Nucleic acid compound

Abstract

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

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 protein formation 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, numerous 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, oncology, infectious diseases, and ocular diseases.

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

[0005] According to the first aspect of the present application, the present application provides a nucleic acid for inhibiting B4GALT1 expression, which comprises a double-stranded region, the double-stranded region comprising a first chain and a second chain at least partially complementary to the first chain, wherein the first chain is: (i) at least partially complementary to a portion of RNA transcribed from the B4GALT1 gene, and (ii) comprises at least 17 consecutive nucleosides, which differ from any one of the first chain sequences listed in Table 2 by 0 or 1 nucleoside.

[0006] According to the second aspect of the present application, the present application provides a nucleic acid for inhibiting B4GALT1 expression, which comprises a double-stranded region, the double-stranded region comprising a first chain and a second chain at least partially complementary to the first chain, wherein the first chain is: (i) at least partially complementary to a portion of RNA transcribed from the B4GALT1 gene, and (ii) comprises at least 17 consecutive nucleosides, which differ from any one of the first chain modified sequences listed in Table 3 by 0 or 1 nucleoside.

[0007] A nucleic acid as described herein, wherein the first strand comprises nucleosides 2-18 of any sequence according to the first and second aspects of the present application.

[0008] The nucleic acid according to the above-mentioned first aspect of the present application, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any one of the second strand sequences listed in Table 2 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand in the 17 consecutive nucleosides.

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

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

[0011] The nucleic acid according to the above-mentioned second aspect of the present application, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any one of the second strand modified sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the double-stranded 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 modification sequences listed in Table 3.

[0014] According to the nucleic acid of the first aspect of the present application, the second strand comprises any one of the second strand sequences 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 modification sequences listed in Table 4.

[0016] The nucleic acid according to the above-mentioned first aspect of the present application, wherein the first chain comprises any one of the following sequences: SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQID NO:40, SEQ ID NO:41.

[0017] The nucleic acid according to the above-mentioned second aspect of the present application, wherein the first chain comprises any one of the following sequences: SEQID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQID NO:80, SEQ ID NO:81.

[0018] The nucleic acid according to the first aspect of the present application, wherein the second chain comprises any one of the following sequences: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQID NO:60, SEQ ID NO:61.

[0019] The nucleic acid according to the above-mentioned second aspect of the present application, wherein the second chain comprises any one of the following sequences: SEQID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQID NO:100, SEQ ID NO:101.

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

[0021] Unmodified first strand Unmodified second strand SEQ ID NO:22 SEQ ID NO:42 SEQ ID NO:23 SEQ ID NO:43 SEQ ID NO:24 SEQ ID NO:44 SEQ ID NO:25 SEQ ID NO:45 SEQ ID NO:26 SEQ ID NO:46 SEQ ID NO:27 SEQ ID NO:47 SEQ ID NO:28 SEQ ID NO:48 SEQ ID NO:29 SEQ ID NO:49 SEQ ID NO:30 SEQ ID NO:50 SEQ ID NO:31 SEQ ID NO:51 SEQ ID NO:32 SEQ ID NO:52 SEQ ID NO:33 SEQ ID NO:53 SEQ ID NO:34 SEQ ID NO:54 SEQ ID NO:35 SEQ ID NO:55 SEQ ID NO:36 SEQ ID NO:56 SEQ ID NO:37 SEQ ID NO:57 SEQ ID NO:38 SEQ ID NO:58 SEQ ID NO:39 SEQ ID NO:59 SEQ ID NO:40 SEQ ID NO:60 SEQ ID NO:41 SEQ ID NO:61

[0022] A nucleic acid comprising a first and a second strand, the first and second strands comprising, consisting of, or consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0023] Unmodified first strand Unmodified second strand SEQ ID NO:202 SEQ ID NO:302 SEQ ID NO:205 SEQ ID NO:305 SEQ ID NO:217 SEQ ID NO:317 SEQ ID NO:228 SEQ ID NO:328

[0024] A nucleic acid comprising a first and a second strand, the first and second strands comprising, consisting of, or consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0025] Modified first strand Modified second strand SEQ ID NO:62 SEQ ID NO:82 SEQ ID NO:63 SEQ ID NO:83 SEQ ID NO:64 SEQ ID NO:84 SEQ ID NO:65 SEQ ID NO:85 SEQ ID NO:66 SEQ ID NO:86 SEQ ID NO:67 SEQ ID NO:87 SEQ ID NO:68 SEQ ID NO:88 SEQ ID NO:69 SEQ ID NO:89 SEQ ID NO:70 SEQ ID NO:90 SEQ ID NO:71 SEQ ID NO:91 SEQ ID NO:72 SEQ ID NO:92 SEQ ID NO:73 SEQ ID NO:93 SEQ ID NO:74 SEQ ID NO:94 SEQ ID NO:75 SEQ ID NO:95 SEQ ID NO:76 SEQ ID NO:96 SEQ ID NO:77 SEQ ID NO:97 SEQ ID NO:78 SEQ ID NO:98 SEQ ID NO:79 SEQ ID NO:99 SEQ ID NO:80 SEQ ID NO: 100 SEQ ID NO:81 SEQ ID NO:101

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

[0027] Modified first strand Modified second strand SEQ ID NO:502 SEQ ID NO:610 SEQ ID NO:503 SEQ ID NO:611 SEQ ID NO:504 SEQ ID NO:612 SEQ ID NO:505 SEQ ID NO:613 SEQ ID NO:506 SEQ ID NO:614 SEQ ID NO:507 SEQ ID NO:615 SEQ ID NO:508 SEQ ID NO:616 SEQ ID NO:509 SEQ ID NO:617 SEQ ID NO:510 SEQ ID NO:611 SEQ ID NO:511 SEQ ID NO:613 SEQ ID NO:512 SEQ ID NO:615 SEQ ID NO:513 SEQ ID NO:617

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

[0029]

[0030]

[0031] A nucleic acid comprising a first and a second strand, the first and second strands comprising, consisting of, or consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0032]

[0033]

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

[0035] Unmodified first strand Unmodified second strand SEQ ID NO:202 SEQ ID NO:302 SEQ ID NO:205 SEQ ID NO:305

[0036] A nucleic acid comprising a first strand and a second strand comprising, consisting of, or consisting essentially of a sequence The difference between the first and second sequences is 0 or 1 nucleotides. The nucleotide sequence is composed of:

[0037] Modified first strand Modified second strand SEQ ID NO:502 SEQ ID NO:610 SEQ ID NO:503 SEQ ID NO:611 SEQ ID NO:510 SEQ ID NO:611 SEQ ID NO:800 SEQ ID NO:611 SEQ ID NO:801 SEQ ID NO:611 SEQ ID NO:802 SEQ ID NO:611 SEQ ID NO:803 SEQ ID NO:611 SEQ ID NO:804 SEQ ID NO:611 SEQ ID NO:805 SEQ ID NO:611 SEQ ID NO:806 SEQ ID NO:611

[0038] A nucleic acid comprising a first and a second strand, the first and second strands comprising, consisting of, or consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0039] Modified first strand Modified second strand SEQ ID NO:504 SEQ ID NO:612 SEQ ID NO:505 SEQ ID NO:613 SEQ ID NO:511 SEQ ID NO:613 SEQ ID NO:807 SEQ ID NO:613 SEQ ID NO:808 SEQ ID NO:613 SEQ ID NO:809 SEQ ID NO:613 SEQ ID NO:810 SEQ ID NO:613 SEQ ID NO:811 SEQ ID NO:613 SEQ ID NO:812 SEQ ID NO:613 SEQ ID NO:813 SEQ ID NO:613 SEQ ID NO:814 SEQ ID NO:613

[0040] A nucleic acid comprising a first and a second strand, the first and second strands comprising, consisting of, or consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0041] Modified first strand Modified second strand SEQ ID NO:503 SEQ ID NO:611 SEQ ID NO:505 SEQ ID NO:613

[0042] A conjugate for inhibiting the expression of a B4GALT1 target gene in a cell, the conjugate comprising a nucleic acid as disclosed herein and one or more ligand moieties.

[0043] A pharmaceutical composition comprising the nucleic acid disclosed herein and a pharmaceutically acceptable excipient or carrier.

[0044] A nucleic acid or pharmaceutical composition for use in treatment.

[0045] A nucleic acid or pharmaceutical composition for preventing or treating diabetes.

[0046] A nucleic acid or pharmaceutical composition for use in preventing or treating cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1: The linker and ligand portions of the constructs suitable for use in this application include tether 1a. Although Figure 1 Linkers conjugated to oligonucleotides are described, but it is to be understood that the present application also encompasses conjugates of the same linkers as the oligonucleosides disclosed herein.

[0048] It should also be understood that although Figure 1 Based on the linker and ligand parts ( Figure 1 As shown, these parts are connected to the oligonucleoside part described in this application) specifically describe a product molecule, which product optionally further comprises, or consists essentially of the following molecules: wherein the linker and ligand parts are essentially as Figure 1 tethered to the oligonucleoside moiety as shown, but with the F substituent on the cyclooctyl ring replaced by a substituent generated by hydrolytic displacement (e.g., an OH substituent). Thus, (a) tethered 1a constructs can essentially consist of molecules having Figure 1 The linker and ligand moieties specifically described in the preceding paragraphs have an F substituent on the cyclooctyl ring; or (b) the tethered 1a construct may consist essentially of a molecule having Figure 1 The linker and ligand moieties specifically described in (a) and (b) may comprise a mixture of molecules as defined in (a) and / or (b), except that the F substituent on the cyclooctyl ring is substituted with a substituent generated by hydrolytic displacement (e.g., an OH substituent), or (c) the tether 1a construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0049] Figure 2 : The linker and ligand portions of the constructs suitable for use in the present application, including tether 1b. Although Figure 2 Linkers conjugated to oligonucleotides are described, but it should be understood that the present application also encompasses conjugates of the same linkers as the oligonucleosides disclosed herein.

[0050] about Figure 1 Comments and Figure 1 The situation where the F substituent on the cyclooctyl ring may be replaced by a substituent generated by hydrolytic displacement (e.g., an OH substituent) is also applicable to the tether 1b construct. Thus, (a) the tether 1b construct can essentially consist of a molecule having Figure 2 The linker and ligand moieties specifically described in the foregoing have an F substituent on the cyclooctyl ring; or (b) the tethered 1b construct may consist essentially of a molecule having Figure 2 The linker and ligand portions specifically described in (a) and (b) may comprise a mixture of molecules as defined in (a) and / or (b), except that the F substituent on the cyclooctyl ring is substituted with a substituent generated by hydrolytic displacement (e.g., an OH substituent), or (c) the tethered 1b construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0051] Figure 3 : The linker and ligand portions of the constructs suitable for use in the present application, including tether 2a. Figure 3 Linkers conjugated to oligonucleotides are described, but it should be understood that the present application also encompasses conjugates of the same linkers as the oligonucleosides disclosed herein.

[0052] Figure 4 : The linker and ligand portions of the constructs suitable for use in the present application, including tether 2b. Figure 4 Linkers conjugated to oligonucleotides are described, but it should be understood that the present application also encompasses conjugates of the same linkers as the oligonucleosides disclosed herein.

[0053] Figure 5 : The structural formula described in statements 1-101 disclosed herein.

[0054] Figure 6 : The structural formula described in clauses 1-56 disclosed herein

[0055] Figure 7a and 7b : A reverse abasic construct that can be used with the nucleic acid sequences described herein according to the present application. Figure 7a , GalNAC linker was connected to the 5' end region of the sense strand used ( Figure 7a Not shown in ). Figure 7b , GalNAC linker was connected to the 3' end region of the sense strand used ( Figure 7b not shown in the figure).

[0056] like Figure 7a wherein iaia as shown in the 3' terminal region of the sense strand indicates: (i) two abasic nucleosides provided as the penultimate and terminal nucleosides of the 3' terminal region of the sense strand, (ii) wherein a 3'-3' inverted bond is provided between the penultimate nucleoside of the sense strand (i.e., position 21 of the sense strand, wherein position 1 is the 5' terminal 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'.

[0057] like Figure 7b wherein the iaia shown in the 5'-terminal region of the sense strand indicates: (i) two abasic nucleosides provided as the penultimate and terminal nucleosides of the 5'-terminal 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 of the 5'-terminal region of the sense strand as numbered by nucleoside positions on the sense strand) and the adjacent penultimate abasic residue, and (iii) when reading toward the 5'-terminal region including the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'-5'.

[0058] Figure 8 : According to the double-stranded structure of Table 5.

[0059] Figure 9 Results of RNAi screening for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Each bar represents the mean relative expression of B4GALT1 mRNA after treatment with 5 nM of a single siRNA construct, compared to untreated wells.

[0060] Figure 10 : Results of RNAi molecular screening for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Each bar represents the mean relative expression of B4GALT1 mRNA after treatment with 0.1 nM of a single siRNA construct compared to untreated wells.

[0061] Figure 11 Results of a dose-response experiment for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Dots represent the mean relative expression of B4GALT1 mRNA in untreated wells following treatment with the indicated concentrations of siRNA constructs 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 of untreated wells on the same plate.

[0062] Figure 12 : Time-course inhibition of B4GALT1 expression in mice.

[0063] Figure 13 :ETXM1200(ETXS2400&ETXS2399), ETXM1201(ETXS2402&ETXS2401), ETXM1217(ETXS2434&ETXS2401), E TXM1764(ETXS3528&ETXS2401), ETXM1765(ETXS3530&ETXS2401), ETXM1766(ETXS3532&ETXS2401), ETXM 1767(ETXS3534&ETXS2401),ETXM Inhibition of B4GALT1 expression by ETXM1768 (ETXS3536 & ETXS2401), ETXM1769 (ETXS3538 & ETXS2401), and ETXM1770 (ETXS3540 & ETXS2401).

[0064] Figure 14:ETXM1203(ETXS2406&ETXS2405), ETXM1204(ETXS2408&ETXS2407), ETXM1218(ETXS2436&ETXS2407), E TXM1772(ETXS3544&ETXS2407), ETXM1773(ETXS3546&ETXS2407), ETXM1774(ETXS3548&ETXS2407), ETXM Inhibition of ZPI expression by ETXM1775 (ETXS3550 & ETXS2407), ETXM1776 (ETXS3552 & ETXS2407), ETXM1777 (ETXS3554 & ETXS2407), and ETXM1778 (ETXS3556 & ETXS2407).

[0065] Figure 15 : Selection of active GalNAc-siRNA with EC50 values ​​less than 100 nM. In primary mouse hepatocytes, the dose-response relationship for B4GALT1 gene knockdown was measured using GalNAc-siRNA targeting mouse B4GALT1, diluted 10 times from 1000 nM to the lowest concentration, followed by incubation for 48 hours. EC50 was determined by fitting the data to a 4-parameter sigmoidal dose-response (variable slope) equation using GraphPad Prism. 50 Four active GalNAc-siRNAs, ETXM619, ETXM624, ETXM628, and ETXM633, were screened and used for in vivo pharmacology.

[0066] Figure 16 Summary of the knockdown effect of B4GALT1 mRNA in mouse liver tissue following multiple administrations of GalNAc-siRNA, ETXM619, ETXM624, ETXM628, and ETXM633 (10 mg / kg). The y-axis values ​​represent relative mRNA expression relative to the untreated group (n=5). Each data point represents the mean ± standard deviation of relative mRNA expression from n=3 experiments. The red arrow at the top of the graph indicates the day the test article was administered.

[0067] Figure 17 Effects of B4GALT1 mRNA knockdown on plasma LDL-c, glucose, and fibrinogen levels. Following three doses of ETXMs (10 mg / kg, subcutaneously) on days 0, 3, and 7, plasma samples were collected on day 14. ETXM-treated (n=12) normal C57BL / 6 mice showed significant reductions in LDL-c, glucose, and fibrinogen levels compared to the untreated group (n=5). Data presented here are mean ± SD.

[0068] definition

[0069] "First strand" is also referred to herein as the antisense strand or guide strand, which are used interchangeably herein and 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., an mRNA). For example, 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, mismatches may typically occur in the interior or terminal regions of the molecule. In some embodiments, a double-stranded nucleic acid, such as an siRNA agent of the present application, includes nucleoside mismatches in the antisense strand.

[0070] "Second strand" (also referred to herein as the sense strand or passenger strand, and used interchangeably herein) refers to a nucleic acid strand, such as an siRNA, that includes a region that is substantially complementary to a region of the antisense strand as defined herein.

[0071] In the context of molecules comprising a nucleic acid with a ligand moiety, optionally also with a linker moiety, the nucleic acid of the present application may be referred to as an oligonucleoside or an oligonucleoside moiety.

[0072] 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 non-phosphodiester bond are also considered. For example, the bond between nucleosides can be a phosphorothioate bond. Therefore, the term "oligonucleoside" as used herein encompasses oligonucleotide and other nucleoside oligomers. Oligonucleoside is a nucleic acid having at least a portion that is preferably an oligonucleotide according to the application. According to the application, the oligonucleoside with one or more or most of phosphodiester backbone bonds between nucleosides is also preferred. According to the application, the oligonucleoside with one or more or most of phosphodiester backbone bonds between nucleosides, and the oligonucleoside with one or more phosphorothioate backbone bonds (usually in the terminal region of the first and / or second chain) between nucleosides is also preferred.

[0073] It is preferred herein that nucleic acid according to the application is a double-stranded oligonucleoside comprising one or more phosphorothioate backbone bonds between nucleosides. Therefore, in all cases where the application relates to oligonucleotides, particularly in the chemical structures disclosed herein, the oligonucleotides may be oligonucleosides defined herein equally.

[0074] In some embodiments, the double-stranded nucleic acid (eg, siRNA) reagents of the present application include nucleoside mismatches in the sense strand. In some embodiments, the nucleoside mismatches are, for example, within 5, 4, 3, 2, or 1 nucleosides of the 3' end of the nucleic acid (eg, siRNA).

[0075] In another embodiment, the nucleoside is mismatched, for example, in the 3'-terminal nucleoside of the nucleic acid (eg, siRNA).

[0076] "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 processing of the primary transcript RNA.

[0077] The target sequence can be about 10-35 nucleosides in length, such as about 15-30 nucleosides. For example, the target sequence can be about 15-30 nucleosides, 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, 19-29 , 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 nucleosides in length. Ranges and lengths between the above-recited ranges and lengths are also considered part of this application.

[0078] The term "ribonucleoside" or "nucleoside" may also refer to modified nucleosides, as described in detail below.

[0079] The nucleic acid may be DNA or RNA and may contain modified nucleosides. RNA is the preferred nucleic acid.

[0080] The terms "iRNA," "siRNA," "RNAi agent," and "iRNA agent," "RNA interfering agent" are used interchangeably herein to refer to an RNA-containing agent 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).

[0081] Double-stranded RNA is referred to herein as a "double-stranded siRNA (dsiRNA) agent," "double-stranded siRNAs (dsiRNAs) molecules," "double-stranded RNA (dsRNA) agent," "double-stranded RNA (dsRNA) molecule," "dsiRNA agent," "dsiRNA molecule," or "dsiRNA," which refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as "sense" and "antisense" orientations relative to the target RNA.

[0082] The majority of nucleosides in each strand of a nucleic acid, such as a dsRNA molecule, are preferably ribonucleosides, but in this case, each or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides or modified nucleosides. In addition, as used herein, "siRNA" may include chemically modified ribonucleosides.

[0083] The term "modified nucleoside" refers to a nucleoside having independently modified sugar moieties, modified internucleoside linkages, or modified nucleobases, or any combination thereof. Thus, the term modified nucleoside includes substitutions, additions, or removals of, for example, functional groups or atoms, to internucleoside linkages, sugar moieties, or nucleobases. For purposes of this specification and claims, any such modifications used in siRNA-type molecules are encompassed by "iRNA," "RNAi agent," "siRNA," or "siRNAi agent."

[0084] The two strands forming the double-stranded structure can be different parts of a larger molecule or separate molecules, such as RNA molecules.

[0085] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the double-stranded structure of a double-stranded nucleic acid. A nucleic acid according to the present application may comprise at least one nucleoside overhang; alternatively, the overhang may comprise at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more. The nucleoside overhang may comprise or consist of nucleosides / nucleoside analogs, including deoxynucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleoside may be located at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand.

[0086] In some embodiments, the antisense strand has a 1-10 nucleoside overhang at the 3' end or the 5' end, e.g., a 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.

[0087] "Blunt" or "blunt end" means that there is no unpaired nucleoside at that end of the double-stranded nucleic acid, that is, no nucleoside overhang. The nucleic acid of the present application includes nucleic acids that have no nucleoside overhang at one end or both ends.

