoside of the 5' and 3' terminal regions of the first strand is linked to the corresponding 5' and 3' adjacent penultimate nucleosides, respectively, by a phosphorothioate internucleoside bond, and each 5' and 3' penultimate nucleoside is linked to the corresponding 5' and 3' adjacent penultimate nucleoside by a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties at the 3' terminal region of the second strand.

49. The nucleic acid according to claim 47, wherein (i) the first strand and the second strand are each 23 nucleotides in length; (ii) two phosphorothioate internucleoside bonds are respectively located between three consecutive positions in the 3' proximal region of the second strand, wherein the first phosphorothioate internucleoside bond is located between the adjacent first base nucleoside in (a) and the adjacent second base nucleoside in the 3' proximal region of the second strand, and the second phosphorothioate internucleoside bond is located between the adjacent second base nucleoside and the adjacent third base nucleoside in the 3' proximal region of the second strand; (iii) two phosphorothioate internucleoside bonds are located between three consecutive positions of the 5' and 3' terminal regions of the first strand, respectively, whereby each terminal nucleoside of the 5' and 3' terminal regions of the first strand is linked to the corresponding 5' and 3' adjacent penultimate nucleosides, respectively, by a phosphorothioate internucleoside bond, and each 5' and 3' penultimate nucleoside is linked to the corresponding 5' and 3' adjacent penultimate nucleoside by a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties at the 5' terminal region of the second strand.

50. The nucleic acid of any one of the preceding claims, wherein the nucleic acid comprises one or more phosphorothioate internucleoside linkages.

51. A nucleic acid according to claim 50, wherein the one or more phosphorothioate internucleoside bonds are located between at least three consecutive positions of the 5' or 3' proximal terminal region of the second strand, respectively, whereby the proximal terminal region is preferably adjacent to the terminal region, wherein at least according to claim 45 the one or more abasic nucleosides of the second strand are located in the terminal region.

52. The nucleic acid of claim 50 or 51, wherein the one or more phosphorothioate internucleoside bonds are located between at least three consecutive positions of the 5' and / or 3' terminal region of the first strand, respectively, whereby preferably the terminal positions of the 5' and / or 3' terminal region of the first strand are linked to their adjacent positions via phosphorothioate internucleoside bonds.

53. The nucleic acid of claim 33, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') of any one of: Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me.

54. The nucleic acid of claim 33, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') of any one of: (Me) 8 –(F) 3 –(Me) 10 。 55. The nucleic acid of claim 53, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') of any one of: Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or Me(s)Me(s)Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me–Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or Me–Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me–Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me–Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, Among them (s) is a sulfuric acid hydroxide key.

56. Basic requirement 54 predicated nucleic acid, which includes the second predicated predicated modification nucleic acid, and the following arbitrary one modification model (5'-3'): Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , Among them (s) is the sulfur-based acid oxidation nucleus.

57. Basic requirement 53 predicated nucleic acid, including the second predicated predicated nucleic acid, and the following optional modification formula (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or ia–ia-Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or ia–ia-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, or ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or ia–ia-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, or Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, Among them, the ia-ia representative has an anti-directionless red base nucleus, and the ia-ia representative has an anti-directionless red base nucleus at its second 3' end, and has two protruding ends.

58. Basic requirement 54 predicated nucleic acid, which includes the second predicated modification of the nucleic acid, and the following arbitrary one modification model (5'-3'): him-him-(Me) 8 –(F) 3 -(Along with) 10 , Among them, the representative of IA is unopposed.

59. Basic requirement 53 predicated nucleic acid, which includes the second predicated modification of the nucleic acid, and the following arbitrary one modification model (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia–ia-Me(s)Me(s)Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me–Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia, or Me–Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or Me–Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or Me–Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, in: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is at the 3' end of the second strand, the inverted abasic nucleoside is at a 2-nucleoside overhang.

60. The nucleic acid of claim 54, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') of any one of: ia-ia-Me(s)Me(s)(Me) 6 –(F) 3 –(Me) 10 , in: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside.

61. The nucleic acid of claim 33, wherein the modified nucleosides comprise any one of the following modification patterns: Modification mode 1: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; Or modifier mode 2: Second strand (5'-3'): Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Or modifier mode 3: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Or in modifier mode 4: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First pin (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; Modification model 5: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; Modification model 6: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First pin (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me.