[0088] Unless otherwise indicated, the term "complementary" when used to describe a first nucleoside sequence relevant to a second nucleoside sequence refers to the ability of an oligonucleoside comprising the first nucleoside sequence to hybridize with an oligonucleoside comprising the second nucleoside sequence and to form a double-stranded 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: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 ℃ or 70 ℃ for 12-16 hours, followed by washing (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).

[0089] For example, as described herein, the complementary sequence in a nucleic acid (e.g., dsiRNA) comprises base pairing of an oligonucleoside of a first nucleoside sequence with an oligonucleoside of 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 a first sequence is referred to herein as "substantially complementary" or "partially complementary" to a 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 no more than 5, while maintaining the ability to hybridize under conditions most relevant to their final application, such as inhibiting gene expression via the RISC pathway. In determining complementarity, overhangs should not be considered mismatches. For example, a nucleic acid, such as a dsiRNA, comprises an oligonucleoside of 17 nucleosides in length and an oligonucleoside of 19 nucleosides in length, wherein the longer oligonucleoside comprises a 17-nucleoside sequence that is completely complementary to the shorter oligonucleoside, and may still be referred to as "completely complementary."

[0090] For example, as used herein, a "complementary" sequence may also include, or be formed entirely of, non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, as long as the above-mentioned requirements for hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0091] The terms "complementary," "fully complementary," and "substantially / partially complementary" as used herein may refer to base pairing 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.

[0092] In the present application, the second strand of a nucleic acid according to the present application, particularly a dsiRNA for inhibiting B4GALT1, is at least partially complementary to the first strand of the nucleic acid. In some embodiments, the first and second strands of a nucleic acid described herein are partially complementary if they form a double-stranded region that is at least 17 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0093] In some embodiments, the first and second strands of a nucleic acid described herein are partially complementary if they form a double-stranded region that is 19 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs. In some embodiments, the first and second strands of a nucleic acid described herein are partially complementary if they form a double-stranded region that is 21 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0094] Alternatively, the first and second strands of the nucleic acids described herein are partially complementary if they form a double-stranded region of at least 17 base pairs in length, wherein at least 14, 15, 16 or 17 of the base pairs are complementary base pairs, in particular Watson Crick base pairs.

[0095] In some embodiments, the first and second strands of a nucleic acid described herein are partially complementary if they form a double-stranded region of 19 base pairs in length, wherein at least 14, 15, 16, 17, 18, or all 19 base pairs are complementary base pairs, particularly Watson Crick base pairs. In some embodiments, the first and second strands of a nucleic acid described herein are partially complementary if they form a double-stranded region of 21 base pairs in length, wherein at least 16, 17, 18, 19, 20, or all 21 base pairs are complementary base pairs, particularly Watson Crick base pairs.

[0096] For example, 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 contiguous portion of a target mRNA (e.g., an mRNA encoding a gene). In some embodiments, the contiguous portion of the mRNA is a sequence listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201. For example, a nucleic acid is complementary to at least a portion of a target gene mRNA if the sequence is substantially or partially complementary to a non-interrupted portion of an mRNA encoding a target gene.

[0097] Thus, in some preferred embodiments, the antisense oligonucleotides disclosed herein are fully complementary to the target gene sequence.

[0098] 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 the equivalent region of the target RNA sequence over its entire length, for example, at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0099] In some embodiments, the first (antisense) strand of a nucleic acid according to the present application is partially or fully complementary to a contiguous portion of RNA transcribed from the B4GALT1 gene. In some embodiments, the first strand of a nucleic acid according to the present application is partially or fully complementary to a contiguous portion of at least 17 nucleosides of B4GALT1 mRNA. In some embodiments, the first strand of a nucleic acid according to the present application is partially or fully complementary to a contiguous portion of 17, 18, 19, 20, 21, 22, or 23 nucleosides of B4GALT1 mRNA. In some embodiments, the first strand of a nucleic acid according to the present application is partially or fully complementary to a contiguous portion of 17, 18, or 19 nucleosides of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201.

[0100] In some embodiments, if the first (antisense) strand of a nucleic acid according to the present application comprises a contiguous nucleoside sequence of at least 17 nucleosides, and wherein at least 14, 15, 16, or 17 nucleosides of the contiguous nucleoside sequence are complementary to a contiguous portion of B4GALT1 mRNA, then it is partially complementary to a contiguous portion of B4GALT1 mRNA. In some embodiments, the first strand of a nucleic acid according to the present application comprises a contiguous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16, or 17 nucleosides of the contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201. In some embodiments, the first strand of a nucleic acid according to the present application comprises a contiguous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18, or all 19 nucleosides of the contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201.

[0101] In some embodiments, the nucleic acids described herein, such as siRNAs, comprise a sense strand that is substantially or partially complementary to an antisense oligonucleotide, which in turn is complementary to a target gene sequence and comprises a contiguous nucleotide sequence. The nucleotide sequence of the sense strand is typically at least about 80% complementary to the equivalent region of the nucleotide sequence of the antisense strand over its entire length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0102] In some embodiments, the nucleic acids of the present application, such as siRNA, comprise an antisense strand that is substantially or partially complementary to a target sequence and comprises a contiguous nucleotide sequence that is at least 80% complementary to the target sequence over its entire length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0103] As used herein, when the target gene sequence has sufficient complementarity with a nucleic acid (e.g., siRNA) reagent to promote target knockdown, "subject" refers to an animal, such as a mammal, including primates (e.g., humans, non-human primates, such as monkeys and chimpanzees), or non-primates or birds, that endogenously or heterologously expresses the target gene. In some preferred embodiments, the subject is a human.

[0104] The terms "treating" or "treatment" refer 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 as compared to expected survival in the absence of treatment. Treatment may include preventing the development of complications, such as reducing liver damage in a subject with a liver infection.

[0105] As used herein, a "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 affect the treatment of the disease (e.g., by reducing, ameliorating, or maintaining the existing disease or one or more symptoms of the disease or its associated complications).

[0106] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, pursuant to a reasonable benefit / risk ratio.

[0107] As used herein, the term "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, that is involved in carrying or transporting the test 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 injurious to the subject being treated.

[0108] When a value or range of values ​​is mentioned for a parameter, the values ​​and ranges between the stated values ​​are also intended to be part of this application.

[0109] As used herein, the articles "a" and "an" refer to one or more than one (ie, to at least one) of the grammatical object of the article.

[0110] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0111] Unless the context clearly indicates otherwise, the term "or" is used herein to mean and can be used interchangeably with the term "and / or." For example, "the sense strand or the antisense strand" is understood to mean "the sense strand or the antisense strand or the sense strand and the antisense strand."

[0112] As used herein, the term "about" means within the typical tolerance range in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In some embodiments, about means +10%. In some embodiments, about means +5%. When about appears before a series of numbers or a range, it is understood that "about" can modify each number in the series or range.

[0113] For example, the context clearly indicates that the term "at least" preceding a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can be logically included. For example, the number of nucleosides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides in a nucleic acid molecule of 21 nucleosides" means that 18, 19, 20, or 21 nucleosides have the specified property. When at least appears before a series of numbers or a range, it is understood that "at least" can modify each number in the series or range.

[0114] For example, as used herein, "not more than" or "less than" refers to the value adjacent to the phrase and the value or integer logically lower to zero, depending on the logic of the context. For example, a duplex having "not more than 2 nucleoside" overhangs has 2, 1, or 0 nucleoside overhangs. When "not more than" appears before a series of numbers or a range, it is understood that "not more than" can modify each number in the series or range.

[0115] The terminal region of a chain is the last five nucleotides at the 5' or 3' end.

[0116] Those skilled in the art can appropriately combine various embodiments of the present application.

[0117] Abasic nucleosides

[0118] In some embodiments, there is one, such as two, such as three, such as four or more abasic nucleosides in the nucleic acids according to the present application. Abasic nucleosides are modified nucleosides because they lack the base normally found at position 1 of the sugar moiety. Typically, there will be a hydrogen at position 1 of the sugar moiety of the abasic nucleosides present in the nucleic acids according to the present application.

[0119] The abasic nucleoside is located in the terminal region of the second strand, preferably within the terminal 5 nucleosides of the strand end. The terminal region may be the terminal 5 nucleosides, which include the abasic nucleoside.

[0120] As preferred features, the second chain may include (all such combinations are specifically contemplated unless mutually exclusive):

[0121] The terminal region of the second strand has 2 or more abasic nucleosides; and / or

[0122] The second strand has 2 or more abasic nucleosides in the 5' or 3' region; and / or

[0123] The second strand has two or more abasic nucleosides in the 5' or 3' terminal region, wherein the abasic nucleosides are present in an overhanging form as described herein; and / or

[0124] The terminal region of the second strand has two or more consecutive abasic nucleosides, wherein preferably one of such abasic nucleosides is the terminal nucleoside; and / or

[0125] There are two 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

[0126] In the terminal region of the second strand, at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside bond; and / or

[0127] In the 5' or 3' terminal region of the second strand, at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside bond; and / or

[0128] At the penultimate nucleoside position is an abasic nucleoside linked via an inverted linkage to a nucleoside other than the terminal nucleoside (referred to herein as the penultimate nucleoside); and / or

[0129] an abasic nucleoside which appears as two terminal nucleosides joined via a 5'-3' bond when the chain is read in the direction of the end containing the terminal nucleoside;

[0130] an abasic nucleoside which appears as two terminal nucleosides joined via a 3'-5' bond when the chain is read in the direction of the end containing the terminal nucleoside;

[0131] at the terminal 2 positions is an abasic nucleoside, wherein the penultimate nucleoside is linked to the penultimate nucleoside via the reverse bond, and wherein the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond;

[0132] At the terminal 2 positions are abasic nucleosides, wherein the penultimate nucleoside is linked to the penultimate nucleoside via the reverse bond, and wherein either

[0133] (1) the reverse bond is a 5-5' reverse bond, and when read toward the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 3'-5'; or

[0134] (2) The reverse bond is a 3-3' reverse bond, and when read toward the end including the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 5'-3'.

[0135] Preferably, there is an abasic nucleoside at the terminus of the second strand.

[0136] In the terminal region of the second strand, preferably at the terminal and penultimate position, there are preferably 2 or more abasic nucleosides.

[0137] Preferably, two or more abasic nucleosides are consecutive, for example, all abasic nucleosides can be consecutive. For example, the terminal 1, 2, 3, or terminal 4 nucleosides can be abasic nucleosides.

[0138] Abasic nucleosides may also be linked to adjacent nucleosides via 5'-3' phosphodiester bonds or reverse linkages, unless there is only one abasic nucleoside at the end, in which case it is reverse-linked to the adjacent nucleoside.

[0139] Inverted linkages (also referred to as inverted linkages, as also found in the art) include 5'-5', 3-'3', 3'-2', or 2'-3' phosphodiester bonds between adjacent sugar moieties of nucleosides.

[0140] Non-terminal abasic nucleosides will have two phosphodiester bonds, one to each adjacent nucleoside, and these bonds may be inverted, 5'-3' phosphodiester bonds, or one of each.

[0141] Preferred embodiments include comprising two abasic nucleosides at the terminal and penultimate positions of the second strand, and wherein the reversed internucleoside linkage is located between the penultimate (abasic) nucleoside and the penultimate nucleoside.

[0142] Preferably, there are two abasic nucleosides at the terminal and penultimate positions of the second strand, the penultimate nucleoside being 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 towards the end of the molecule).

[0143] 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 nucleotide is linked to an adjacent basic nucleoside via an inverted internucleoside bond.

[0144] Typically, the second strand comprises two consecutive abasic nucleosides in the 5'-terminal region of the second strand, 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 abasic nucleoside in the adjacent 5' proximal region by an inverted internucleoside bond; and (b) the inverted bond is a 5-5' inverted bond; and (c) when reading towards the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 3'-5'. More typically, (i) the first and second strands are each 23 nucleosides in length; and (ii) there are two phosphorothioate internucleoside bonds between three consecutive positions in the 5' proximal region of the second strand, wherein, in the region near the 5' end of the second strand, there is a first phosphorothioate internucleoside bond between the adjacent first basic nucleoside and the adjacent second basic nucleoside, and in the region near the 5' end of the second strand, there is a second phosphorothioate internucleoside bond between the adjacent second basic nucleoside and the adjacent third basic nucleoside. (iii) there are two phosphorothioate internucleoside bonds between three consecutive positions in the 5' and 3' terminal regions of the first strand, such that the terminal nucleoside of each of the 5' and 3' terminal regions of the first strand is linked to the respective 5' and 3' adjacent penultimate nucleosides via a phosphorothioate internucleoside bond, and the first penultimate nucleoside of each 5' and 3' is linked to the corresponding 5' and 3' adjacent penultimate nucleoside via a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is coupled directly or indirectly to one or more ligand moieties at its 3' terminal region.

[0145] Alternatively, the second strand comprises two consecutive abasic nucleosides, preferably pendant from the 3'-terminal region of the second strand, wherein one of such abasic nucleosides 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 in the adjacent 3' proximal region by an inverted internucleoside bond; and (b) the inverted bond is a 3-3' inverted bond; and (c) when reading towards the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and the penultimate basic nucleoside is 5'-3'. More typically, (i) the first and second strands are each 23 nucleosides in length; and (ii) there are two phosphorothioate internucleoside bonds between three consecutive positions in the region near the 3' end of the second strand, wherein, in the region near the 3' end of the second strand, there is a first phosphorothioate internucleoside bond between the adjacent first basic nucleoside and the adjacent second basic nucleoside in (a), and in the region near the 3' end of the second strand, there is a second phosphorothioate internucleoside bond between the adjacent second basic nucleoside and the adjacent third basic nucleoside. (iii) there are two phosphorothioate internucleoside bonds between three consecutive positions in the 5' and 3' terminal regions of the first strand, such that the terminal nucleoside of each of the 5' and 3' terminal regions of the first strand is linked to the second-to-last nucleoside of the respective 5' and 3' adjacent positions by a phosphorothioate internucleoside bond, and the first second-to-last nucleoside of each 5' and 3' adjacent position is linked to the third-to-last nucleoside of the respective 5' and 3' adjacent positions by a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is coupled directly or indirectly to one or more ligand moieties at its 5' terminal region.

[0146] Examples of structures are as follows (the specific RNA nucleoside shown is not limiting and can be any RNA nucleoside):

[0147] A 3'-3' reverse bond (also shows the 5'-3' orientation of the last phosphodiester bond between two abasic molecules read toward the ends of the molecule)

[0148]

[0149] B illustrates the reverse 5'-5' bond (and shows the 3'-5' orientation of the last phosphodiester bond between two abasic molecules read toward the ends of the molecule).

[0150]

[0151] The one or more abasic nucleosides existing in the nucleic acid provide in the case of having one or more reverse internucleosides key, i.e. 5'-5' or 3'-3' reverse internucleosides key. Reverse connection is owing to the orientation change of adjacent nucleoside sugar and occurs, so that sugar group has 3'-5' direction, rather than traditional 5'-3' direction (with reference to the numbering of nucleoside sugar ring atoms). As the one or more abasic nucleosides existing in the nucleic acid of the application preferably include this reverse nucleoside sugar.

[0152] In some embodiments, the terminal nucleoside of nucleic acid has a reverse orientation.If the terminal nucleoside has reverse orientation, then this will cause " reverse " terminal configuration of whole nucleic acid.Although some structure that this paper draws and quotes has used conventional 5 '-3 ' direction (with reference to the numbering of nucleoside sugar ring atom), it is understandable that there is the terminal nucleoside that a direction changes and a near-end 3 '-3 ' reverse bond, will cause nucleic acid to have overall 5 '-5 ' end structure (being that conventional 3 ' terminal nucleoside becomes 5 ' terminal nucleoside).Perhaps, it is understandable that there is the terminal nucleoside that a direction changes and a near-end 5 '-5 ' reverse bond, will cause nucleic acid to have overall 3 '-3 ' end structure.

[0153] Near-end 3'-3' as described herein or 5'-5' reverse bond can comprise the reverse bond that directly contiguous / is connected to the terminal nucleoside with reverse orientation, for example, has the single terminal nucleoside of reverse orientation.Perhaps, near-end 3'-3' as described herein or 5'-5' reverse bond can comprise the reverse bond adjacent to 2 or more nucleosides with reverse orientation, for example, 2 or more terminal region nucleosides with reverse orientation, for example, terminal and penultimate nucleoside.In this way, reverse bond can be connected on the penultimate nucleoside with reverse orientation.Although it will be appreciated by those skilled in the art that reverse orientation as described above can cause nucleic acid molecules to have for example integral body 3'-3' as described herein or 5'-5' end structure, it should also be appreciated that, in the case of having one or more extra reverse connections and / or nucleosides with reverse orientation, whole nucleic acid may have the 3'-5' end structure corresponding to the 5' / 3' end of conventional positioning.

[0154] 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.

[0155] Thus, those skilled in the art will clearly understand that 5'-5', 3'-3' and 3'-5' (reading in this terminal direction) terminal variants of the more conventional 5'-3' structure (numbered with reference to the ring atoms on the terminal nucleoside sugar) described herein are included within the scope of this application, wherein one or more reversed bonds are present.

[0156] For example, between reverse nucleosides, bond and / or one or more nucleosides with reverse orientation produce reverse end, and when the relative position of connection (for example with joint) or the position of internal feature (nucleosides of for example modification) with respect to 5 ' or 3 ' end definition of nucleic acid, then described 5 ' or 3 ' end refer to conventional 5 ' or 3 ' end when not having reverse connection, and wherein said conventional 5 ' or 3 ' end is determined by considering the directionality and / or nucleoside orientation of most internal nucleoside bonds in nucleic acid.Can judge from the direction of these internal bonds and / or nucleosides, when not having reverse connection, which ends of nucleic acid will constitute conventional 5 ' and 3 ' end (with reference to the ring atom numbering on the terminal nucleoside sugar) of molecule.

[0157] In some embodiments, the second (sense) strand of the nucleic acid according to the present application comprises two consecutive abasic nucleosides in the 5' end region, as shown in the following 5' end motif:

[0158]

[0159] in:

[0160] B represents nucleoside base,

[0161] T represents H, OH or 2' ribose modification,

[0162] Z represents the remaining nucleosides of the second strand.

[0163] In some embodiments, the second (sense) strand of the nucleic acid according to the present application comprises two consecutive abasic nucleosides in the 5' end region, as shown in the following 5' end motif:

[0164]

[0165] in:

[0166] B represents nucleoside base,

[0167] T represents H, OH or 2' ribose modification (preferably 2' ribose modification, more preferably 2'Me or 2'F ribose modification),

[0168] V represents O or S (preferably O),

[0169] R stands for H or C 1-4 Alkyl (preferably H),

[0170] Z represents the remaining nucleosides of the second chain,

[0171] More preferably, the following 5' end motif

[0172]

[0173] in:

[0174] B stands for nucleobase

[0175] T represents a 2' ribose modification (preferably a 2'Me or 2'F ribose modification),

[0176] Z represents the remaining nucleosides of the second strand.

[0177] 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.

[0178] GalNAc-siRNA constructs having 5'-GalNAc on the sense strand can have an inverted linkage at the other end of the sense strand.

[0179] The GalNAc-siRNA construct having 3'-GalNAc on the sense strand may have an inverted linkage at the other end of the sense strand.

[0180] In a preferred embodiment, the second (sense) strand of the nucleic acid according to the present application comprises two consecutive abasic nucleosides in the 5' end region, as shown in the following 5' end motif:

[0181]

[0182] in:

[0183] B represents nucleoside base,

[0184] T represents H, OH or 2' ribose modification (preferably 2' ribose modification, more preferably 2'Me or 2'F ribose modification),

[0185] V represents O or S (preferably O),

[0186] R stands for H or C 1-4 Alkyl (preferably H),

[0187] Z comprises 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, and more preferably 19 consecutive nucleosides,

[0188] More preferably, the following 5' end motif

[0189]

[0190] in:

[0191] B represents nucleoside base,

[0192] T represents a 2' ribose modification (preferably a 2'Me or 2'F ribose modification),

[0193] Z comprises 19 consecutive nucleosides.

[0194] Nucleic acid length

[0195] In one aspect, i) the length of the first strand of the nucleic acid is in the range of 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 in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0196] Typically, the double-stranded region of the nucleic acid is between 17 and 30 nucleotides in length, more preferably 19 or 21 nucleotides in length. Likewise, the complementary region between the first strand and the portion of the RNA transcribed from the B4GALT1 gene is between 17 and 30 nucleotides in length.

[0197] Nucleic acid modification

[0198] In some embodiments, the nucleic acids described herein, e.g., RNA, e.g., dsiRNA, do not comprise further modifications, such as chemical modifications or conjugations known in the art and described herein.

[0199] In other preferred embodiments, the nucleic acids described herein, such as RNA, such as dsiRNA, are further chemically modified to enhance stability or other beneficial properties.

[0200] In some embodiments of the present application, substantially all of the nucleosides are modified.

[0201] The nucleic acids herein can be synthesized or modified by methods well known in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, STR et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference.

[0202] Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, coupling, reverse linkage) or 3'-terminal modifications (coupling, DNA nucleosides within RNA or RNA nucleosides within DNA, reverse linkage, etc.); base modifications, such as substitution with stable bases, unstable bases, or bases that can pair with more partners, coupled bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of sugar groups; or backbone modifications, including modification or replacement of phosphodiester bonds.

[0203] Specific nucleic acid examples, such as siRNA compounds useful in the embodiments described herein, include but are not limited to RNAs containing modified backbones or without natural internucleoside bonds. Nucleic acids with modified backbones, such as RNA, include those without phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes cited in the art, modified nucleic acids, such as RNAs without phosphorus atoms in their internucleoside backbones, may also be considered oligonucleosides. In some embodiments, modified nucleic acids (e.g., siRNAs) have phosphorus atoms in their internucleoside backbones.

[0204] Modified nucleic acids, such as 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, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters and boronates with normal 3'-5' linkages, 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 5'-3' or 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0205] Modified nucleic acids, such as RNA, can also contain one or more substituted sugar moieties. Nucleic acids described herein, such as siRNAs, such as dsiRNAs, can 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 the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted. 2'-O-methyl and 2'-F are preferred modifications.

[0206] In some preferred embodiments, the nucleic acid comprises at least one modified nucleoside.

[0207] The nucleic acid of the present application may comprise one or more modified nucleosides on the first strand and / or the second strand.

[0208] In some embodiments, substantially all nucleosides of the sense strand and all nucleosides of the antisense strand comprise modifications.

[0209] In some embodiments, all nucleosides of the sense strand and substantially all nucleosides of the antisense strand comprise modifications.

[0210] In some embodiments, all nucleosides of the sense strand and all nucleosides of the antisense strand comprise modifications.

[0211] 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 constrained nucleosides, constrained ethyl nucleosides, abasic nucleosides, 2'-amino-modified nucleosides, 2'-O-allyl modified nucleosides, 2'-O-alkyl-modified nucleosides, 2'-hydroxyl-modified nucleosides, 2'-methoxyethyl-modified nucleosides, 2'-O-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates, non-natural bases comprising nucleosides, tetrahydropyran modified nucleosides, 1,5-anhydrohexitol modified nucleosides, cyclohexenyl modified nucleosides, nucleosides comprising thiophosphate groups, nucleosides comprising methylphosphonate groups, nucleosides comprising 5'-phosphates, and nucleosides comprising 5'-phosphate mimetics. In another embodiment, the modified nucleosides comprise a short sequence of 3'-terminal deoxythymidine (dT).

[0212] The modification of the nucleoside may preferably be selected from the group consisting of, 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 a 2'-O-methyl ("2'-Me") or 2'-fluoro modification.

[0213] One preferred modification is modification of the 2'-OH group of the ribose sugar group, optionally selected from 2'-Me or 2'-F modifications.

[0214] Preferred nucleic acids comprise one or more nucleosides on the first strand and / or the second strand that are modified to form modified nucleosides as follows:

[0215] A nucleic acid, wherein the modification is a modification on the 2'-OH group of the ribose sugar moiety, optionally selected from a 2'-Me or 2'-F modification.

[0216] A nucleic acid wherein the first strand comprises a 2'-F modification at position 2, position 6, position 14, or any combination thereof, counting from position 1 of the first strand.

[0217] A nucleic acid wherein the second strand comprises a 2'-F modification at position 7, position 9, position 11, or any combination thereof, counting from position 1 of the second strand.

[0218] A nucleic acid, wherein the first strand and the second strand each comprise a 2'-Me and a 2'-F modification.

[0219] A nucleic acid comprising at least one thermally labile modification, suitably located at one or more positions 1 to 9 of the first strand, counted starting from position 1 of the first strand, and / or located at one or more positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the labile modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid, more preferably (S)-glycol nucleic acid.

[0220] A nucleic acid, counting from position 1 of the first strand, comprises at least one thermolabile modification at position 7 of the first strand.

[0221] A nucleic acid, which is an siRNA oligonucleotide, counting from position 1 of the second strand, wherein the siRNA oligonucleotide comprises three or more 2'-F modifications at positions 6 to 12 of the second strand, for example, 4, 5, 6 or 7 2'-F modifications at positions 6 to 12 of the second strand.

[0222] A nucleic acid which is a siRNA oligonucleotide, counting from position 1 of the second strand, wherein the second strand comprises at least 3, such as 4, 5 or 6, 2'-Me modifications at positions 1-6 of the second strand.

[0223] A nucleic acid which is an siRNA oligonucleotide, wherein the first strand comprises at least five 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 from the terminal nucleoside in the 3' terminal region.

[0224] A nucleic acid which is a siRNA oligonucleotide, wherein the first strand comprises at least 7 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region.

[0225] A nucleic acid which is an 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 to: (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 to: (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 in a blunt-ended oligonucleoside, wherein each of the first and second strands is 19 nucleosides in length.

[0226] 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 joined to the adjacent nucleoside (at position 2) by an internal 3' to 5' bond, referenced to the bonds between the backbone sugar moieties, and read in a direction away from the end of the molecule.

[0227] Thus, it can be seen that "position 1 of the sense strand" is the nucleoside most 5' to the conventional 5' terminus of the sense strand (excluding abasic nucleosides). Typically, the nucleoside at position 1 of the sense strand will be identical to the 5' nucleoside of the selected target nucleic acid sequence. More generally, starting from position 1 of the sense strand, the nucleosides of the sense strand will be identical to those of the target nucleic acid sequence, while also allowing for acceptable mismatches between sequences.

[0228] For example, as used herein, "position 1 of the antisense strand" is the nucleoside closest to the 5' end of the conventional 5' terminus of the antisense strand (excluding abasic nucleosides). As described above, there will be a region of complementarity between the sense and antisense strands, and thus the antisense strand will also have a region complementary to the target nucleic acid sequence described above.

[0229] In some embodiments, the nucleic acid, such as an siRNA agent, further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage. For example, the phosphorothioate or methylphosphonate internucleoside linkage can be located at the 3'-end or at the terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands, the sense strand and the antisense strand.

[0230] In some embodiments, the phosphorothioate or methylphosphonate internucleoside linkage is located at the 5' end or terminal region of one strand (ie, the sense strand or the antisense strand); or at the termini of both strands, the sense strand and the antisense strand.

[0231] In some embodiments, the phosphorothioate or methylphosphonate internucleoside linkages are located at the 5' and 3'-ends, or at the terminal region of one strand (ie, the sense strand or the antisense strand); or at the termini of both strands, the sense strand and the antisense strand.

[0232] Any nucleic acid can comprise one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside linkages at a chain terminus.

[0233] At least one oligoribonucleoside chain preferably comprises at least two consecutive phosphorothioate modifications within the last three nucleosides of the oligonucleoside.

[0234] Therefore, the present application also relates to: a nucleic acid disclosed herein, which comprises a phosphorothioate internucleoside bond between at least two or three consecutive positions, for example in the 5' and / or 3' terminal region and / or the proximal terminal region of the second chain, wherein the proximal terminal region is preferably adjacent to the terminal region, and in the second chain, the one or more abasic nucleosides are located in the terminal region.

[0235] The present application discloses a nucleic acid comprising phosphorothioate internucleoside bonds between at least two or three consecutive positions in the 5' and / or 3' end region of the first chain, wherein preferably, the terminal position of the 5' and / or 3' end region of the first chain is connected to its adjacent position via a phosphorothioate internucleoside bond.

[0236] The nucleic acid chain can be RNA comprising phosphorothioate internucleoside linkages between three nucleosides adjacent to two terminal abasic nucleosides.

[0237] 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 moiety comprising one or more GalNAc ligand moieties at the opposite 3' end of the second strand. More preferably, reading from position 1 of the second strand, the same nucleic acid may further comprise a phosphorothioate bond between nucleotides at positions 3-4 and 4-5 of the second strand. More preferably, the same nucleic acid may further comprise a 2'F modification at positions 7, 9, and 11 of the second strand.

[0238] Preferably modified as follows:

[0239] A nucleic acid wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any of the following:

[0240] Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0241] Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0242] Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0243] Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0244] Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me.

[0245] A nucleic acid wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any of the following:

[0246] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0247] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0248] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0249] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0250] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0251] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or

[0252] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0253] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0254] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0255] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me.

[0256] where (s) is a phosphorothioate internucleoside bond.

[0257] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0258] ia-ia-Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0259] ia-ia-Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0260] ia-ia-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0261] ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0262] ia-ia-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0263] Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, or

[0264] Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or

[0265] Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or

[0266] Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or

[0267] Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia,

[0268] Among them, ia-ia indicates the opposite direction without the red base nucleus, so ia-ia indicates the opposite direction without the red base nucleus at the second position.

[0269] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0270] ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0271] ia-ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0272] 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, or

[0273] 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, or

[0274] 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, or

[0275] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, or

[0276] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or

[0277] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or

[0278] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or

[0279] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia,

[0280] in:

[0281] (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is located at the 3' end of the second chain, the inverted abasic nucleoside exists in a pendant form of two nucleosides.

[0282] A nucleic acid wherein the modified nucleosides comprise any of the following modification patterns:

[0283] 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;

[0284] 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;

[0285] Or modification 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;

[0286] 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, 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;

[0287] 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;

[0288] 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.

[0289] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0290] 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;

[0291] Or modification pattern 2: Second strand (5'-3'): Me(s)Me(s)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;

[0292] 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;

[0293] 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;

[0294] 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;

[0295] 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;

[0296] Where (s) is a phosphorothioate internucleoside bond.

[0297] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0298] 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;

[0299] 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;

[0300] 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;

[0301] 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;

[0302] 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;

[0303] 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;

[0304] Where (s) is a phosphorothioate internucleoside bond.

[0305] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0306] 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;

[0307] 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;

[0308] 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;

[0309] 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;

[0310] Or modification pattern 5: 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, 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;

[0311] Or modification pattern 6: Second strand (5'-3'): ia-ia-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;

[0312] Where ia represents an inverted abasic nucleoside.

[0313] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0314] 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-Me-Me-ia-ia, 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;

[0315] 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-Me-Me-ia-ia, 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;

[0316] 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-ia-ia, 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;

[0317] 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-ia-ia, 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;

[0318] 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;

[0319] 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-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me;

[0320] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is located at the 3' end of the second strand, the inverted abasic nucleoside is present as a 2-nucleoside overhang.

[0321] A nucleic acid wherein the modified nucleosides comprise any of the following modification patterns:

[0322] 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;

[0323] Or modification 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;

[0324] Modification model 3: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-M e, 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;

[0325] Modification model 4: Second pin (5'-3'): ia-ia-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;

[0326] Modification model 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-M e, 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;

[0327] 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;

[0328] Among them:

[0329] (s) This is the sulfur substitute for the acid, the ia is the opposite.

[0330] One type of nucleic acid, including the modified nucleic acid, and the following modified model:

[0331] 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-ia-ia, 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;

[0332] 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-ia-ia, 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;

[0333] 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-ia-ia, 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;

[0334] 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-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;

[0335] 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-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;

[0336] 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-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;

[0337] wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is located at the 3' end of the second strand, the inverted abasic nucleoside is present as a 2-nucleoside overhang.

[0338] Particularly preferred is a nucleic acid wherein the modified nucleosides comprise any of the following modification patterns:

[0339] Modification pattern 5: Second strand (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 , 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;

[0340] Wherein: (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside.

[0341] 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 4 or 6 2'F modifications.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0346] Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3

[0347] wherein X2, X3, and X4 are selected from 2'Me and 2'F sugar modifications, with the proviso that for X2, X3, and X4, at least one is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications.

[0348] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0349] Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3

[0350] wherein X2 is a 2'F sugar modification, and X3 and X4 are 2'Me sugar modifications.

[0351] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0352] Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3

[0353] wherein X3 is a 2'F sugar modification, and X2 and X4 are 2'Me sugar modifications.

[0354] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0355] Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3

[0356] wherein X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications.

[0357] 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 7 2'F modifications.

[0358] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0359] Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3;

[0360] wherein X2, X3, and X4 are selected from 2'Me and 2'F sugar modifications, provided that for X2, X3, and X4, at least one is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications.

[0361] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0362] Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3;

[0363] wherein X2 is a 2'F sugar modification, and X3 and X4 are 2'Me sugar modifications.

[0364] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0365] Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3;

[0366] wherein X3 is a 2'F sugar modification, and X2 and X4 are 2'Me sugar modifications.

[0367] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0368] Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3;

[0369] wherein X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications.

[0370] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0371] Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7;

[0372] Wherein X1 is a thermally unstable modification.

[0373] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0374] Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7;

[0375] Wherein X1 is a thermally unstable modification.

[0376] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0377] (Me)8-(F)3-(Me) 10 .

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

[0379] (Me)8-(F)3-(Me) 10 ,as well as

[0380] 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 4 or 6 2'F modifications.

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

[0382] (Me)8-(F)3-(Me) 10 ,as well as

[0383] 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, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0384] A nucleic acid comprising a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a double-stranded 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'):

[0385] (Me)8-(F)3-(Me) 10 ,as well as

[0386] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0387] Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, where X1 is a thermally labile modification.

[0388] 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 and second strands form a double-stranded 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'):

[0389] (Me)8-(F)3-(Me)10 ,as well as

[0390] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0391] (Me-F)3-(Me)7-F-Me-F-(Me)7.

[0392] 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 and second strands form a double-stranded 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'):

[0393] (Me)8-(F)3-(Me) 10 ,as well as

[0394] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0395] Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5.

[0396] 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 and second strands form a double-stranded 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'):

[0397] (Me)8-(F)3-(Me) 10 ,as well as

[0398] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0399] Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3.

[0400] 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 and second strands form a double-stranded 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'):

[0401] (Me)8-(F)3-(Me) 10 ,as well as

[0402] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0403] Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, where X1 is a thermally labile modification.

[0404] 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 and second strands form a double-stranded 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'):

[0405] (Me)8-(F)3-(Me) 10 ,as well as

[0406] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0407] (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6.

[0408] 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 and second strands form a double-stranded 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'):

[0409] (Me)8-(F)3-(Me) 10 ,as well as

[0410] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0411] Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5.

[0412] 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 and second strands form a double-stranded 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'):

[0413] (Me)8-(F)3-(Me) 10 ,as well as

[0414] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0415] Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3.

[0416] A nucleic acid wherein the second strand comprises the following 2' sugar and abasic modification pattern (5'-3'):

[0417] ia-ia-(Me)8-(F)3-(Me) 10

[0418] Where ia represents an inverted abasic nucleoside.

[0419] A nucleic acid wherein the second strand comprises the following 2' sugar and abasic modification pattern (5'-3'):

[0420] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0421] 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 4 or 6 2'F modifications.

[0422] A nucleic acid wherein the second strand comprises the following 2' sugar and abasic modification pattern (5'-3'):

[0423] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0424] 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, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0425] 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 and second strands form a double-stranded 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'):

[0426] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0427] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0428] Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, where X1 is a thermally labile modification.

[0429] 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 and second strands form a double-stranded 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'):

[0430] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0431] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0432] (Me-F)3-(Me)7-F-Me-F-(Me)7.

[0433] 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 and second strands form a double-stranded 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'):

[0434] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0435] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0436] Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5.

[0437] 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 and second strands form a double-stranded 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'):

[0438] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0439] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

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

[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 and second strands form a double-stranded 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

[0443] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0444] Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7,

[0445] Wherein X1 is a thermally unstable modification.

[0446] 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 and second strands form a double-stranded 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'):

[0447] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0448] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0449] (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6.

[0450] 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 and second strands form a double-stranded 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'):

[0451] ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0452] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0453] Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5.

[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 and second strands form a double-stranded 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

[0456] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0457] Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3.

[0458] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0459] ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 ,

[0460] wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage.

[0461] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0462] 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

[0463] 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 4 or 6 2'F modifications.

[0464] A nucleic acid, wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0465] 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

[0466] 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.

[0467] 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 and second strands form a double-stranded 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'):

[0468] 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

[0469] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0470] Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me, wherein X1 is a thermally labile modification.

[0471] 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 and second strands form a double-stranded 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'):

[0472] 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

[0473] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0474] Me(s)F(s)Me-F-Me-F-(Me)7-F-Me-F-(Me)5(s)Me(s)Me.

[0475] 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 and second strands form a double-stranded 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'):

[0476] 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

[0477] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0478] Me(s)F(s)(Me)3-F-(Me)7-(F-Me)2-F-(Me)3(s)Me(s)Me.

[0479] 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 and second strands form a double-stranded 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'):

[0480] 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

[0481] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0482] Me(s)F(s)(Me)3-F-(Me)7-F-Me-F-(Me)3-F-Me(s)Me(s)Me.

[0483] 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 and second strands form a double-stranded 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'):

[0484] 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

[0485] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0486] Me(s)F(s)(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)5(s)Me(s)Me, wherein X1 is a thermally labile modification.

[0487] 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 and second strands form a double-stranded 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'):

[0488] 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

[0489] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0490] Me(s)F(s)Me-F-Me-F-Me-(F)2-(Me)4-(F-Me)2-(Me)4(s)Me(s)Me.

[0491] 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 and second strands form a double-stranded 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'):

[0492] 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

[0493] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0494] Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)3(s)Me(s)Me.

[0495] 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 and second strands form a double-stranded 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'):

[0496] 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

[0497] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0498] Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-Me(s)Me(s)Me.

[0499] Preferred modifications are as follows:

[0500] Modification mode 1:

[0501] Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[0502] First strand (5'-3'): Me-F-Me-Me-Me-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a thermally unstable modification;

[0503] Or modified mode 2:

[0504] Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[0505] 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;

[0506] Or modifier mode 3:

[0507] 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,

[0508] First 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;

[0509] Modification model 4:

[0510] 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,

[0511] 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;

[0512] Modification model 5:

[0513] 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,

[0514] 1st pin (5'-3'): Me-F-Me-Me-Me-X1-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me, of which X1 is heat failure fixing;

[0515] Modification model 6:

[0516] 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,

[0517] 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;

[0518] Modification model 7:

[0519] 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,

[0520] First strand (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;

[0521] Or modification pattern 8:

[0522] 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,

[0523] First strand (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

[0524] Where ia represents an inverted abasic nucleoside.

[0525] More preferably, the modification is as follows:

[0526] Modification pattern 1:

[0527] 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,

[0528] First strand (5’-3’): Me(s)F(s)Me–Me–Me–X1–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, where X1 is a thermally labile modification;

[0529] Or modification pattern 2:

[0530] 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,

[0531] 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;

[0532] Modification model 3:

[0533] 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,

[0534] 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;

[0535] Modification model 4:

[0536] 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,

[0537] 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;

[0538] Modification model 5:

[0539] 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,

[0540] 1st pin (5'-3'): Me(s)F(s)Me–Me-Me–X1–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, in which X1 is heat failure fixation;

[0541] Modification model 6:

[0542] 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,

[0543] 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;

[0544] Or modifier mode 7:

[0545] 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,

[0546] 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;

[0547] Or modifier mode 8:

[0548] 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,

[0549] 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;

[0550] wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside.

[0551] Coupling

[0552] Another modification of the nucleic acids (e.g., RNA, e.g., siRNA) of the present application involves linking the nucleic acid (e.g., siRNA) to one or more ligand moieties, e.g., to enhance the activity, cellular distribution, or cellular uptake of the nucleic acid (e.g., siRNA), e.g., entry into cells.

[0553] In some embodiments, the ligand moiety can be attached to a nucleic acid (e.g., an 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, such as covalently connecting two parts of a compound.

[0554] The ligand can be linked to the 3' or 5' end of the sense strand.

[0555] The ligand is preferably coupled to the 3' end of the sense strand of a nucleic acid, such as an siRNA agent.

[0556] Therefore, in another aspect, the present application relates to a conjugate for inhibiting the expression of a target gene in a cell, wherein the conjugate comprises a nucleic acid portion and one or more ligand portions, wherein the nucleic acid portion comprises the nucleic acid disclosed herein.

[0557] In one aspect, the second strand of the nucleic acid is coupled directly or indirectly (eg, via a linker) to one or more ligand moieties, wherein the ligand moiety is typically located at the terminal region of the second strand, preferably at its 3' terminal region.

[0558] In some embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative attached to a nucleic acid (eg, dsiRNA) via a linker.

[0559] Therefore, the present application relates to a conjugate, wherein the ligand portion comprises

[0560] i) one or more GalNAc ligands; and / or

[0561] ii) one or more GalNAc ligand derivatives; and / or

[0562] iii) one or more GalNAc ligands coupled to the nucleic acid via a linker.