62. Basic requirement 33 Predicated nucleic acid, which includes a predicated nucleic acid, any one of the following modified models: Ceremony model 1: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me–F–Me–Me–Me–X 1 –Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me–Me-Me, where X 1 It is a thermal destabilization modification; A modification model 2: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me; Modification model 3: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me–Me–Me; Modification model 4: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me-Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–F–Me–Me-Me; Modification model 5: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me–F–Me–Me-Me–X 1 –Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, where X 1 It is a thermal destabilization modification; Modification model 6: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–F–Me-F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me; Modification model 7: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me–Me–Me; Modification model 8: Second pin (5'-3'): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First pin (5'-3'): Me–F–Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–F–Me–Me–Me.

63. Basic requirement 61 Predicated nucleic acid, which includes a predetermined nucleic acid, and any one of the following modified models: Ceremony model 1: Second pin (5'-3'): Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; A modification model 2: Second pin (5'-3'): Me(s)Me(s)Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Modification model 3: Second pin (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Modification model 4: Second pin (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Modification model 5: Second pin (5'-3'): Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Modification model 6: Second pin (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Among them (s) is the sulfur-based acid oxidation nucleus.

64. Rooting requirements 62 Predicated nucleic acid, which includes a predicated nucleic acid, any one of the following modified models: Ceremony model 1: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me(s)F(s)Me–Me–Me–X 1 –Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, where X 1 It is a thermal destabilization modification; A modification model 2: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me; Modification model 3: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–F–Me–Me–Me(s)Me(s)Me; Modification model 4: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me-Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–F–Me(s)Me(s)Me; Modification model 5: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me(s)F(s)Me–Me-Me–X 1 –Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, where X 1 It is a thermal destabilization modification; Modification model 6: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–F–Me-F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me; Modification model 7: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me(s)Me(s)Me; Modification model 8: Second pin (5'-3'): Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–F–Me(s)Me(s)Me; Among them (s) is the sulfur-based acid oxidation nucleus.

65. Rooting requirements 61 Predicated nucleic acid, it includes a predicated nucleic acid, and any one of the following modified models: Ceremony model 1: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modification pattern 2: Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modification pattern 3: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modification pattern 4: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modification pattern 5: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modification pattern 6: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; where (s) is a phosphorothioate internucleoside bond.

66. Rooting requirements 61 Predicated nucleic acid, which includes a predicated nucleic acid, any one of the following modified models: Ceremony model 1: Second pin (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First pin (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; A modification model 2: Second pin (5'-3'): ia–ia-Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First pin (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Modification model 3: Second pin (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First pin (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Modification model 4: Second pin (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First pin (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Modification model 5: Second pin (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; Modification model 6: Second pin (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; Among them, the representative of IA is unopposed.

67. Rooting requirements 62 Predicated nucleic acid, which includes a predicated nucleic acid, any one of the following modified models: Ceremony model 1: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me–F–Me–Me–Me–X 1 –Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me–Me-Me, where X 1 It is a thermal destabilization modification; A modification model 2: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me; Modification model 3: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me–Me–Me; Modification model 4: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me-Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–F–Me–Me-Me; Modification model 5: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me–F–Me–Me-Me–X 1 –Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, where X 1 It is a thermal destabilization modification; Modification model 6: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–F–Me-F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me; Modification model 7: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me–Me–Me; Modification model 8: Second pin (5'-3'): ia–ia–Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me–F–Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–F–Me–Me–Me, Among them, the representative of IA is unopposed.

68. Rooting requirements 61 Predicated nucleic acid, which includes a predicated nucleic acid, and any one of the following modified models: Ceremony model 1: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, First pin (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; A modification model 2: Second pin (5'-3'): Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First pin (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Modification model 3: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First pin (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Modification model 4: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First pin (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Modification model 5: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, 1st pin (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; Modification model 6: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me; wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is at the 3' end of the second strand, the inverted abasic nucleoside is at a 2-nucleoside overhang.

69. The nucleic acid of claim 61, wherein the modified nucleosides comprise any one of the following modification patterns: Modification mode 1: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modifier mode 2: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modifier mode 3: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or in modifier mode 4: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Or modifier mode 5: Second pin (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 1st pin (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Modification model 6: Second pin (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, 1st pin (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me; Among them: (s) This is the base of the base, the representative of the base.