[0563] The GalNAc ligand can be coupled directly or indirectly to the 5' or 3' region of the sense strand of the nucleic acid, preferably to the 3' region thereof.

[0564] GalNAc ligands are well known in the art and are described inter alia in EP 3 775 207 A1.

[0565] In some embodiments, the GalNAc ligand is contained in Figures 1 to 4 or Figure 5 In any of the linkers shown in (Formula XI), the "oligonucleotide" may be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may contain other bonds in addition to 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 coupled to the second strand, more preferably to the 3' terminal region of the second strand, via a phosphodiester bond.

[0566] In some embodiments, the GalNAc ligand is contained in Figure 3In the linker shown, the "oligonucleotide" can be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may contain other bonds in addition to 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 coupled to the second strand, more preferably to the 3' end region of the second strand, via a phosphodiester bond.

[0567] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), the "oligonucleotide" can be any nucleic acid disclosed herein. Therefore, the "oligonucleotide" may contain other bonds in addition to 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 coupled 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 Figures 1 to 4 or Figure 5 In any one of the linkers shown in (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified or unmodified second chain, the second chain comprising or consisting of SEQ ID NO: 302 or SEQ ID NO: 305, preferably, wherein the linker is coupled to the 3' terminal region of the second chain, i.e., the 3' terminal region of SEQ ID NO: 302 or SEQ ID NO: 305, via a phosphodiester bond.

[0569] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified or unmodified second chain, the second chain comprising or consisting of SEQ ID NO: 302 or SEQ ID NO: 305, preferably, wherein the linker is coupled to the 3' end region of the second chain, i.e., the 3' end region of SEQ ID NO: 302 or SEQ ID NO: 305, via a phosphodiester bond.

[0570] In some embodiments, the GalNAc ligand is contained in Figure 5In the linker shown in (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified or unmodified second chain, the second chain comprising or consisting of SEQ ID NO: 302 or SEQ ID NO: 305, preferably, wherein the linker is coupled to the 3'-terminal region of the second chain, i.e., the 3'-terminal region of SEQ ID NO: 302 or SEQ ID NO: 305, via a phosphodiester bond.

[0571] 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 the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified second chain, the second chain comprising or consisting of SEQ ID NO: 611 or SEQ ID NO: 613, preferably, wherein the linker is coupled to the 3' terminal region of the second chain, i.e., the 3' terminal region of SEQ ID NO: 611 or SEQ ID NO: 613, via a phosphodiester bond.

[0572] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified second chain, the second chain comprising or consisting of SEQ ID NO: 611 or SEQ ID NO: 613, preferably, wherein the linker is coupled to the 3' end region of the second chain, i.e., the 3' end region of SEQ ID NO: 611 or SEQ ID NO: 613, via a phosphodiester bond.

[0573] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified second chain, the second chain comprising or consisting of SEQ ID NO: 611 or SEQ ID NO: 613, preferably, wherein the linker is coupled to the 3'-terminal region of the second chain, i.e., the 3'-terminal region of SEQ ID NO: 611 or SEQ ID NO: 613, via a phosphodiester bond.

[0574] 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 the "oligonucleotide" represents the nucleic acid described in 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 comprising or consisting of SEQ ID NO: 503, and the second chain comprising or consisting of SEQ ID NO: 611, preferably, wherein the linker is coupled to the 3' terminal region of the second chain, i.e., the 3' terminal region of SEQ ID NO: 611, via a phosphodiester bond.

[0575] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, the "oligonucleotide" represents the nucleic acid described in 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 comprising or consisting of SEQ ID NO: 503, and the second chain comprising or consisting of SEQ ID NO: 611, preferably, wherein the linker is coupled to the 3' end region of the second chain, i.e., the 3' end region of SEQ ID NO: 611, via a phosphodiester bond.

[0576] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in 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 or consists of SEQ ID NO: 503, and the second chain comprises or consists of SEQ ID NO: 611, preferably, wherein the linker is coupled to the 3' end region of the second chain, i.e., the 3' end region of SEQ ID NO: 611, via a phosphodiester bond.

[0577] 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 the "oligonucleotide" represents the nucleic acid described in 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 comprising or consisting of SEQ ID NO: 505, and the second chain comprising or consisting of SEQ ID NO: 613, preferably, wherein the linker is coupled to the 3' terminal region of the second chain, i.e., the 3' terminal region of SEQ ID NO: 613, via a phosphodiester bond.

[0578] In some embodiments, the GalNAc ligand is contained in Figure 3In the linker shown, the "oligonucleotide" represents the nucleic acid described in 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 comprising or consisting of SEQ ID NO: 505, and the second chain comprising or consisting of SEQ ID NO: 613, preferably, wherein the linker is coupled to the 3' end region of the second chain, i.e., the 3' end region of SEQ ID NO: 613, via a phosphodiester bond.

[0579] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker shown in (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in 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 or consists of SEQ ID NO: 505, and the second chain comprises or consists of SEQ ID NO: 613, preferably, wherein the linker is coupled to the 3' end region of the second chain, i.e., the 3' end region of SEQ ID NO: 613, via a phosphodiester bond.

[0580] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, the first strand comprising or consisting of SEQ ID NO: 503, the second strand comprising or consisting of SEQ ID NO: 611, wherein the second strand has the following structure

[0581]

[0582] in:

[0583] T represents 2'Me ribose modification,

[0584] B represents the nucleoside bases of the first two nucleosides in the 5' end region of SEQ ID NO: 611, and

[0585] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO: 611.

[0586] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker represented by (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, the first strand comprising or consisting of SEQ ID NO: 503, the second strand comprising or consisting of SEQ ID NO: 611, wherein the second strand has the following structure

[0587]

[0588] in:

[0589] T represents 2'Me ribose modification,

[0590] B represents the nucleoside bases of the first two nucleosides in the 5' end region of SEQ ID NO: 611, and

[0591] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO: 611.

[0592] In some embodiments, the GalNAc ligand is contained in Figure 3 In the linker shown, the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, the first strand comprising or consisting of SEQ ID NO: 505, the second strand comprising or consisting of SEQ ID NO: 613, wherein the second strand has the following structure

[0593]

[0594] in:

[0595] T represents 2'Me ribose modification,

[0596] B represents the nucleobases of the first two nucleosides in the 5' end region of SEQ ID NO: 613, and

[0597] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO: 613.

[0598] In some embodiments, the GalNAc ligand is contained in Figure 5 In the linker represented by (Formula XI), wherein the "oligonucleotide" represents the nucleic acid described in the present application, wherein the nucleic acid according to the present application comprises a modified first strand and a modified second strand, the first strand comprising or consisting of SEQ ID NO: 505, the second strand comprising or consisting of SEQ ID NO: 613, wherein the second strand has the following structure

[0599]

[0600] in:

[0601] T represents 2'Me ribose modification,

[0602] B represents the nucleobases of the first two nucleosides in the 5' end region of SEQ ID NO: 613, and

[0603] Z represents the remaining 19 consecutive base nucleosides of SEQ ID NO: 613.

[0604] Vectors and cells

[0605] In one aspect, the present application provides a cell containing a nucleic acid, such as an inhibitory RNA [RNAi] as described herein.

[0606] In one aspect, the present application provides a cell comprising the vector described herein.

[0607] Pharmaceutically acceptable compositions

[0608] In one aspect, the present application provides a pharmaceutical composition for inhibiting target gene expression, the composition comprising the nucleic acid disclosed herein.

[0609] Pharmaceutically acceptable compositions may comprise excipients and / or carriers.

[0610] 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, ethylcellulose, and cellulose acetate; (4) 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) 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 formulations.

[0611] 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, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium carboxymethyl starch, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0612] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not adversely react with nucleic acids can also be used to formulate the compositions of the present application. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

[0613] Preparations for topical administration of nucleic acid can include aseptic and non-sterile aqueous solutions, common solvents such as non-aqueous solutions in alcohol, or solutions of nucleic acid in liquid or solid oil bases. The solution can also contain buffer, diluent and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients can be used, which are suitable for non-parenteral administration and which do not have a deleterious reaction with the nucleic acid used.

[0614] In one embodiment, the nucleic acid or composition is administered in a buffer-free solution. In certain embodiments, the buffer-free solution is saline or water. In other embodiments, the nucleic acid (e.g., siRNA reagent) is administered in a buffered solution. In these embodiments, the buffered solution can include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. For example, the buffered solution can be phosphate buffered saline (PBS).

[0615] dose

[0616] The pharmaceutical composition of the present application can be administered with a dosage sufficient to inhibit gene expression. Typically, the suitable dosage of the nucleic acid (e.g., siRNA) of the present application will be in the range of approximately 0.001 to approximately 200.0 milligrams per kilogram of recipient body weight per day, typically in the range of approximately 1 to 50 milligrams per kilogram of body weight per day. Typically, the suitable dosage of the nucleic acid (e.g., siRNA of the present application) will be in the range of approximately 0.1 mg / kg to approximately 5.0 mg / kg, for example, approximately 0.3 mg / kg and approximately 3.0 mg / kg.

[0617] Repeated dosage regimens can include administering a therapeutic amount of nucleic acid, such as siRNA, regularly, such as every other day or yearly. In some embodiments, the nucleic acid (e.g., siRNA) is administered about once a month to about once a quarter (i.e., administered about every three months).

[0618] In some embodiments, the nucleic acid (e.g., siRNA) agent is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered at a dosage of about 10 mg / kg to about 30 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered at a dosage selected from about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered once weekly, monthly, every two months, or quarterly (i.e., once every three months) at a dosage of about 0.1 mg / kg to about 5.0 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered to a subject once a week. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered to a subject once a month. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered to a subject once quarterly (i.e., once every three months).

[0619] After the initial treatment regimen, the frequency of treatment can be reduced. For example, after three months of weekly or biweekly dosing, the dosing can be repeated monthly for six months or a year, or longer.

[0620] The pharmaceutical composition can be administered once a day, or in one day with appropriate time intervals with twice, three or more divided doses, or even can be carried out continuous infusion or administration by controlled release formulations. In this case, in order to reach the daily total dose, the nucleic acid (for example siRNA) comprising in each divided dose must correspondingly reduce. Dosage unit also can be configured to send in a few days, for example, using conventional sustained release formulations, which provide the sustained release of nucleic acid (for example siRNA) within a few days. Sustained release formulations are well-known in the art, and are particularly suitable for delivering reagents at specific locations, for example, can be used together with the reagent of the present application. In this embodiment, dosage unit comprises the corresponding multiple of daily dose.

[0621] In other embodiments, the pharmaceutical composition of a single dose can have a long-lasting effect so that subsequent doses are administered at intervals no more than 3, 4 or 5 days, or at intervals 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 administered once a week. In other embodiments of the application, the pharmaceutical composition of the application of a single dose is administered once every two months. In some embodiments, the siRNA is administered once per month to approximately once per quarter (i.e., approximately once every three months), or even administered once every 6 months or 12 months.

[0622] As is known in the art, estimates of effective dosages and in vivo half-lives of individual nucleic acids encompassed by the present application (eg, siRNAs) can be made using conventional methods or based on in vivo testing using appropriate animal models.

[0623] The pharmaceutical composition of the present application can be administered in a variety of ways, depending on whether local or systemic treatment is needed and depending on the area to be treated. Administration can be local (for example, by transdermal patch), pulmonary, for example, by inhalation or blowing into powder or aerosol, including by 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 administration, for example, by intracerebral parenchyma, intrathecal or intraventricular administration. In some preferred embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by subcutaneous injection.

[0624] In one embodiment, the nucleic acid (eg, agent) is administered subcutaneously to the subject.

[0625] Nucleic acids, such as siRNA, can be delivered in a manner that targets specific tissues, such as liver cells in particular.

[0626] Methods for inhibiting B4GALT1 gene expression

[0627] The present application also provides a method for inhibiting the expression of the B4GALT1 gene in a cell. The method comprises contacting the cell with a nucleic acid of the present application (e.g., an siRNA reagent, such as a double-stranded siRNA) in an amount sufficient to effectively inhibit the expression of the B4GALT1 gene in the cell, thereby inhibiting the expression of the B4GALT1 gene in the cell. It should be noted that the nucleic acid "for inhibiting the expression of B4GALT1" is a nucleic acid capable of inhibiting the expression of B4GALT1, preferably as described below.

[0628] The contact of cells with nucleic acids (e.g., siRNA, e.g., double-stranded siRNA reagents) can be carried out in vitro or in vivo. Contacting cells with nucleic acids in vivo includes contacting cells or cell groups in a subject (e.g., a human subject) with nucleic acids (e.g., siRNA). A combination of in vitro and in vivo methods of cell contact is also possible. 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 directs the siRNA reagent to the target site.

[0629] The term "inhibit," as used herein, is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any degree of inhibition.

[0630] In some embodiments of the methods of the present application, B4GALT1 gene expression is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or below the level of detection as determined, preferably when measured by qPCR as described herein and / or when siRNA is introduced into the target cells by transfection. In some embodiments, the methods include clinically relevant inhibition of B4GALT1 target gene expression, for example, as demonstrated by clinically relevant results following treatment of a subject with an agent that reduces target gene expression.

[0631] In some embodiments, when transfected into cells, the nucleic acids described herein inhibit expression of the B4GALT1 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 as determined by reverse transcriptase (RT)-qPCR, as described herein.

[0632] In a preferred embodiment, when transfected into cells, the nucleic acids described herein inhibit the expression of the B4GALT1 gene with an IC50 value of less than 2500 pM. In a more preferred embodiment, when transfected into cells, the nucleic acids described herein inhibit the expression of the B4GALT1 gene with an IC50 value of less than 1000 pM. In a more preferred embodiment, when transfected into cells, the nucleic acids described herein inhibit the expression of the B4GALT1 gene with an IC50 value of less than 500 pM. In a most preferred embodiment, when transfected into cells, the nucleic acids described herein inhibit the expression of the B4GALT1 gene with an IC50 value of less than 100 pM.

[0633] Inhibition of B4GALT1 gene expression can be quantified by the following methods:

[0634] Huh7 cells (a cell line derived from human hepatocytes, obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2. Cells can then be transfected with siRNA duplexes targeting B4GALT1 mRNA or negative control siRNA (siRNA-control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 934), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 933)) using 10x3-fold serial dilutions over a final duplex concentration range of 20nM to 1pM. Transfection can be performed by adding 9.7μL Opti-MEM (ThermoFisher) plus 0.3μL Lipofectamine RNAiMAX (ThermoFisher) to 10μL of each siRNA duplex. The mixture can be incubated at room temperature for 15 minutes, followed by the addition of 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells can be incubated at 37°C / 5% CO2 for 24 hours, followed by purification of total RNA using the RNeasy 96 kit (Qiagen). In a single experiment, each duplex can be tested by transfection in duplicate in a well.

[0635] cDNA synthesis can be performed using the FastQuant RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the TaqMan Gene Expression Assay Kit (ThermoFisher Scientific) with primers specific for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1).

[0636] qPCR can be performed in duplicate on cDNA from each well, and the mean threshold cycle (Ct) is calculated. Relative expression of B4GALT1 relative to untreated cells can be calculated from the mean Ct values ​​using the comparative Ct (ΔΔCt) method and normalized to GAPDH. The maximal percentage inhibition of B4GALT1 expression and IC50 values ​​can be calculated using a four-parameter (variable slope) model in GraphPad Prism 9.

[0637] Alternatively or additionally, the inhibitory potential of the nucleic acids of the present invention can be quantified without prior transfection of target cells with said nucleic acids.

[0638] Thus, in some embodiments, when cells are incubated with the nucleic acids of the present application, the nucleic acids of the present application inhibit the expression of the B4GALT1 gene with an EC50 value of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM or 100 nM, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0639] In a preferred embodiment, when cells are incubated with the nucleic acids of the present application, the nucleic acids of the present application inhibit the expression of the B4GALT1 gene with an EC50 value of less than 1000 nM. In a more preferred embodiment, when cells are incubated with the nucleic acids of the present application, the nucleic acids of the present application inhibit the expression of the B4GALT1 gene with an EC50 value of less than 500 nM. In an even more preferred embodiment, when cells are incubated with the nucleic acids of the present application, the nucleic acids of the present application inhibit the expression of the B4GALT1 gene with an EC50 value of less than 200 nM. In a most preferred embodiment, when cells are incubated with the nucleic acids of the present application, the nucleic acids of the present application inhibit the expression of the B4GALT1 gene with an EC50 value of less than 100 nM.

[0640] Inhibition of B4GALT1 gene expression in the presence of free nucleic acids can be quantified using the following method:

[0641] Primary C57BL / 6 mouse hepatocytes (PMHs) can be freshly isolated by a two-step collagenase liver perfusion. Cells can be maintained in DMEM (Gibco-11995-092) supplemented with FBS, penicillin / streptomycin, HEPES, and L-glutamine. Cells can be cultured in a humidified incubator at 37°C, 5% CO2. Within 2 hours of isolation, PMHs can be plated at a density of 36,000 cells / well in conventional 96-well tissue culture plates. Dose-response analysis in PMHs can be performed by directly incubating cells with GalNAc siRNA at final concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, and 1.95 nM in a physiological, free-uptake environment. In control wells, cells can be incubated without GalNAc siRNA. After 48 hours of incubation, cells can be harvested for RNA extraction. Total RNA can be extracted using an RNeasy kit according to the manufacturer's instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR can be performed using an ABIPrism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH. The expression of the target gene in each test sample can be determined by relative quantification using the comparative Ct (ΔΔCt) method. This method measures the Ct difference (ΔCt) between the target gene and the housekeeping gene. The formula is as follows: ΔCt = average Ct of B4GALT1 - average Ct of GAPDH, ΔΔCt = ΔCt (sample) - average ΔCt, relative expression of target gene mRNA = 2 -ΔΔCt .

[0642] Alternatively or additionally, inhibition of B4GALT1 gene expression may be characterized by a decrease in the mean relative expression of the B4GALT1 gene.

[0643] In some embodiments, when cells are transfected with 0.1 nM of a nucleic acid described herein, the average relative expression of B4GALT1 is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4, as described herein, preferably as determined by qPCR, more preferably as determined by reverse transcriptase (RT)-qPCR.

[0644] In some embodiments, when cells are transfected with 5 nM of a nucleic acid described herein, as described herein, the average relative expression of B4GALT1 is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, preferably as determined by qPCR, more preferably as determined by reverse transcriptase (RT)-qPCR.

[0645] The average relative expression of the B4GALT1 gene can be quantified by the following method:

[0646] Huh7 cells (a cell line derived from human hepatocytes, obtained from JCRB Cell Bank) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37° C. and 5% CO 2 . Cells were transfected with siRNA duplexes targeting B4GALT1 mRNA or negative control siRNA (siRNA-control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 934), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 933)) at final duplex concentrations between 5 nM and 0.1 nM. Transfections were performed by adding 9.7 μL Opti-MEM (Thermo Fisher Scientific) plus 0.3 μL Lipofectamine RNAiMAX (Thermo Fisher Scientific) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes, followed by the addition of 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells were incubated at 37°C / 5% CO2 for 24 hours, and total RNA was then purified using the RNeasy 96 kit (Qiagen). Each duplex was tested by duplicate transfections in two independent experiments.

[0647] cDNA synthesis was performed using the FastQuant RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the TaqMan Gene Expression Assay Kit (Thermo Fisher Scientific) with specific primers for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1).

[0648] qPCR was performed in duplicate on cDNA from each well, and the mean Ct was calculated. The relative expression of B4GALT1 relative to untreated cells was calculated from the mean Ct values ​​using the comparative Ct (ΔΔCt) method and normalized to GAPDH.

[0649] Inhibition of B4GALT1 gene expression may be manifested as a decrease in the amount of target B4GALT1 mRNA compared to a suitable control.

[0650] In other embodiments, inhibition of B4GALT1 gene expression can be assessed based on a reduction in a parameter functionally related to gene expression, such as protein expression or signaling pathways.

[0651] Methods for treating or preventing diseases associated with B4GALT1 gene expression

[0652] The present application also provides methods for reducing or inhibiting B4GALT1 gene expression in cells or reducing target expression or function using nucleic acids (e.g., siRNAs of the present application) or compositions containing nucleic acids (e.g., siRNAs of the present application). The methods include contacting cells with nucleic acids (e.g., dsiRNAs of the present application) and maintaining the cells for a sufficient period of time to obtain degradation of mRNA transcripts of the B4GALT1 gene, thereby inhibiting B4GALT1 gene expression in the cells. Reduction in gene expression can be assessed by any method known in the art.

[0653] In the methods of the present application, cells can be contacted in vitro or in vivo, ie, the cells can be within the body of a subject.

[0654] Cells suitable for treatment using the methods of the present application can be any cells that express target genes associated with diabetes and cardiovascular diseases.

[0655] 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) comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of the B4GALT1 gene of the mammal to be treated.

[0656] The application also provides a method for treating a subject in need. The method of treatment of the present application includes administering a nucleic acid of the present application (e.g., siRNA, such as an siRNA targeting B4GALT1) or a pharmaceutical composition comprising a nucleic acid targeting B4GALT1 to a subject (e.g., a subject who would benefit from reducing or inhibiting the expression of the B4GALT1 gene) in a therapeutically effective amount. The disease to be treated is diabetes or cardiovascular disease.

[0657] As used herein, the term "diabetes" refers to a group of metabolic diseases in which a subject's blood sugar levels rise, either because the body does not produce enough insulin or because the cells do not respond to the insulin that is produced. There are three main types of diabetes: (1) Type 1 diabetes (T1D): This condition requires insulin injections because the body does not produce insulin. (Also known as insulin-dependent diabetes mellitus, IDDM, and juvenile diabetes.) (2) Type 2 diabetes (T2D): This condition is caused by insulin resistance, a condition in which cells cannot use insulin properly, sometimes accompanied by an absolute insulin deficiency. (Previously known as non-insulin-dependent diabetes mellitus, NIDDM, and adult-onset diabetes.) (3) Gestational diabetes (GD): This condition occurs when a woman who has never had diabetes before has high blood sugar levels during pregnancy. It may precede the development of T2D.

[0658] In some embodiments, the nucleic acid according to the present application or the pharmaceutical composition comprising the nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D).

[0659] In some embodiments, the nucleic acid according to the present application or the pharmaceutical composition comprising the nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in LDL cholesterol (LDL-c) levels in the blood.

[0660] In some embodiments, the nucleic acid according to the present application or the pharmaceutical composition comprising the nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in fasting blood glucose levels.

[0661] In some embodiments, the nucleic acid according to the present application or the pharmaceutical composition comprising the nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a decrease in the level of fibrinogen in the blood.

[0662] As used herein, the term "cardiovascular disease" refers to any disease, disorder or disease state associated with, caused by or resulting from structural or functional abnormalities of the heart or the blood vessels supplying the heart, which can impair its normal function. Cardiovascular disease may include coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina pectoris, deep vein thrombosis, stroke, congestive heart failure or arrhythmia. In a preferred embodiment, the cardiovascular disease is coronary artery disease. In some embodiments, the nucleic acid according to the application or a pharmaceutical composition comprising the nucleic acid is used to treat cardiovascular disease, preferably coronary artery disease.

[0663] In some embodiments, the nucleic acid according to the present application or the pharmaceutical composition comprising the nucleic acid is used for the treatment of diabetes, preferably coronary artery disease, wherein the treatment results in a decrease in low-density lipoprotein cholesterol (LDL-c) levels in the blood.

[0664] In some embodiments, the nucleic acid according to the present application or the pharmaceutical composition comprising the nucleic acid is used for the treatment of diabetes, preferably coronary artery disease, wherein the treatment results in a decrease in the level of fibrinogen in the blood.

[0665] The nucleic acid of the present application, such as siRNA, can be administered as "free" nucleic acid or "free" siRNA in the absence of a pharmaceutical composition. Naked nucleic acid can be in a suitable buffer solution. The buffer solution can contain 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.

[0666] Alternatively, the nucleic acids of the present application, such as siRNA, can be administered as a pharmaceutical composition, such as a dsiRNA liposome formulation.

[0667] In one embodiment, the method comprises administering a composition featured herein such that expression of the B4GALT1 gene is reduced, for example, 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 B4GALT1 target gene is reduced for an extended duration, for example, 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.

[0668] A therapeutic amount of a nucleic acid, such as siRNA, can be administered to a subject, for example, from about 0.01 mg / kg to about 200 mg / kg, to treat a disease associated with diabetes or cardiovascular disease.

[0669] Nucleic acids (e.g., siRNA) can be administered periodically over a period of time by intravenous infusion. In some embodiments, after the initial treatment regimen, the frequency of dosing can be reduced. Administration of siRNA can reduce the level of gene product of the B4GALT1 target gene, for example, by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% in patient cells or tissues, or to a level below that detected by the detection method used. In some embodiments, administration results in clinical stabilization or, preferably, a clinically relevant reduction in at least one sign or symptom of a B4GALT1 gene-related disease.

[0670] Alternatively, nucleic acid such as siRNA can be administered by subcutaneous injection, i.e., subcutaneous injection. One or more injections can be used to deliver the nucleic acid of the required daily dose to the experimenter, such as siRNA. Injection can be repeated over a period of time. Administration can be repeated regularly. In some embodiments, after the initial treatment regimen, the administration frequency can be reduced. Repeated dosing regimens can include the nucleic acid of a regular administration treatment amount, such as every other day or to once a year. In some embodiments, the nucleic acid is administered approximately once a month to approximately once per quarter (i.e., approximately once every three months).

[0671] In one aspect, the present application is applicable to the compounds, methods, compositions or uses of the following statements numbered 1-101, wherein any structural formula mentioned in statements 1-101 refers only to those structural formulas defined in statements 1-110. These structural formulas are as follows: Figure 5 Specifically, the oligonucleoside moiety represented by Z in any of the following sentences may comprise a nucleic acid for inhibiting B4GALT1 expression, as defined in any of the following sentences.

[0672] 1. A compound comprising the following structure:

[0673]

[0674] in:

[0675] R1 is independently selected at each occurrence from the group consisting of hydrogen, methyl and ethyl;

[0676] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups;

[0677] X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur at each occurrence;

[0678] m is an integer from 1 to 6;

[0679] n is an integer from 1 to 10;

[0680] q, r, s, t, and v are independently integers from 0 to 4, provided that:

[0681] (i) q and r cannot be 0 at the same time; and

[0682] (ii) s, t and v cannot be 0 at the same time;

[0683] Z is an oligonucleoside moiety.

[0684] 2. The compound according to statement 1, wherein R1 is hydrogen at each occurrence.

[0685] 3. The compound according to claim 1, wherein R1 is methyl.

[0686] 4. The compound according to statement 1, wherein R1 is ethyl.

[0687] 5. The compound according to any one of clauses 1 to 4, wherein R2 is hydroxy.

[0688] 6. A compound according to any one of clauses 1 to 4, wherein R2 is halogen.

[0689] 7. The compound according to clause 6, wherein R2 is fluoro.

[0690] 8. The compound according to clause 6, wherein R2 is chloro.

[0691] 9. The compound according to clause 6, wherein R2 is bromo.

[0692] 10. The compound according to clause 6, wherein R2 is iodine.

[0693] 11. The compound according to clause 6, wherein R2 is nitro.

[0694] 12. The compound according to any one of clauses 1 to 11, wherein X1 is methylene.

[0695] 13. A compound according to any one of clauses 1 to 11, wherein X1 is oxygen.

[0696] 14. A compound according to any one of clauses 1 to 11, wherein X1 is sulfur.

[0697] 15. A compound according to any one of clauses 1 to 14, wherein X2 is methylene.

[0698] 16. A compound according to any one of clauses 1 to 15, wherein X2 is oxygen.

[0699] 17. A compound according to any one of clauses 1 to 16, wherein X2 is sulfur.

[0700] 18. The compound according to any one of clauses 1 to 17, wherein m=3.

[0701] 19. The compound according to any one of clauses 1 to 18, wherein n=6.

[0702] 20. The compound according to clauses 13 and 15, wherein X1 is oxygen and X2 is methylene, preferably wherein:

[0703] q=1, r=2, s=1, t=1, v=1.

[0704] 21. Compounds according to clauses 12 and 15, wherein X1 and X2 are both methylene, preferably wherein:

[0705] q=1, r=3, s=1, t=1, v=1.

[0706] 22. A compound according to any one of clauses 1 to 21, wherein Z is:

[0707]

[0708] in:

[0709] Z1, Z2, Z3, Z4 at each occurrence are independently oxygen or sulfur; and

[0710] The bond between P and Z2 and one bond between P and Z3 is a single bond and the other bond is a double bond.

[0711] 23. The compound according to clause 22, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0712] 24. A compound according to statement 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 each of the first strand and the second strand has a 5' and a 3' end.

[0713] 25. The compound according to clause 24, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[0714] 26. The compound according to clause 24, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[0715] 27. A compound of formula (II):

[0716]

[0717] 28. A compound of formula (III):

[0718]

[0719] 29. A compound according to statement 27 or 28, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0720] 30. A composition comprising a compound of formula (II) as defined in statement 27, and a compound of formula (III) as defined in statement 28, optionally subject to statement 29.

[0721] 31. The composition according to clause 30, wherein the compound of formula (III) as defined in clause 28 is present in an amount of 10 to 15% by weight of the composition.

[0722] 32. A compound of formula (IV):

[0723]

[0724] 33. A compound of formula (V):

[0725]

[0726] 34. A compound according to statement 32 or 33, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0727] 35. A composition comprising a compound of formula (IV) as defined in statement 32, and a compound of formula (V) as defined in statement 33, optionally subject to statement 34.

[0728] 36. The composition according to clause 35, wherein the compound of formula (V) as defined in clause 33 is present in an amount of 10 to 15% by weight of the composition.

[0729] 37. A compound as defined in any one of clauses 1 to 29 or 32 to 34, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex further comprising one or more ribose sugars modified at the 2' position, preferably a plurality of ribose sugars modified at the 2' position.

[0730] 38. The compound according to clause 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0731] 39. The compound according to any one of clauses 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.

[0732] 40. A compound according to statement 39, wherein the one or more degradation protecting moieties are not located at the end of the oligonucleoside chain carrying the ligand moiety, and / or wherein the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and reversed abasic nucleosides, wherein the reversed abasic nucleoside is located at the distal end of the chain carrying the ligand moiety.

[0733] 41. The compound according to any one of statements 1 to 29, or 32 to 34, or 37 to 40, wherein the ligand moiety as described by formula (I) in statement 1 comprises one or more ligands.

[0734] 42. The compound according to clause 41, wherein the ligand moiety as described in clause 1 as formula (I) comprises one or more carbohydrate ligands.

[0735] 43. The compound according to clause 42, wherein the one or more carbohydrates can be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide.

[0736] 44. A compound according to clause 43, wherein the one or more carbohydrates comprises one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties and / or one or more mannose moieties.

[0737] 45. A compound according to clause 44, wherein said one or more carbohydrates comprises one or more N-acetylgalactosamine moieties.

[0738] 46. ​​A compound according to clause 45, comprising two or three N-acetylgalactosamine moieties.

[0739] 47. The compound according to any one of clauses 41 to 46, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[0740] 48. The compound of clause 47, wherein the one or more ligands are linked in a bifurcated or trifurcated branched configuration.

[0741] 49. The compound according to clauses 46 to 48, wherein the linker described in formula (I) of clause 1:

[0742]

[0743] is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0744]

[0745] in:

[0746] AI is hydrogen, or a suitable hydroxy protecting group;

[0747] a is the integer 2 or 3; and

[0748] b is an integer from 2 to 5; or

[0749]

[0750] in:

[0751] A I is hydrogen or a suitable hydroxy protecting group;

[0752] a is the integer 2 or 3; and

[0753] c and d are independently an integer from 1 to 6; or

[0754]

[0755] in:

[0756] A I is hydrogen or a suitable hydroxy protecting group;

[0757] a is the integer 2 or 3; and

[0758] e is an integer from 2 to 10.

[0759] 50. The compound according to clauses 46 to 48, wherein the moiety described in formula (I) of clause 1:

[0760]

[0761] Formula (VII)

[0762]

[0763] in:

[0764] A I It is hydrogen;

[0765] a is an integer 2 or 3.

[0766] 51. The compound according to clause 49 or 50, wherein a=2.

[0767] 52. The compound according to clause 49 or 50, wherein a=3.

[0768] 53. The compound according to clause 49, wherein b=3.

[0769] 54. A compound of formula (VIII):

[0770]

[0771] 55. A compound of formula (IX):

[0772]

[0773] 56. A compound according to statement 54 or 55, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has a 5' and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[0774] 57. A composition comprising a compound of formula (VIII) as defined in statement 54, and a compound of formula (IX) as defined in statement 55, optionally subject to statement 56.

[0775] 58. The composition according to clause 57, wherein the compound of formula (IX) as defined in clause 55 is present in an amount of 10 to 15% by weight of the composition.

[0776] 59. A compound of formula (X):

[0777]

[0778] 60. A compound of formula (XI):

[0779]

[0780] 61. A compound according to statement 59 or 60, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has a 5' and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[0781] 62. A composition comprising a compound of formula (X) as defined in statement 59, and a compound of formula (XI) as defined in statement 60, optionally subject to statement 61.

[0782] 63. The composition according to clause 62, wherein the compound of formula (XI) as defined in clause 60 is present in an amount of 10 to 15% by weight of the composition.

[0783] 64. A compound according to any one of clauses 54 to 63, wherein the oligonucleoside comprises an RNA duplex, further comprising one or more ribose sugars modified at the 2' position, preferably a plurality of ribose sugars modified at the 2' position.

[0784] 65. The compound according to clause 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0785] 66. The compound of any one of clauses 54 to 65, wherein the oligonucleoside further comprises one or more degradation protecting moieties at one or more termini.

[0786] 67. A compound according to statement 66, wherein the one or more degradation protecting moieties are not located at the end of the oligonucleoside chain carrying the ligand moiety, and / or wherein the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and inverted abasic nucleosides, wherein the inverted abasic nucleosides are located at the distal end of the chain carrying the ligand moiety, as shown in any of formulas (VIII), (IX), (X) or (XI) of any of statements 54, 55, 59 or 60.

[0787] 68. A method of preparing a compound according to any one of clauses 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 clauses 30, 31, 35, 36, 57, 58, 62, 63, comprising reacting compounds of formula (XII) and (XIII):

[0788]

[0789] in:

[0790] R1 is independently selected at each occurrence from the group consisting of hydrogen, methyl and ethyl;

[0791] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups;

[0792] X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur at each occurrence;

[0793] m is an integer from 1 to 6;

[0794] n is an integer from 1 to 10;

[0795] q, r, s, t, and v are independently integers from 0 to 4, provided that:

[0796] (i) q and r cannot be 0 at the same time; and

[0797] (ii) s, t and v cannot all be 0 at the same time;

[0798] Z is an oligonucleoside moiety;

[0799] The ligand is then deprotected and / or the second strand of the oligonucleotide moiety is annealed, as appropriate.

[0800] 69. The method according to clause 68, wherein the compound of formula (XII) is prepared by reacting a compound of formula (XIV) with a compound of formula (XV):

[0801]

[0802]

[0803] R1 is independently selected at each occurrence from the group consisting of hydrogen, methyl and ethyl;

[0804] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups;

[0805] X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur at each occurrence;

[0806] q, r, s, t, and v are independently integers from 0 to 4, provided that:

[0807] (i) q and r cannot be 0 at the same time; and

[0808] (ii) s, t and v cannot all be 0 at the same time;

[0809] Z is an oligonucleoside moiety.

[0810] 70. The method of claim 68, wherein:

[0811] The compound of formula (XII) is formula (XIIa):

[0812]

[0813] And the compound of formula (XIII) is formula (XIIIa):

[0814]

[0815] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0816] 71. The method of claim 68, wherein:

[0817] The compound of formula (XII) is formula (XIIb):

[0818]

[0819] And the compound of formula (XIII) is formula (XIIIa)

[0820]

[0821] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0822] 72. The method of claim 68, wherein:

[0823] The compound of formula (XII) is formula (XIIc):

[0824]

[0825]

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

[0827]

[0828] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0829] 73. The method of claim 68, wherein:

[0830] The compound of formula (XII) is formula (XIId):

[0831]

[0832] And the compound of formula (XIII) is formula (XIIIa)

[0833]

[0834] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first and second strands has 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. 74. The method of any one of clauses 70 to 73, wherein:

[0835] The compound of formula (XIIIa) is formula (XIIIb):

[0836]

[0837] 75. The method according to clause 69, which is subject to clauses 70 to 73, wherein: the compound of formula (XIV) is formula (XIVa) or formula (XIVb):

[0838]

[0839] and the compound of formula (XV) is formula (XVa) or formula (XVb)

[0840]

[0841] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has a 5' and a 3' end, and wherein (i) the RNA duplex is linked at the 5' end of the second strand to adjacent phosphate groups in Formula (XVa), or (ii) the RNA duplex is linked at the 3' end of the second strand to adjacent phosphate groups in Formula (XVb).

[0842] 76. A compound of formula (XII):

[0843]

[0844] in,

[0845] R1 is independently selected at each occurrence from the group consisting of hydrogen, methyl and ethyl;

[0846] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups;

[0847] X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur at each occurrence;

[0848] q, r, s, t, and v are independently integers from 0 to 4, provided that:

[0849] (i) q and r cannot be 0 at the same time; and

[0850] (ii) s, t and v cannot all be 0 at the same time;

[0851] Z is an oligonucleoside moiety.

[0852] 77. A compound of formula (XIIa):

[0853]

[0854] 78. A compound of formula (XIIb):

[0855]

[0856] 79. A compound of formula (XIIc):

[0857]

[0858] 80. A compound of formula (XIId)

[0859]

[0860] 81. A compound of formula (XIII)

[0861]

[0862] in:

[0863] R1 is independently selected at each occurrence from the group consisting of hydrogen, methyl and ethyl;

[0864] m is an integer from 1 to 6;

[0865] n is an integer from 1 to 10.

[0866] 82. A compound of formula (XIIIa):

[0867]

[0868] Formula (XIIIa).

[0869] 83. A compound of formula (XIIIb):

[0870]

[0871] 84. A compound of formula (XIV):

[0872]

[0873] in:

[0874] R1 is selected from the group consisting of hydrogen, methyl and ethyl;

[0875] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups;

[0876] X2 is selected from the group consisting of methylene, oxygen, and sulfur;

[0877] s, t, and v are independently integers from 0 to 4, provided that s, t, and v cannot all be 0 at the same time.

[0878] 85. A compound of formula (XIVa):

[0879]

[0880] 86. A compound of formula (XIVb)

[0881]

[0882] 87. A compound of formula (XV)

[0883]

[0884] in:

[0885] R1 is independently selected at each occurrence from the group consisting of hydrogen, methyl and ethyl;

[0886] X1 is selected from the group consisting of methylene, oxygen and sulfur;

[0887] q and r are independently integers from 0 to 4, provided that q and r cannot be 0 at the same time;

[0888] Z is an oligonucleoside moiety.

[0889] 88. A compound of formula (XVa):

[0890]

[0891] 89. A compound of formula (XVb)

[0892]

[0893] 90. Use of a compound according to any one of clauses 76, 81 to 84, 87 for the preparation of a compound according to any one of clauses 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 clauses 30, 31, 35, 36, 57, 58, 62 and 63.

[0894] 91. Use of a compound according to clause 85 for preparing a compound according to any one of clauses 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 clauses 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2 = F.

[0895] 92. Use of a compound according to clause 86 for preparing a compound according to any one of clauses 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 clauses 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2 = OH.

[0896] 93. Use of a compound according to clause 77 for the preparation of a compound according to any one of clauses 20, 25, 27, 29, 54, 56, and / or a composition according to any one of clauses 30, 31, 57, 58.

[0897] 94. Use of a compound according to clause 78 for the preparation of a compound according to any one of clauses 20, 25, 28, 29, 55, 56, and / or a composition according to any one of clauses 30, 31, 57, 58.

[0898] 95. Use of a compound according to clause 79 for the preparation of a compound according to any one of clauses 21, 26, 32, 34, 59, 61, and / or a composition according to any one of clauses 35, 36, 62, 63.

[0899] 96. Use of a compound according to clause 80 for preparing a compound according to any one of clauses 21, 26, 33, 34, 59, 61, and / or a composition according to any one of clauses 35, 36, 62, 63.

[0900] 97. Use of a compound according to clause 88 for preparing a compound according to any one of clauses 20, 25, 27 to 29, 54 to 56, and / or a composition according to any one of clauses 30, 31, 57, 58.

[0901] 98. Use of a compound according to clause 89 for preparing a compound according to any one of clauses 21, 26, 32 to 34, 59 to 61, and / or a composition according to any one of clauses 35, 36, 62, and 63.

[0902] 99. A compound or composition obtained or obtainable by a method according to any one of clauses 68 to 75.

[0903] 100. A pharmaceutical composition comprising a compound according to any one of statements 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 statements 30, 31, 35, 36, 57, 58, 62, and 63, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0904] Use of a compound according to any one of statements 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 statements 30, 31, 35, 36, 57, 58, 62 and 63 in therapy.