70. Basic requirement 62 Predicated nucleic acid, which includes a predicated nucleic acid, any one of the following modified models: Ceremony model 1: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me(s)F(s)Me–Me–Me–X 1 –Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, where X1 is a thermal destabilization modification; A modification model 2: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me; Modification model 3: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–F–Me–Me–Me(s)Me(s)Me; Modification model 4: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me-Me–F–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–F–Me(s)Me(s)Me; Modification model 5: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, First strand (5'-3'): Me(s)F(s)Me–Me-Me–X 1 –Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, where X1 is a thermally destabilizing modification; Modification model 6: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–F–Me-F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me; Modification model 7: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–F–Me–Me–Me(s)Me(s)Me; Modification model 8: Second pin (5'-3'): ia–ia–Me(s)Me(s)Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me, 1st pin (5'-3'): Me(s)F(s)Me–Me–Me–F–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–F–Me(s)Me(s)Me; Among them: (s) This is the sulfuric acid, nuclear, and nuclear key. The representative of IA is the opposite direction. 71.Nucleic acid with basic requirements 61, the following modification model has the following modifications: Ceremony model 1: Second pin (5'-3'): Me–Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me A modification model 2: Second pin (5'-3'): Me–Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modifier mode 3: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or in modifier mode 4: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modifier mode 5: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modifier mode 6: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me in: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is at the 3' end of the second strand, the inverted abasic nucleoside is at a 2-nucleoside overhang.

72. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, optionally wherein the ligand moiety is in the terminal region of the second strand, preferably in the 3' terminal region thereof.

73. The nucleic acid of claim 72, wherein the ligand moiety comprises: (i) one or more N-acetylgalactosamine (GalNAc) ligands, and / or (ii) one or more N-acetylgalactosamine (GalNAc) ligand derivatives, and / or (iii) one or more N-acetylgalactosamine (GalNAc) ligands and / or derivatives thereof conjugated to the nucleic acid via a linker.

74. The nucleic acid of claim 73, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are conjugated directly or indirectly to the 5' or 3' terminal region of the second strand of the nucleic acid, preferably to the 3' terminal region thereof.

75. A nucleic acid according to any one of claims 72 to 74, wherein the ligand portion comprises the structure:

76. A nucleic acid according to any one of claims 72 to 75, comprising the structure: in: R 1 is independently selected at each occurrence from the group consisting of hydrogen, methyl, and ethyl; R 2 Selected from the group consisting of hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro; X 1 and X 2 is independently selected at each occurrence from: methylene, oxygen, and sulfur; m is an integer from 1 to 6; n is an integer from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, provided that: (i) q and r cannot be 0 at the same time; and (ii) s, t and v cannot be 0 at the same time; Z is an oligonucleoside moiety.

77. The nucleic acid of claim 76, comprising the structure: wherein [oligonucleotide] represents the consecutive nucleosides of the second strand.

78. A nucleic acid according to any one of claims 72 to 75, comprising the structure: in: r and s are independently integers selected from 1 to 16; and Z is an oligonucleoside moiety.

79. The nucleic acid of claim 78, comprising the structure: wherein [oligonucleotide] represents the consecutive nucleosides of the second strand.

80. The nucleic acid of any one of claims 72 to 79, wherein the nucleic acid comprises the following modification pattern: Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, in: (s) is a phosphorothioate internucleoside bond, ia represents the inverted abasic nucleoside, And wherein the nucleic acid is conjugated directly or indirectly to one or more ligand moieties, optionally wherein the ligand moiety is at the terminal region of the second strand, preferably at its 3' terminal region.

81. The nucleic acid of claim 80, wherein the second strand comprises the structure: wherein [oligonucleotide] represents the contiguous nucleosides of the second strand, and wherein the one or more ligand moieties are conjugated to the 3' terminal region of the second strand via a linker.

82. The nucleic acid of claim 80 or 81, wherein the first strand and the second strand comprise, consist of, or consist essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences: 。 83. The nucleic acid of any one of claims 80 to 82, wherein the two consecutive inverted abasic nucleosides in the 5' terminal region of the second strand are as shown in the following 5' terminal motif: in: T represents 2'Me ribose modification, B represents the nucleobases of the first two nucleosides in the 5' terminal region of the second strand, and Z represents the remaining 19 consecutive base nucleosides of the second strand.

84. A pharmaceutical composition comprising the nucleic acid of any one of the preceding claims, and a pharmaceutically acceptable excipient or carrier.

85. A nucleic acid or pharmaceutical composition according to any one of the preceding claims for therapeutic use.

86. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for preventing or treating a disease associated with a hemostatic disorder, such as a disease associated with a hemostatic disorder, such as hemophilia.

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