[0905] In another aspect, the present application may be applied to the compounds, methods, compositions or uses of the following clauses numbered 1-56, wherein any structural formula mentioned in the clause refers only to those structural formulas defined in clauses 1-56. These structural formulas are as follows: Figure 6Specifically, the oligonucleoside moiety represented by Z in any of the following clauses may comprise a nucleic acid for inhibiting B4GALT1 expression, as defined in any of the following sentences.

[0906] 1. A compound comprising the following structure:

[0907]

[0908] in

[0909] r and s are independently integers selected from 1 to 16; and

[0910] Z is an oligonucleoside moiety.

[0911] 2. The compound according to item 1, wherein s is an integer selected from 4 to 12.

[0912] 3. The compound according to clause 2, wherein s is 6.

[0913] 4. The compound according to any one of items 1 to 3, wherein r is an integer selected from 4 to 14.

[0914] 5. The compound according to item 4, wherein r is 6.

[0915] 6. The compound according to item 4, wherein r is 12.

[0916] 7. The compound according to clause 5, which is subject to clause 3.

[0917] 8. The compound according to clause 6, which is subject to clause 3.

[0918] 9. The compound according to any one of items 1 to 8, wherein Z is:

[0919]

[0920] in:

[0921] Z1, Z2, Z3, Z4 at each occurrence are independently oxygen or sulfur; and

[0922] One of the bonds between P and Z2 and between P and Z3 is a single bond and the other is a double bond.

[0923] 10. The compound according to any one of items 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0924] 11. The compound according to clause 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 each of the first strand and the second strand has a 5' and a 3' end.

[0925] 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.

[0926] 13. The compound according to item 11, preferably also subject to items 3 and 5, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[0927] 14. A compound of formula (II), preferably subject to clause 12:

[0928]

[0929] 15. A compound of formula (III), preferably subject to clause 13:

[0930]

[0931] 16. A compound as defined in any one of clauses 1 to 15, wherein the oligonucleoside comprises an RNA duplex, said RNA duplex further comprising one or more ribose sugars modified at the 2' position, preferably a plurality of ribose sugars modified at the 2' position.

[0932] 17. The compound according to clause 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0933] 18. The compound according to any one of items 1 to 17, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0934] 19. A compound according to clause 18, wherein the one or more degradation protecting moieties are not located at the termini of the oligonucleoside chain carrying the linker / ligand moiety, and / or wherein the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are located at the distal end of the same chain carrying the linker / ligand moiety.

[0935] 20. The compound according to any one of clauses 1 to 19, wherein the ligand moiety as described by formula (I) in clause 1 comprises one or more ligands.

[0936] 21. The compound according to clause 20, wherein the ligand moiety as described by formula (I) in clause 1 comprises one or more carbohydrate ligands.

[0937] 22. The 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.

[0938] 23. The compound according to clause 22, wherein the one or more carbohydrates comprises one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0939] 24. The compound according to clause 23, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[0940] 25. A compound according to item 24, comprising two or three N-acetylgalactosamine moieties.

[0941] 26. A compound according to any preceding clause, wherein the one or more ligands are linked in a linear or branched configuration.

[0942] 27. The compound according to clause 26, wherein the one or more ligands are linked in a bifurcated or trifurcated branched configuration.

[0943] 28. The compound according to clauses 20 to 27, wherein the moiety described in formula (I) in clause 1:

[0944]

[0945] is any one of formula (IV), (V) or (VI), preferably formula (IV):

[0946]

[0947] in:

[0948] A I is hydrogen, or a suitable hydroxy protecting group;

[0949] a is the integer 2 or 3; and

[0950] b is an integer from 2 to 5; or

[0951]

[0952] in:

[0953] A I is hydrogen or a suitable hydroxy protecting group;

[0954] a is the integer 2 or 3; and

[0955] c and d are independent integers from 1 to 6; or

[0956]

[0957] in:

[0958] A I is hydrogen or a suitable hydroxy protecting group;

[0959] a is the integer 2 or 3; and

[0960] e is an integer from 2 to 10.

[0961] 29. The compound according to any one of clauses 1 to 28, wherein the moiety described in formula (I) in clause 1:

[0962]

[0963] is formula (VII):

[0964]

[0965] in:

[0966] A I It is hydrogen;

[0967] a is an integer 2 or 3.

[0968] 30. The compound according to item 28 or 29, wherein a=2.

[0969] 31. The compound according to item 28 or 29, wherein a=3.

[0970] 32. The compound according to clause 28, wherein b=3.

[0971] 33. A compound of formula (VIII):

[0972]

[0973] 34. A compound of formula (IX):

[0974]

[0975] 35. The compound according to item 33 or 34, wherein the oligonucleoside comprises an RNA duplex, said RNA duplex further comprising one or more ribose sugars modified at the 2' position, preferably a plurality of ribose sugars modified at the 2' position.

[0976] 36. The compound according to clause 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0977] 37. The 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.

[0978] 38. A compound according to clause 37, wherein the one or more degradation protecting moieties are not located at the termini of the oligonucleoside chain carrying the linker / ligand moiety, and / or wherein the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are located at the distal end of the same chain carrying the linker / ligand moiety.

[0979] 39. A compound according to clause 33, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0980] 40. A compound according to clause 34, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0981] 41. A process for preparing a compound according to any one of clauses 1 to 40, comprising reacting compounds of formula (X) and (XI):

[0982]

[0983] in,

[0984] r and s are independently integers selected from 1 to 16; and

[0985] Z is an oligonucleoside moiety;

[0986] The ligand is then deprotected and / or the second strand of the oligonucleotide is annealed where appropriate.

[0987] 42. A process according to clause 41 for preparing a compound according to any one of clauses 6, 8 to 14, 16 to 33 and 35 to 40, wherein:

[0988] The compound of formula (X) is formula (Xa):

[0989]

[0990] And the compound of formula (XI) is formula (XIa):

[0991]

[0992] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0993] 43. A process according to clause 41 for preparing a compound according to any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40, wherein:

[0994] The compound of formula (X) is formula (Xb):

[0995]

[0996] And the compound of formula (XI) is formula (XIa):

[0997]

[0998] wherein the oligonucleoside comprises an RNA duplex comprising a first 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 each of the first strand and the second strand has 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.

[0999] 44. A method according to clause 42 or 43, wherein

[1000] The compound of formula (XIa) is formula (XIb)

[1001]

[1002] 45. A compound of formula (X)

[1003]

[1004] in:

[1005] r is independently an integer from 0 to 16; and

[1006] Z is an oligonucleoside moiety.

[1007] 46. ​​A compound having the formula (Xa)

[1008]

[1009] 47. A compound of formula (Xb)

[1010]

[1011] 48. A compound of formula (XI)

[1012]

[1013] in,

[1014] s is independently an integer from 0 to 16; and

[1015] Z is an oligonucleoside moiety.

[1016] 49. A compound of formula (XIa)

[1017]

[1018] 50. A compound of formula (XIb)

[1019]

[1020] 51. Use of a compound according to item 45 and any one of items 48 to 50 for the preparation of a compound according to any one of items 1 to 40.

[1021] 52. Use of a compound according to item 46 for preparing a compound according to any one of items 6, 8 to 14, 16 to 33, and 35 to 40.

[1022] 53. Use of a compound according to item 47 for preparing a compound according to any one of items 5, 7, 9-13, 15-32, and 34-40.

[1023] 54. A compound or composition obtained or obtainable by a method according to any one of clauses 41 to 44.

[1024] 55. A pharmaceutical composition comprising a compound according to any one of clauses 1 to 40, and a pharmaceutically acceptable carrier, diluent or excipient.

[1025] 56. A compound according to any one of clauses 1 to 40 for use in therapy. Example

[1026] The present application will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present application. It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and will be suggested to those skilled in the art to make various modifications or changes thereto, and will be included within the scope of the spirit and scope of the present application and the appended clauses.

[1027] Example 1: Synthesis of Tether 1

[1028] General experimental conditions:

[1029] Thin layer chromatography (TLC) was performed on aluminum plates coated with silica using a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under UV light (254 nm) or by spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent followed by heating according to Stahl (from Sigma-Aldrich). A Biotage Isola One flash chromatograph equipped with a dual variable UV wavelength detector (200-400 nm) was used, using a Biotage Flash chromatography was performed using Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1030] All moisture-sensitive reactions were performed under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK Scientific.

[1031] HPLC / ESI-MS was performed using a Dionex UltiMate 3000RS UHPLC system and a ThermoScientific MSQ Plus mass spectrometer. The chromatographic column was an Acquity UPLC Protein BEH C4 column ( 1.7 μm, 2.1 x 100 mm) with a column temperature of 60°C. The solvent system consisted of solvent A in H2O containing 0.1% formic acid, and solvent B in acetonitrile (ACN) containing 0.1% formic acid. A gradient of 5-100% B was used over 15 minutes at a flow rate of 0.4 mL / min. Detector and conditions: Corona supercharged aerosol detection (from the European Space Agency (esa)). Nebulizer temperature: 25°C. N2 pressure: 35.1 psi. Filter: Corona.

[1032] 1 H and 13C NMR spectra were obtained at room temperature on a Varian spectrometer at 500 MHz (1H 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, 13 CNMR δ is 77.2ppm; DMSO-d6-1HNMR: δ is 2.50ppm, 13 C NMR δ is 39.5 ppm. Coupling constants are reported in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[1033] Synthetic route of the coupling building block TriGalNAc_tether 1:

[1034]

[1035] Preparation of compound 2: D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reactants were stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with a cold saturated NaHCO aqueous solution (100 mL) and water (100 ml). The organic layer was separated, dried over NaSO and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM, 10 CV).

[1036] The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1037]

[1038] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq) and azide-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 to the solution. The mixture was stirred at room temperature for 1 h. TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered, and the filtrate was diluted with DCM (100 mL) and washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 ml). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM, 10CV) to give the title product as a light yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: calculated value is C 20 H 32 N4O 11 The m / z value was 504.21. The found value was 505.4. 1H NMR (500 MHz, CDCl3) δ 6.21-6.14 (m, 1H), 5.30 (dd, J = 3.4, 1.1 Hz, 1H), 5.04 (dd, J = 11.2, 3.4 Hz, 1H), 4.76 (d, J = 8.6 Hz, 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 = 4.2 Hz, 6H). 13 C NMR (125MHz, CDCl3) 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(CH2), 68.5(CH2), 66.6(CH2), 61.5(CH2), 23.1(CH3), 20.7(3xCH3).

[1039]

[1040] 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 using 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 give the title compound (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)) as a colorless oil. The compound was used without further purification. MS: calculated value was C 20 H34 N2O 11 The measured value is 479.4.

[1041]

[1042] Preparation of compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM / water (40 mL 1:1 v / v) and stirred vigorously with NaCO (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 the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CHCl (100 mL) and washed with water (100 ml). The organic layer was separated and dried over NaSO. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane over 12 CV) to afford the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane). MS: calculated for C 33 H 53 NO 11 639.3. Found 640.9. 1H NMR (500 MHz, DMSO-d6) δ 7.38-7.26 (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 (125MHz, DMSO-d6) δ170.3(3xC), 154.5(C), 137.1(C), 128.2(2xCH), 127.7(CH), 127.6(2x CH), 79.7(3xC), 68.4(3xCH2), 66.8(3xCH2), 64.9(C), 58.7(CH2), 35.8(3xCH2), 27.7(9xCH3).

[1043]

[1044] Preparation of compound 8: Cbz-NH-tris-Boc ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to obtain the compound in the form of a TFA salt (0.183 g, 98%). The compound was used without further purification. MS: calculated value was C 21 H 29 The NO value is 471.6. The measured value is 472.4.

[1045]

[1046] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq) were dissolved in N,N-dimethylformamide (DMF) (25 mL). N,N,N',N'-tetramethyl-O-(1H-benzotriazol-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 then added to the solution and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO 3 (100 mL). The organic layer was dried over Na 2 SO 4 , the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 14 CV). The product was obtained as a light yellow oil (1.2 g, 43%, rf=0.20 (5% MeOH in DCM)). MS: calculated value: C 81 H 125 N7O 41of 1852.9. Found: 1854.7. 1H NMR (500 MHz, DMSO-d6) δ 7.90-7.80 (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 (125MHz, DMSO-d6) 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(CH2), 69.0(CH2), 68.2(CH2 ), 67.2(CH2), 66.7(CH2), 61.4(CH2), 22.6(CH2), 22.4(3xCH3), 20.7(9xCH3).

[1047]

[1048] Preparation of compound 10: trident 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 using vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. After completion of the reaction, mass spectrometry was performed and the resulting mixture was filtered through a thin pad of diatomaceous earth. The solvent was evaporated and the obtained residue was dried under high vacuum and used in the next step without further purification. A light yellow oily product (0.24 g, quantitative yield) was obtained. MS: calculated value of C 73 H 119 N7O 39 The measured value is 1718.8. The actual value is 1719.3.

[1049]

[1050] 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. The reactants were stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 16 CV). The product was obtained as a white solid (1.54 g, 43%, rf=0.71 (5% MeOH in DCM)). MS: calculated value was C 15 H 23 The N5O5 value is 353.4. The measured value is 354.3.

[1051]

[1052] Preparation of TriGalNAc (12): Trigalnac 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 dried over Na2SO4. The solvent was evaporated and the crude material was purified by flash chromatography (elution gradient: 0-10% MeOH in DCM, 20CV) to give the title compound (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)) as a white fluffy solid. MS: calculated value: C 84 H 137 N 11 O4 is 1957.1. The measured value is 1959.6.

[1053] Coupling of tether 1 to siRNA strand: 5'- or 3'-terminal monofluorocyclooctyne (MFCO) coupling

[1054] 5' end MFCO coupling

[1055]

[1056] 3' end MFCO coupling

[1057]

[1058] General conditions for MFCO coupling: Amine-modified single strands were dissolved in 50 mM carbonate / bicarbonate buffer pH 9.6 / dimethyl sulfoxide (DMSO) 4:6 (v / v) at a concentration of 700 OD / mL, and one molar equivalent of a 35 mM solution of MFCO-C6-NHS ester (Berry & Associates, Cat. # LK 4300) in DMF was added to the solution. The reaction was carried out at room temperature, and after 1 h another molar equivalent of MFCO solution was added. The reaction was allowed to proceed for another hour and monitored by LC / MS. At least two molar equivalents of excess MFCO NHS ester reagent were required to achieve quantitative consumption of the starting material relative to the amino-modified oligonucleotide. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius, and then Purification was by reverse phase (RP HPLC) on a Pure instrument (GE Healthcare).

[1059] Purification was performed using a Waters XBridge C18 Prep 19 x 50 mm column. Buffer A was 100 mM TEAAc pH 7, and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60°C. UV traces were recorded at 280 nm. A gradient of 0-100% buffer B was applied over 60 column volumes.

[1060] Fractions containing the full-length conjugated oligonucleotide were pooled, precipitated in the refrigerator with 3 M 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 speed vacuum concentrator to obtain the conjugated oligonucleotide in an isolated yield of 40-80%.

[1061] 5'-GalNAc-T1 conjugate

[1062]

[1063] 3'-GalNAc-T1 conjugate

[1064]

[1065] General procedure for TriGalNAc conjugation: MFCO-modified single-stranded polymer was dissolved in water at a concentration of 2000 OD / mL, and an equivalent solution of compound 12 (10 mM) in DMF was added to the solution. The reaction was carried out at room temperature, and after 3 h, 0.7 molar equivalents of compound 12 solution were added. The reaction was allowed to proceed overnight and monitored for completion 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 RP HPLC on a Pure instrument (GE Healthcare).

[1066] RP HPLC purification was performed using a Waters XBridge C18 Prep 19 x 50 mm column. Buffer A was 100 mM triethylammonium acetate, pH 7, and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60°C. UV traces were recorded at 280 nm. A gradient of 0-100% buffer B was applied over 60 column volumes.

[1067] The fractions containing the full-length coupled oligonucleotides were combined and precipitated in a refrigerator using 3M NaOAc, pH 5.2, and 85% ethanol. The collected precipitate was dissolved in water to obtain an oligonucleotide solution of approximately 1000 OD / mL. The O-acetate was removed by adding 20% ​​aqueous ammonia. Quantitative removal of these protecting groups was verified by LC-MS.

[1068] exist The conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on a Pure (GE Healthcare) instrument to provide the conjugated oligonucleotides in isolated yields of 50-70%.

[1069] The following scheme further illustrates the synthetic route: Option 1:

[1070]

[1071] Option 2:

[1072]

[1073] Option 3: 5' end MFCO coupling

[1074] 3' end MFCO coupling

[1075]

[1076] Option 4:

[1077]

[1078] Option 5:

[1079]

[1080] Example 2: Double-strand annealing

[1081] To generate the desired siRNA duplex, the two complementary strands were annealed by mixing 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 over 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[1082] The HPLC was carried out on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system by Superdex TM Duplexes were analyzed by analytical SEC HPLC using a 75 Increase 5 / 150GL column, 5x153-158 mm (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 1.5 mL / min. UV traces were recorded at 260 and 280 nm. 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).

[1083] Example 3: Synthesis of Tether 2

[1084] General experimental conditions:

[1085] Thin layer chromatography (TLC) was performed on aluminum plates coated with silica using a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under UV light (254 nm) or by spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent followed by heating according to Stahl (from Sigma-Aldrich). A Biotage Isola One flash chromatograph equipped with a dual variable UV wavelength detector (200-400 nm) was used, using a Biotage Flash chromatography was performed using Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1086] All moisture-sensitive reactions were performed under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK Technology.

[1087] HPLC / ESI-MS was performed using a Dionex UltiMate 3000RS UHPLC system and a ThermoScientific MSQ Plus mass spectrometer. The chromatographic column was a Waters Acquity UPLC Protein BEH C4 column ( 1.7 μm, 2.1x100 mm), column temperature 60 ° C. The solvent system consists of solvent A in H2O containing 0.1% formic acid, and solvent B in acetonitrile (ACN) containing 0.1% formic acid. A gradient of 5-100% B in 15 minutes at a flow rate of 0.4 mL / min was used. Detector and conditions: Corona supercharged aerosol detection (from the European Space Agency (esa)). Nebulizer temperature: 25 ° C. N2 pressure: 35.1 psi. Filter: Corona.

[1088] 1 H and 13 C NMR spectra were obtained at room temperature on a Varian spectrometer at 500 MHz (1H 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, 13 CNMR δ is 77.2ppm; DMSO-d6-1H NMR: δ is 2.50ppm, 13 C NMR δ is 39.5 ppm). Coupling constants are in Hertz.

[1089] Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[1090]

[1091] Preparation of compound 2: D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reactant was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with a cold saturated NaHCO aqueous solution (100 mL) and water (100 ml). The organic layer was separated, dried over Na SO and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM, 10 CV).

[1092] The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1093]

[1094] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq) and azide-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 to the solution. The mixture was stirred at room temperature for 1 h. 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), washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 ml). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM, 10CV) to give the title product as a light yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: calculated value is C 20 H 32 N4O 11 The m / z value was 504.21. The found value was 505.4. 1H NMR (500 MHz, CDCl3) δ 6.21-6.14 (m, 1H), 5.30 (dd, J = 3.4, 1.1 Hz, 1H), 5.04 (dd, J = 11.2, 3.4 Hz, 1H), 4.76 (d, J = 8.6 Hz, 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 = 4.2 Hz, 6H).13 C NMR (125MHz, CDCl3) 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(CH2), 68.5(CH2), 66.6(CH2), 61.5(CH2), 23.1(CH3), 20.7(3xCH3).

[1095]

[1096] 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 using 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 give the title compound (0.95 g, quantitative yield, rf=0.25 (10% MeOH in DCM)) as a colorless oil. The compound was used without further purification. MS: calculated value was C 20 H 34 N2O 11 The measured value is 479.4.

[1097]

[1098] Preparation of compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM / water (40 mL 1:1 v / v) and stirred vigorously with NaCO (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 the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CHCl (100 mL) and washed with water (100 ml). The organic layer was separated and dried over NaSO. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane over 12 CV) to afford the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane). MS: calculated for C 33 H 53 NO 11639.3. Found 640.9. 1H NMR (500 MHz, DMSO-d6) δ 7.38-7.26 (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 (125MHz, DMSO-d6) δ170.3(3xC), 154.5(C), 137.1(C), 128.2(2xCH), 127.7(CH), 127.6(2x CH), 79.7(3xC), 68.4(3xCH2), 66.8(3xCH2), 64.9(C), 58.7(CH2), 35.8(3xCH2), 27.7(9xCH3).

[1099]

[1100] Preparation of compound 8: Cbz-NH-tris-Boc ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to obtain the compound in the form of a TFA salt (0.183 g, 98%). The compound was used without further purification. MS: calculated value was C 21 H 29 The NO value is 471.6. The measured value is 472.4.

[1101]

[1102] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq) were dissolved in N,N-dimethylformamide (DMF) (25 mL). N,N,N',N'-tetramethyl-O-(1H-benzotriazol-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 then added to the solution and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO 3 (100 mL). The organic layer was dried over Na 2 SO 4 , the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 14 CV). The product was obtained as a light yellow oil (1.2 g, 43%, rf=0.20 (5% MeOH in DCM)). MS: calculated value: C 81 H 125 N7O 41 of 1852.9. Found: 1854.7. 1H NMR (500 MHz, DMSO-d6) δ 7.90-7.80 (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). 13C NMR (125MHz, DMSO-d6) 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(CH2), 69.0(CH2), 68.2(CH2 ), 67.2(CH2), 66.7(CH2), 61.4(CH2), 22.6(CH2), 22.4(3xCH3), 20.7(9xCH3).

[1103]

[1104] Preparation of compound 10: trident 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 using vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. After completion of the reaction, mass spectrometry was performed and the resulting mixture was filtered through a thin pad of diatomaceous earth. The solvent was evaporated and the obtained residue was dried under high vacuum and used in the next step without further purification. A light yellow oily product (0.24 g, quantitative yield) was obtained. MS: calculated value was C 73 H 119 N7O 39 The measured value is 1718.8. The actual value is 1719.3.

[1105]

[1106] Preparation of Compound 14: Under argon, tridentate 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). To this mixture was added dropwise a solution of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) in DCM (5 mL). 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 dried over Na2SO4. The solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 20 CV). The product was obtained as a white, fluffy solid (0.25 g, 48%, rf = 0.4 (10% MeOH in DCM)). MS: calculated value is C88 H 137 N7O 42 The measured value is 1965.1.

[1107]

[1108] Preparation of TriGalNAc (15): Trident 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 using 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 pad of celite. The solvent was removed under reduced pressure and the resulting residue was dried under high vacuum overnight. The residue was used for coupling with oligonucleosides without further purification (0.28 g, quantitative yield). MS: calculated value: C 81 H 131 N7O 42 The measured value is 1875.3.

[1109] Coupling of tether 2 to siRNA strand: TriGalNAc tether 2 (GalNAc-T2) at the 5'-end or 3'-end

[1110] 5'-GalNAc-T2 conjugate

[1111]

[1112] 3'-GalNAc-T2 conjugate

[1113]

[1114] Preparation of TriGalNAc tether 2 NHS ester: To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 μmol) in DMF (2.1 mL) was added N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol). The solution was stirred at room temperature for 18 hours and used in the subsequent coupling reaction without purification.

[1115] General procedure for coupling of TriGalNAc tether 2: The amine-modified single chain was dissolved in 50 mM carbonate / bicarbonate buffer pH 9.6 / DMSO 4:6 (v / v) at a concentration of 700 OD / mL, and one molar equivalent of tether 2 NHS ester (57 mM) solution in DMF was added to the solution. The reaction was carried out at room temperature and another molar equivalent of NHS ester solution was added after 1 h. The reaction was allowed to proceed for another hour and the progress of the reaction was monitored by LCMS. At least two molar equivalents of excess NHS ester reagent were required to achieve quantitative consumption of the starting material relative to the amino-modified oligonucleoside. The reaction mixture was diluted 15-fold with water, filtered once through a 1.2 μm filter from Sartorius, and then Purification was performed by reverse phase (RP HPLC) on a Pure (GE Healthcare) instrument.

[1116] 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 was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60°C. UV traces were recorded at 280 nm. A gradient of 0-100% buffer B was applied over 60 column volumes.

[1117] The fractions containing the full-length coupled oligonucleotides were combined, precipitated in a refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and then dissolved in water at 1000 OD / mL. O-acetate was removed with 20% aqueous ammonium hydroxide solution until complete (monitored by LC-MS).

[1118] exist The conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on a Pure (GE Healthcare) instrument to generate the conjugated oligonucleotides in isolated yields of 60-80%.

[1119] The conjugates were characterized by HPLC-MS analysis on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics) using a 2.1 x 50 mm XBridge C18 column (Waters). Buffer A consisted of 16.3 mM triethylamine, 100 mM HFIP in 1% aqueous MeOH, and buffer B consisted of 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 280 nm. A gradient of 1-100% B was applied over 31 minutes.

[1120] The following scheme further illustrates the synthetic route: Option 6:

[1121]

[1122] Option 7:

[1123]

[1124] Option 8:

[1125]

[1126] Option 9:

[1127]

[1128] Example 4: Double-strand annealing

[1129] To generate the desired siRNA duplex, the two complementary strands were annealed by mixing 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 over 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[1130] The HPLC was carried out on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system by Superdex TM Duplexes were analyzed by analytical SEC HPLC using a 75 Increase 5 / 150GL column, 5x153-158 mm (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 1.5 mL / min. UV traces were recorded at 260 and 280 nm. 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).

[1131] Example 5: Alternative synthetic route for coupling building block TriGalNAc_tether 2:

[1132]

[1133]

[1134] Coupling of tether 2 to siRNA strand: TriGalNAc tether 2 (GalNAc-T2) at the 5'-end or 3'-end

[1135] Coupling conditions

[1136]

[1137] Preactivation: To a solution of compound 15 (16 μmol, 4 eq.) in DMF (160 μL) at 25° C., add TFA-O-PFP (15 μL, 21 eq.) followed by DIPEA (23 μL, 32 eq.). Shake the tube at 25° C. for 2 h. Quench the reaction with H O (10 μL).

[1138] Coupling: The resulting mixture was diluted with DMF (400 μl) and then an oligoamine solution (4.0 μmol in 10x PBS, pH 7.4, 500 μl; final oligomer concentration in organic and aqueous solutions: 4 μmol / ml = 4 mM) was added. The tube was shaken at 25°C for 16 h and the reaction analyzed by LCMS. The resulting mixture was treated with 28% NH4OH (4.5 ml) and shaken at 25°C for 2 h. The mixture was analyzed by LCMS, concentrated, and purified by IP-RP HPLC to produce an oligonucleotide coupled to the tethered 2-GalNAc.

[1139] 5'-GalNAc-T2 conjugate

[1140]

[1141] 3'-GalNAc-T2 conjugate

[1142]

[1143] Example 6: Solid Phase Synthesis: Scale ≤ 1 μmol

[1144] Synthesis of siRNA sense and antisense strands was performed on a MerMade 192X synthesizer with 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).

[1145] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1146] 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-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1147] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1148] 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). 0.02 M iodine in acetonitrile / pyridine / HO (DNAchem) was used as the oxidizing agent. Thiolation of phosphorothioate bonds was performed using 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 0.25 M mM 5-ethylmercaptotetrazole (ETT) in acetonitrile was used as the activator solution.

[1149] The reverse abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or MegaVitamin B (OP-040).

[1150] In each cycle, DMT was removed by 3% TCA deblocking solution in DCM (DNAchem).

[1151] The coupling time was 180 seconds. The oxidant contact time was set to 80 seconds, and the thiolation time was 2*100 seconds.

[1152] At the end of the synthesis, the oligonucleotides were cleaved from the solid support using a 4:1 (v / v) NH4OH:EtOH solution at 45°C (TCI) for 20 h. The solid support was then filtered off, the filter washed thoroughly with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[1153] Oligonucleotides were treated to form sodium salts by ultracentrifugation using an Amicon Ultra-2 centrifugal filter device, PBS buffer (10x, Teknova, pH 7.4, sterile) or by EtOH precipitation from 1 M sodium acetate.

[1154] Single-strand identity was assessed by MS ESI-ELISA followed by annealing in water to form the final double-stranded siRNA, and double-strand purity was assessed by size exclusion chromatography.

[1155] Example 7: Solid Phase Synthesis: Scale ≥ 5 μmol

[1156] The synthesis of siRNA sense and antisense strands was performed on a MerMade 12 synthesizer at a scale of 5 μMOL with a commercially available solid support made of controlled pore glass with a universal linker (universal CPG with a loading of 40 μmol / g; LGC Biosearch or Glen Research). The sense strand for 3' coupling was synthesized on a solid support at a concentration of 12 μmol.

[1157] RNA phosphoramidites were purchased from ChemGenes or MegaVideo.

[1158] The 2'-O-methylphosphoramidites used were as follows: 5'-(4,4'-dimethoxytrityl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1159] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1160] The reverse abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or MegaVitamin B (OP-040).

[1161] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemistry) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite, which was dissolved in DMF / MeCN (1:4, v / v). 0.02 M iodine in acetonitrile / pyridine / H2O (DNAchem) was used as the oxidant. Thiolation of phosphorothioate bonds was performed using 0.2 M PAD S (TCI) in acetonitrile / pyridine (1:1 v / v). 0.25 M mM 5-ethylmercaptotetrazole (ETT) in acetonitrile was used as the activator solution.

[1162] In each cycle, DMT was removed by 3% TCA deblocking solution in DCM (DNAchem).

[1163] For chains synthesized on universal CPG, coupling was performed for 130 seconds using 8 equivalents of amidite, with oxidation time of 47 seconds and thiolation time of 210 seconds.

[1164] For chains synthesized on 3'-PT-Amino-Modifier C6 CPG, coupling was performed with 8 equivalents of phosphoramidite for 2 x 150 seconds. Oxidation time was 47 seconds, and thiolation time was 250 seconds.

[1165] At the end of the synthesis, the oligonucleotides were cleaved from the solid support using a 4:1 (v / v) NH4OH:EtOH solution at 45°C (TCI) for 20 h. The solid support was then filtered off, the filter washed thoroughly with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[1166] Oligonucleotides were treated with EtOH precipitation using 1 M sodium acetate to form the sodium salt.

[1167] Single-stranded oligonucleotides were purified by Xbridge BEH C18 5 μm. Purification was performed by IP-RP HPLC on a 19x150 mm (Waters) column using a gradient of increasing 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.

[1168] Single-chain purity and identity were assessed by UPLC / MS ESI on an Xbridge BEH C18 2.5 μm, 3x50 mm (Waters) column using an increasing gradient 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).

[1169] The sense strand was coupled according to the protocol provided in any one of Examples 1, 3, and 5.

[1170] The sense and antisense strands were then annealed in water to form the final double-stranded siRNA, and the double-strand purity was assessed by size exclusion chromatography.

[1171] Example 8: Nucleic acid sequence:

[1172] The siRNA oligonucleotides according to the present application target B4GALT1. The complete DNA sequence of the B4GALT1 target is as follows (SEQ ID NO: 1):

[1173]

[1174]

[1175]

[1176]

[1177]

[1178]

[1179]

[1180]

[1181]

[1182]

[1183]

[1184]

[1185]

[1186]

[1187]

[1188]

[1189]

[1190]

[1191]

[1192]

[1193]

[1194]

[1195]

[1196] Table 1 below provides oligonucleotide mRNA target sequences for B4GALT1, and the corresponding positions in transcript NM_001497.4. It will be understood that SEQ ID NOs: 2-20, 102-201 and 622-680 relate to human (human) mRNA sequences.

[1197] Table 1

[1198]

[1199]

[1200]

[1201]

[1202]

[1203] Table 2 provides the unmodified first (antisense) and corresponding unmodified second (sense) strand sequences of the siRNA oligonucleotides according to the present application, and the corresponding positions in the entire gene sequence of SEQ ID NO: 1, as shown below.

[1204] Table 2

[1205]

[1206]

[1207]

[1208]

[1209]

[1210]

[1211]

[1212] Table 3 provides the modified first (antisense) sequences of the siRNA oligonucleotides according to the present application, and the corresponding unmodified first (antisense) sequences, as follows.

[1213] Table 3

[1214]

[1215]

[1216]

[1217]

[1218]

[1219]

[1220]

[1221]

[1222] Table 4 provides the modified second (sense) sequences of the siRNA oligonucleotides according to the present application, and the corresponding unmodified second (sense) sequences, as follows.

[1223] Table 4

[1224]

[1225]

[1226]

[1227]

[1228]

[1229]

[1230]

[1231] As shown in Table 4 above, some modified second strand sequences include a preferred 5'iaia motif (or sequence). 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'aia motif (or sequence).

[1232] Table 5 lists the duplexes and duplex IDs, which reference the modified antisense and sense IDs in Tables 3 and 4 above.

[1233] Table 5

[1234]

[1235]

[1236]

[1237]

[1238]

[1239]

[1240] Definitions provided in the table above:

[1241] A-adenosine

[1242] B-Cytidine

[1243] G-Guanosine

[1244] T-thymidine

[1245] m-2'-O-methyl

[1246] f–2'floro

[1247] S-phosphorothioate bond

[1248] o-Thermal-labile nucleosides

[1249] ia-inverted abasic nucleoside

[1250] Example 9: Inhibition screening of B4GALT1 expression in human Huh7 cells

[1251] Huh7 cells (a cell line derived from human hepatocytes, obtained from the JCRB cell bank) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2. Cells were transfected with siRNA duplexes targeting B4GALT1 mRNA or negative control siRNA (siRNA-control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 934)', antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 933)) at final duplex concentrations between 5 nM and 0.1 nM. Transfection was performed by adding 9.7 μL Opti-MEM (ThermoFisher) plus 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% CO2 for 24 hours, and then total RNA was purified using the RNeasy 96 kit (Qiagen). In two independent experiments, each duplex was tested by transfection in duplicate in the wells.

[1252] cDNA synthesis was performed using the FastQuant RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the FastStart Universal Probe Master Kit (Roche) with primers specific for human B4GALT1 and human GAPDH (Hs02786624_g1).

[1253] qPCR can be performed in duplicate on cDNA from each well, and the average Ct can be calculated. The relative expression of B4GALT1 relative to untreated cells can be calculated from the average Ct value using the comparative Ct method and normalized to GAPDH. Based on the results of the initial screening, siRNA duplexes that showed good activity were selected for subsequent dose-response studies. The results are shown in Figure 2. Figure 9 and Figure 10 The sequences of the RNAi molecules are shown in Table 5.

[1254] Table 6. Relative mRNA expression of B4GALT1

[1255]

[1256]

[1257]

[1258]

[1259] Example 10: Dose response of B4GALT1 inhibition in human Huh7 cells

[1260] Huh7 cells (a cell line derived from human hepatocytes, obtained from JCRB Cell Bank) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37° C. and 5% CO 2 . Cells were transfected with siRNA duplexes targeting B4GALT1 mRNA or negative control siRNA (siRNA-control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 934), antisense strand 5'-5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 933)) using 10x3-fold serial dilutions over a final duplex concentration range of 20 nM to 1 pM. Transfections were performed by adding 9.7 μL Opti-MEM (Thermo Fisher Scientific) plus 0.3 μL Lipofectamine RNAiMAX (Thermo Fisher Scientific) 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. Cells were incubated at 37°C / 5% After incubation for 24 hours under CO2, total RNA was purified using the RNeasy96 kit (Qiagen). In a single experiment, each duplex was tested by transfection in duplicate in a well.

[1261] cDNA synthesis was performed using the FastQuant RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the TaqMan Gene Expression Assay Kit (Thermo Fisher Scientific) with specific primers for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1).

[1262] qPCR was performed in duplicate on cDNA from each well, and the average Ct was calculated. The relative expression of B4GALT1 relative to untreated cells can be calculated from the average Ct value using the comparative Ct (ΔΔCt) method and normalized to GAPDH. The maximum inhibition percentage and IC50 value of B4GALT1 expression can be calculated using a four-parameter (variable slope) model in GraphPad Prism 9. The results are shown in Figure 11 The sequences of the RNAi molecules are described in the relevant tables of this article.

[1263] Example 11: Dose response of B4GALT1 inhibition in human Huh7 cells

[1264] Huh7 cells (a cell line derived from human hepatocytes, obtained from the JCRB cell bank) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2. siRNA duplexes designed for target or negative control siRNA were transfected into cells at 0.1nM and 1nM. Transfection was performed by adding 9.7μL Opti-MEM (Thermo Fisher Scientific) plus 0.3μL Lipofectamine RNAiMAX (Thermo Fisher Scientific) to 10μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes, and then 100μL of complete growth medium containing 20,000Huh7 cells was added. The cells were incubated at 37°C / 5% CO2 for 24 hours, and then total RNA was purified using the RNeasy 96 kit (Qiagen). In a single experiment, each duplex was tested by transfection in duplicate in the wells, and the experiment was repeated three times.

[1265] cDNA synthesis was performed using the FastKing RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the TaqMan Gene Expression Assay Kit (Thermo Fisher Scientific) with specific primers for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1).

[1266] qPCR was performed in duplicate on cDNA from each well, and the mean Ct was calculated. Relative target expression relative to untreated cells was calculated from the mean Ct values ​​using the comparative Ct (ΔΔCt) method and normalized to GAPDH.

[1267] To inhibit B4GALT1, siRNA duplexes ETXM1200, ETXM1201, ETXM1217, ETXM1764, ETXM1765, ETXM1766, ETXM1767, ETXM1768, ETXM1769, and ETXM1770 ( Figure 13 ).

[1268] In the second experiment, siRNA duplexes ETXM1203, ETXM1204, ETXM1218, ETXM1772, ETXM1773, ETXM1774, ETXM1775, ETXM1776, ETXM1777, and ETXM1778 were tested for inhibition of B4GALT1. Figure 14 ).

[1269] Example 12: B4GALT1 Pharmacology Study

[1270] ETX's in-house computational biology analysis identified B4GALT1, encoding β-1,4-galactosyltransferase 1, as a gene associated with type 2 diabetes (T2D). Here, the inventors established B4GALT1 as a potential therapeutic target for T2D. In silico-designed GalNAc-siRNA targeting B4GALT1 in mouse liver was synthesized and tested to assess the plausibility of the hypothesis that significant knockdown of B4GALT1 mRNA in the liver would reduce plasma levels of LDL-c, fibrinogen, and fasting glucose.

[1271] In vitro dose-response assays for selecting potent molecules

[1272] An in vitro dose-response experiment was performed to measure gene knockdown in primary mouse hepatocytes (PMHs) to test 20 GalNAc siRNAs targeting liver B4GALT1. Primary C57BL / 6 mouse hepatocytes (PMHs) were freshly isolated by two-step collagenase liver perfusion. Cells were maintained in DMEM (Gibco-11995-092) supplemented with FBS, penicillin / streptomycin, HEPES, and L-glutamine. Cells were cultured in a humidified incubator at 37°C and 5% CO2. Within 2 hours of isolation, PMHs were seeded at a density of 36,000 cells / well on conventional 96-well tissue culture plates. The dose-response analysis in PMH can be achieved by directly incubating cells with GalNAc siRNA at a final concentration of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM in a physiological free uptake environment. In the control wells, cells were incubated in the absence of GalNAc siRNA. After incubation for 48 hours, cells were harvested for RNA extraction. Total RNA was extracted using an RNeasy kit (Qiagen, Shanghai, China) according to the manufacturer's instructions. After reverse transcription, real-time quantitative PCR was performed using an ABIPrism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH. The expression of the target gene in each test sample was determined by relative quantification using the comparative Ct (ΔΔCt) method. The method measures the Ct difference (ΔCt) between the target gene and the housekeeping gene. The formula is as follows: ΔCt = average Ct of B4GALT1 - average Ct of GAPDH, ΔΔCt = ΔCt (sample) - average ΔCt, relative expression of target gene mRNA = 2 -ΔΔCt Based on the results of the in vitro free uptake experiment, GalNAc siRNAs that showed good activity were selected using a 10-point concentration curve for EC 50 Determination ( Figure 15 ).

[1273] In vivo pharmacology of four selected GalNAc siRNAs

[1274] The pharmacodynamic activity of four selected B4GALT1 GalNAc-siRNAs was measured in vivo. Twelve C57BL / 6 male mice were assigned to each of the GalNAc-siRNAs, ETXM619, ETXM624, ETXM628, and ETXM633. Five mice were assigned to a no-treatment control group. On day 0 (i.e., the day the mice were first dosed), day 3, and day 7, the mice were subcutaneously injected with ETXM (10 mg / kg). On days 3, 7, 10, and 14, three mice per treatment group were sacrificed. After termination, liver tissue and plasma samples were collected for further analysis. The day 3 sample was used to assess the single-dose effect of ETXM administered on day 0. The day 7 sample represented the repeated-dose effect of ETXM administered on days 0 and 3. Similarly, the day 10 and day 14 samples represented the repeated-dose effect of ETXM administered on days 0, 3, and 7. On day 14, five mice assigned to the control group were sacrificed.

[1275] B4GALT1 gene knockdown in mouse liver

[1276] Collected liver samples were used to measure B4GALT1 mRNA knockdown levels by RT-qPCR. After collection, each tissue was treated with RNAlater and stored at 4°C overnight, then stored at -80°C until further analysis. Liver tissue was homogenized with TRIZOL for RNA extraction. RNA samples adjusted to 400 ng / μL were reverse transcribed into cDNA using the FastKing RT kit manufactured by TIANGEN. After the gDNA removal step, the purified cDNA samples were used for RT-qPCR. The RT-qPCR method and relative mRNA expression calculation were as described above. Figure 16 All tested articles demonstrated >50% gene knockdown efficiency on days 3, 7, 10, and 14.

[1277] Terminal plasma collection and measurement of plasma biomarkers using a biochemistry analyzer

[1278] After a 4-5 hour fast, a terminal plasma sample was collected via the submandibular vein. Blood samples were collected in sodium heparin-coated tubes and then centrifuged at 7000 g for 10 minutes at 4°C to obtain plasma samples. Plasma samples were used to measure AST, ALT, albumin, ALP, BUN, CREA, TBIL, glucose, total cholesterol, LDL-c, HDL-c, triglycerides, and NEFA (free fatty acids) using a biochemistry analyzer.

[1279] Plasma insulin and fibrinogen levels were measured using ELISA kits

[1280] Blood samples were collected in K2EDTA-coated tubes and then centrifuged at 7000 g for 10 minutes at 4°C to obtain plasma samples. Plasma insulin levels were measured using a mouse insulin ELISA kit (Mercodia, 10-1247-01) according to the manufacturer's protocol. Fibrinogen plasma levels were measured using a mouse fibrinogen antigen detection kit (Innovative Research, IMSFBGKTT).

[1281] The role of B4GALT1 gene silencing in biomarker regulation

[1282] The two-tailed t-test was used to test the equality of the means of the untreated control group (n=5) and the treatment groups on day 14, which included groups that received subcutaneous injections of ETXM619, ETXM624, ETXM628, or ETXM633 on days 0, 3, and 7 (n=3 per group, total n=12). Statistically significant differences in the mean efficacy biomarker readings were detected, including an 18.8% reduction in LDL-C (p<0.05), a 21.0% reduction in fasting glucose (p<0.05), and a 29.6% reduction in fibrinogen (p<0.01) ( Figure 17 ).

[1283] The scope of the present application is not limited to disclosed specific embodiments, for example, these embodiments are provided to illustrate the various aspects of the present application. According to the description and teaching of this paper, the various modifications to the compositions and methods will become apparent. Such changes can be implemented without departing from the true scope of the application and the spirit, and these changes are intended to fall within the scope of the present application.

[1284] If there is any ambiguity between the sequence in this specification and the sequence in the attached sequence listing, the sequence provided in this specification shall be deemed to prevail.

Claims

1. A nucleic acid for inhibiting B4GALT1 expression, comprising a double-stranded region that includes a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is: (i) at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, and (ii) comprises at least 17 consecutive nucleosides that differ from any of the first strand sequences listed in Table 2 by 0 or 1 nucleoside.

2. A nucleic acid for inhibiting B4GALT1 expression, comprising a double-stranded region that includes a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is: (i) at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, and (ii) comprises at least 17 consecutive nucleosides that differ from any of the first strand modified sequences listed in Table 3 by 0 or 1 nucleoside.

3. The nucleic acid according to claim 1 or 2, wherein the first strand comprises nucleosides 2-18 of any of the sequences defined in claim 1 or 2, in particular wherein the first strand comprises nucleosides 2-18 of any of the sequences defined in Table 2 or Table 3.

4. The nucleic acid according to claim 1, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second strand sequences listed in Table 2 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand among the 17 consecutive nucleosides.

5. The nucleic acid according to claim 2, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second strand modified sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand among the 17 consecutive nucleosides.

6. The nucleic acid according to claim 1, wherein the first strand comprises any of the first strand sequences listed in Table 2.

7. The nucleic acid according to claim 2, wherein the first strand comprises any of the first strand modified sequences listed in Table 3.

8. The nucleic acid according to claim 4, wherein the second strand comprises any of the second strand sequences listed in Table 2.

9. The nucleic acid according to claim 5, wherein the second strand comprises any of the second strand modified sequences listed in Table 4.

10. The nucleic acid according to claim 6, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:

41.

11. The nucleic acid according to claim 7, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:

81.

12. The nucleic acid according to claim 8, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:

61.

13. The nucleic acid according to claim 9, wherein the second strand comprises any one of the following sequences: SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:

101.

14. The nucleic acid according to claims 1 and 4, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

15. The nucleic acid according to claims 1 and 4, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

16. The nucleic acid according to claims 1 and 4, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

17. The nucleic acid according to claims 2 and 5, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

18. The nucleic acid according to claims 2 and 5, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

19. The nucleic acid according to claims 2 and 5, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

20. The nucleic acid according to claims 2 and 5, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

21. The nucleic acid according to claims 2 and 5, comprising a first strand and a second strand, the first strand and the second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside.

22. The nucleic acid according to any one of the preceding claims, wherein the length of the first strand is in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides.

23. The nucleic acid according to any one of the preceding claims, wherein the length of the second strand is in the range of 17 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 19 or 21 nucleotides.

24. The nucleic acid according to any one of the preceding claims, wherein the length of the double-stranded region of the nucleic acid is between 17 and 30 nucleotides, and the more preferred length is 19 or 21 nucleotides.

25. The nucleic acid according to any one of the preceding claims, wherein the length of the complementary region between the first strand and a part of the RNA transcribed from the B4GALT1 gene is between 17 and 30 nucleotides.

26. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is blunt-ended or comprises one or more single-stranded nucleotide overhangs, optionally wherein the overhang is located on the first strand or the second strand, preferably at the 3'-end of the first strand or the second strand, and / or wherein the overhang comprises 1 to 4 nucleotides, more preferably 2 nucleotides.

27. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is an siRNA oligonucleotide.

28. The nucleic acid according to any one of the preceding claims, wherein one or more nucleotides on the first strand and / or the second strand are modified to form modified nucleotides.

29. The nucleic acid according to claim 28, wherein one or more nucleotides on the first strand and / or the second strand comprise terminal modifications, base modifications, sugar modifications and / or backbone modifications.

30. The nucleic acid according to claim 28, wherein one or more nucleotides on the first strand and / or the second strand comprise sugar modifications, and the modification is a modification on the 2'-OH group of ribose sugar.

31. The nucleic acid according to claim 30, wherein the sugar modification comprises 2'-Me and / or 2'-F modifications.

32. The nucleic acid according to claim 28, wherein starting from position 1 of the first strand, the first strand comprises 2'-F modifications at position 2, position 6, position 14 or any combination thereof.

33. The nucleic acid according to claim 28, wherein starting from position 1 of the second strand, the second strand comprises 2'-F modifications at position 7, position 9, position 11 or any combination thereof.

34. The nucleic acid according to claim 28, wherein the first and second strands each comprise 2'-Me and 2'-F modifications.

35. The nucleic acid according to claim 28, wherein the nucleic acid comprises at least one thermolabile modification, appropriately located at one or more positions among positions 1 to 9 of the first strand counted 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 labile modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid, more preferably (S)-glycol nucleic acid; more preferably, the nucleic acid comprises at least one thermally labile modification at position 7 of the first strand starting from position 1 of the first strand.

36. The nucleic acid according to claim 28, which is an siRNA oligonucleotide, wherein, Starting from position 1 of the second strand, the siRNA oligonucleotide comprises at least 3 2'-F modifications at positions 6 to 12 of the second strand, for example 4, 5, 6 or 7 2'-F modifications at positions 6 to 12 of the second strand.

37. The nucleic acid according to claim 28, which is an siRNA oligonucleotide, wherein, Starting from position 1 of the second strand, the second strand comprises at least 3, for example 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand.

38. The nucleic acid according to claim 28, which is an 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 from the terminal nucleoside in the 3'-terminal region.

39. The nucleic acid according to claim 28, which is an siRNA oligonucleotide, wherein the first strand comprises 7 consecutive 2'-Me modifications in the 3'-terminal region, preferably including the terminal nucleoside of the 3'-terminal region.

40. The nucleic acid according to claim 28, which is an siRNA oligonucleotide, wherein each of the first and second strands comprises an alternating modification pattern, preferably a fully alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified as: (i) 2'-Me modifications on odd-position nucleosides counted from position 1 of the first strand, and (ii) 2'-F modifications on even-position nucleosides counted from position 1 of the first strand, and the nucleosides of the second strand are modified as: (i) 2'-F modifications on odd-position nucleosides counted from position 1 of the second strand, and (ii) 2'-Me modifications on even-position nucleosides counted from position 1 of the second strand.

41. The nucleic acid according to claim 28, which is an siRNA oligonucleotide, wherein the nucleosides of the first strand comprise a 2'-sugar modification pattern, wherein the modifications are at least selected from 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand does not consist of 4 or 6 2'-F modifications.

42. The nucleic acid according to claim 28, which is an siRNA oligonucleotide, wherein the nucleosides of the first strand comprise a 2'-sugar modification pattern, wherein the modifications are at least selected from 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications.

43. The nucleic acid according to any one of the preceding claims, which further 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 nucleotide is linked to an adjacent base nucleoside by a reverse internucleoside bond.

44. The nucleic acid according to claim 43, wherein the second strand comprises 2 consecutive abasic nucleosides in the 5' end region of the second strand, wherein, One such abasic nucleotide is the terminal nucleoside at the 5'-end of the second strand, and another abasic nucleoside is the penultimate nucleoside in the 5'-terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent first base nucleoside in the adjacent 5'-proximal terminal region by a reverse internucleoside bond; and (b) the reverse bond is a 5-5' reverse bond; and (c) when read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'-5'.

45. The nucleic acid according to claim 43, wherein the second strand comprises 2 consecutive abasic nucleosides, preferably in a hanging shape in the 3' end region of the second strand, wherein, One of such abasic nucleotides is the terminal nucleoside at the 3'-end of the second strand, and another abasic nucleoside is the penultimate nucleoside in the 3'-terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent first base nucleoside in the adjacent 3'-proximal terminal region by a reverse internucleoside bond; and (b) the reverse bond is a 3-3’ reverse bond; and (c) when read towards the end containing the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 5’-3’.

46. The nucleic acid according to claim 44, wherein: (i) each of the first and second strands has a length of 23 nucleosides; (ii) there are two phosphorothioate internucleoside bonds respectively between three consecutive positions in the region near the 5’ end of the second strand, wherein in the region near the 5’ end of the second strand, there is a first phosphorothioate internucleoside bond between the adjacent first nucleoside with a base and the adjacent second nucleoside with a base, and in the region near the 5’ end of the second strand, there is a second phosphorothioate internucleoside bond between the adjacent second nucleoside with a base and the adjacent third nucleoside with a base; (iii) there are two phosphorothioate internucleoside bonds respectively between three consecutive positions in the 5’ and 3’ end regions of the first strand, such that the terminal nucleosides of the 5’ and 3’ end regions of the first strand are respectively connected to their penultimate second nucleosides adjacent to 5’ and 3’ by phosphorothioate internucleoside bonds, and the first penultimate second nucleosides of each 5’ and 3’ are connected to their respective penultimate third nucleosides adjacent to 5’ and 3’ by phosphorothioate internucleoside bonds; and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties at its 3’ end region.

47. The nucleic acid according to claim 45, wherein: (i) each of the first and second strands has a length of 23 nucleosides; (ii) there are two phosphorothioate internucleoside bonds respectively between three consecutive positions in the region near the 3’ end of the second strand, wherein in the region near the 3’ end of the second strand, there is a first phosphorothioate internucleoside bond between the adjacent first nucleoside with a base and the adjacent second nucleoside with a base, and in the region near the 3’ end of the second strand, there is a second phosphorothioate internucleoside bond between the adjacent second nucleoside with a base and the adjacent third nucleoside with a base; (iii) there are two phosphorothioate internucleoside bonds respectively between three consecutive positions in the 5’ and 3’ end regions of the first strand, such that the terminal nucleosides of the 5’ and 3’ end regions of the first strand are respectively connected to their penultimate second nucleosides adjacent to 5’ and 3’ by phosphorothioate internucleoside bonds, and the first penultimate second nucleosides of each 5’ and 3’ are connected to their respective penultimate third nucleosides adjacent to 5’ and 3’ by phosphorothioate internucleoside bonds; and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties at its 5’ end region.

48. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid comprises one or more phosphorothioate internucleoside bonds.

49. The nucleic acid according to claim 48, wherein the one or more phosphorothioate internucleoside bonds are respectively located between at least three consecutive positions in the 5' or 3' proximal end region of the second strand, whereby the proximal end region is preferably adjacent to the terminal region, and wherein the one or more abasic nucleosides of the second strand are located at least according to claim 45.

50. The nucleic acid according to claim 48 or 49, wherein the one or more phosphorothioate internucleoside bonds are respectively located between at least three consecutive positions in the 5' and / or 3' end region of the first strand, and wherein preferably the terminal positions of the 5' and / or 3' end region of the first strand are connected to their adjacent positions by phosphorothioate internucleoside bonds.

51. The nucleic acid according to claim 31, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following: Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me.

52. The nucleic acid according to claim 31, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following: (Me)8-(F)3-(Me) 10 。 53. The nucleic acid according to claim 31, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following: Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, where (s) is a phosphorothioate internucleoside bond.

54. The nucleic acid according to claim 52, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following: Me(s)Me(s)(Me)6-(F )3 -(Me) 10 where (s) is a phosphorothioate internucleoside bond.

55. The nucleic acid according to claim 51, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following: ia-ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia-ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleotide represented by ia-ia is located at the 3'-end of the second strand, the inverted abasic nucleoside exists as a 2-nucleoside overhang.

56. The nucleic acid according to claim 52, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following: ia-ia-(Me)8-(F)3-(Me) 10 , wherein ia represents an inverted abasic nucleoside.

57. The nucleic acid according to claim 51, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following: ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or 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, or 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, or 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, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, Wherein: (s) is a phosphorothioate internucleoside bond; ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is located at the 3' end of the second strand, the inverted abasic nucleoside exists as a 2-nucleoside overhang.

58. The nucleic acid according to claim 52, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following: ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 , Wherein: (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

59. The nucleic acid according to claim 31, wherein the modified nucleoside comprises any one of the following modification patterns: 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-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; 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-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; 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; 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; 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; 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.

60. The nucleic acid according to claim 31, wherein the modified nucleoside comprises any one of the following modification patterns: Modification pattern 1: 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-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a thermally labile modification; Or modification pattern 2: 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-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Or modification pattern 3: 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-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me; Or modification pattern 4: 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-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-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-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a thermally labile modification; Or modification pattern 6: 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-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; Or modification pattern 7: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me; Or modification pattern 8: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me.

61. The nucleic acid according to claim 59, wherein the modified nucleoside comprises any one of the following modification patterns: 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; Or modification pattern 2: Second strand (5’-3’): Me(s)Me(s)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; 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; 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; 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; 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; wherein (s) is a phosphorothioate internucleoside bond.

62. The nucleic acid according to claim 60, wherein the modified nucleoside comprises any one of the following modification patterns: Modification pattern 1: 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-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, wherein X1 is a thermally labile modification; or modification pattern 2: 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-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 3: 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-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me; Or modification pattern 4: 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-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me; 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-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a thermally labile modification; Or modification pattern 6: 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-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 7: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me; Or modification pattern 8: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me; where (s) is a phosphorothioate internucleoside bond.

63. The nucleic acid according to claim 59, wherein the modified nucleoside comprises any one of the following modification patterns: 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; 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.

64. The nucleic acid according to claim 59, wherein the modified nucleoside comprises any one of the following modification patterns: 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; 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; 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; 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; or Modification pattern 5: 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, 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; or Modification pattern 6: Second strand (5’-3’): ia-ia-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-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me; wherein ia represents a reverse abasic nucleoside.

65. The nucleic acid according to claim 60, wherein the modified nucleoside comprises any one of the following modification patterns: Modification pattern 1: 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, First strand (5’-3’): Me-F-Me-Me-Me-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, wherein X1 is a thermally labile modification; or Modification pattern 2: 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, 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; or Modification pattern 3: 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, First strand (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; or Modification pattern 4: 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, First strand(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; or Modification pattern 5: 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, First strand (5’-3’): Me-F-Me-Me-Me-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, wherein X1 is a thermally labile modification; or Modification pattern 6: 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, 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; Or modification pattern 7: 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, First strand (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; Or modification pattern 8: 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, First strand (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; Wherein ia represents an inverted abasic nucleoside.

66. The nucleic acid according to claim 59, wherein the modified nucleoside comprises any one of the following modification patterns: 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-Me-Me-ia-ia, 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; 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-Me-Me-ia-ia, 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; 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-ia-ia, 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; 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-ia-ia, 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; 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-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; 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-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 located at the 3'-end of the second strand, the inverted abasic nucleoside exists as a 2-nucleoside overhang.

67. The nucleic acid according to claim 55, wherein the modified nucleoside comprises any one of the following modification patterns: Modification pattern 1: Second strand (5'-3'): ia-ia-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; Or modification pattern 2: Second strand (5'-3'): ia-ia-Me(s)Me(s)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; 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; 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; 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; 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; Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside.

68. The nucleic acid according to claim 60, wherein the modified nucleoside comprises any one of the following modification patterns: Modification pattern 1: 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-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a thermally labile modification; Or modification pattern 2: 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-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 3: 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-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me; Or modification pattern 4: 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-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me; 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-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a thermally labile modification; Or modification pattern 6: 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-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 7: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me; Or modification pattern 8: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me; wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside.

69. The nucleic acid according to claim 59, wherein the modified nucleoside comprises any one of the following modification patterns: 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-ia-ia, 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-ia-ia, 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-ia-ia, 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-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 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-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 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-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; Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is located at the 3’ end of the second strand, the inverted abasic nucleoside exists as a 2-nucleoside overhang.

70. 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 located in the terminal region of the second strand, preferably in its 3’ end region.

71. The nucleic acid according to claim 70, wherein the ligand moiety comprises: i) one or more N-acetylgalactosamine (GalNAc) ligands; and / or ii) one or more derivatives of N-acetylgalactosamine (GalNAc) ligands; and / or iii) one or more N-acetylgalactosamine (GalNAc) ligands and / or GalNAc ligand derivatives conjugated to the nucleic acid via a linker.

72. The nucleic acid according to claim 71, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly conjugated to the 5’ or 3’ end region of the second strand of the nucleic acid, preferably in its 3’ end region.

73. The nucleic acid according to any one of claims 70 to 72, wherein, The ligand moiety comprises the following structure:

74. The nucleic acid according to any one of claims 70 to 73, which comprises the following structure: Wherein: R1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears; R2 is selected from the group consisting of: hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro; X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur each time they appear; 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 both be 0; and (ii) s, t and v cannot all be 0 at the same time; Z is an oligonucleoside moiety.

75. The nucleic acid according to claim 74, which comprises the following structure: Where [oligonucleotide] represents consecutive nucleosides of the second strand.

76. The nucleic acid according to any one of claims 70 to 73, which comprises the following structure: Wherein: r and s are independently integers selected from 1 to 16; and Z is an oligonucleoside moiety.

77. The nucleic acid according to claim 76, which comprises the following structure: Where [oligonucleotide] represents consecutive nucleosides of the second strand.

78. The nucleic acid according to any one of claims 70 to 77, wherein the nucleic acid comprises the modification pattern: 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; Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and wherein the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, optionally wherein the ligand moiety is located in the terminal region of the second strand, preferably in its 3’-terminal region.

79. The nucleic acid according to claim 78, wherein the second strand comprises the structure: or wherein [oligonucleotide] represents consecutive 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.

80. The nucleic acid according to claim 78 or 79, wherein the first and second strands comprise, consist of, or consist essentially of a nucleoside sequence that differs from either of the following first and second sequences by 0 or 1 nucleoside:

81. The nucleic acid according to any one of claims 78 to 80, wherein 2 consecutive inverted abasic nucleosides in the 5’-terminal region of the second strand are present in the following 5’-terminal motif wherein: T represents a 2’Me ribose modification, B represents the nucleobase of the first two base nucleosides in the 5’-terminal region of the second strand, and Z represents the remaining 19 consecutive base nucleosides of the second strand.

82. A pharmaceutical composition comprising the nucleic acid according to any one of the preceding claims, and a pharmaceutically acceptable excipient or carrier.

83. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for treatment.

84. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for the prevention or treatment of diabetes.

85. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for the prevention or treatment of cardiovascular diseases.

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