NOVEL NUCLEOTIDES AND OLIGONUCLEOTIDES AND RNAi AGENTS COMPRISING THE SAME

Abstract

This invention relates to novel destabilizing molecules (such as butylene glycol moiety), modified nucleosides incorporating butylene glycol moiety, modified nucleotides incorporating butylene glycol moiety, and oligonucleotides and RNAi agents containing such modified nucleotides, which improve selectivity and / or reduce off-target effects while maintaining or improving attenuation of on-target gene expression.

Description

Technical Field

[0001] This invention relates to novel nucleotides comprising a butanediol moiety. This invention also relates to novel nucleosides comprising a butanediol moiety. This invention further relates to oligonucleotides comprising the novel nucleoside or the novel nucleotide. This invention also relates to oligonucleotides comprising novel destabilized nucleotides, such as RNAi agents, wherein the destabilized nucleotide comprises a butanediol moiety. This invention also relates to a method for increasing the selectivity of RNAi agents and / or oligonucleotides by introducing destabilized nucleotides comprising a butanediol moiety. Prior Technology

[0002] Oligonucleotides, which contain molecules that regulate the amount of mRNA in genes, are important for molecular biology research and are increasingly showing promise as therapeutics.

[0003] RNA interference (RNAi) is a highly conserved regulatory mechanism in which RNA molecules participate in the sequence-specific inhibition of gene expression by double-stranded RNA molecules.

[0004] Because RNA is relatively unstable, significant efforts have been made to balance the stability and sequence-specific attenuation capabilities of RNA molecules with any cellular or organ toxicity. The nucleotides of double-stranded RNA have been modified to improve attenuation selectivity by reducing hybridization to off-target RNA sequences. However, nucleotide modifications that reduce off-target hybridization (such as ethylene glycol-containing nucleotides (ethylene glycol nucleic acids, GNAs)) have been shown to reduce both off-target and on-target binding. Therefore, such destabilizing molecules, which are more selective in reducing off-target binding, are still needed. Summary of the Invention

[0005] This article reveals oligonucleotides containing the following formula: , Wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are one or more independently selected nucleotides, X4 is the 3' portion of the oligonucleotide, m and n are independently selected from any integer from 0 to 40, and XB is a butanediol-modified nucleotide.

[0006] This article reveals oligonucleotides containing the following formula: , Wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is the 3' portion of the oligonucleotide, B is a nucleobase, and m and n are any integers independently selected from 0 to 40.

[0007] This article also reveals oligonucleotides containing the following formula: , Wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is the 3' portion of the oligonucleotide, B is a nucleobase, and m and n are any integers independently selected from 0 to 40.

[0008] This article also reveals oligonucleotides containing the following formula: , Wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is the 3' portion of the oligonucleotide, B is a nucleobase, and m and n are any integers independently selected from 0 to 40.

[0009] This article reveals the following oligonucleotide formula: In this system, each B is an independent nucleobase, m and n are independently selected from any integer from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

[0010] This article also reveals the following oligonucleotide formula: In this system, each B is an independent nucleobase, m and n are independently selected from any integer from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

[0011] This article also reveals the following oligonucleotide formula: In this system, each B is an independent nucleobase, m and n are independently selected from any integer from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

[0012] This article also reveals the following compound: , Among them are B-series nucleobases and Y-series organic protecting groups.

[0013] This article also reveals the following compound: , The B-series nucleobases, Y-series organic protecting groups, and Z-series phosphate esters or phosphorous amines represented by the following formula: .

[0014] In some embodiments, the oligonucleotide has a butanediol-modified nucleotide at positions 3, 4, 5, 6, 7, or 8 counting from the 5' end. In other embodiments, the butanediol-modified nucleotide is at position 5, 6, 7, or 8 counting from the 5' end. In other embodiments, the butanediol-modified nucleotide is at position 5 counting from the 5' end. In other embodiments, the butanediol-modified nucleotide is at position 8 counting from the 5' end.

[0015] In some embodiments, the oligonucleotide has a butanediol-modified nucleotide at position 22, counting from the 5' end. In other embodiments, the oligonucleotide has a butanediol-modified nucleotide at position 23, counting from the 5' end. In still other embodiments, the oligonucleotide has butanediol-modified nucleotides at both positions 22 and 23, counting from the 5' end.

[0016] In some embodiments, the oligonucleotide of Formula I is 15 to 30 nucleotides in length. In some embodiments, the oligonucleotide is 18 to 30 nucleotides in length. In other embodiments, the oligonucleotide is 23 nucleotides in length.

[0017] In some embodiments, R in the oligonucleotide of formula I is 2' fluorine at one of the following positions: a. From the 5' end, 2, 3, 7, 14 and 16; b. From the 5' end at points 2, 5, 7, 14, and 16; c. From the 5' end at points 2, 3, 8, 14, and 16; d. From the 5' end at points 2, 5, 8, 14, and 16; e. From the 2nd, 14th, and 16th of the 5' end; or f. From the 5' end at points 2, 6, 14, and 16, And the R is 2'O-methyl at all other positions in the oligonucleotide.

[0018] This article also discloses an RNA agent comprising a sense oligonucleotide and an antisense oligonucleotide, each independently having a length of 15 to 30 nucleotides, wherein the sense oligonucleotide and the antisense oligonucleotide form a double strand, wherein, if desired, one or more sense nucleotides and the antisense nucleotide are independently modified nucleotides, and wherein, if desired, the bond between one or more sense oligonucleotides and the antisense oligonucleotide is a modified nucleotide bond, and the antisense oligonucleotide is an oligonucleotide provided herein.

[0019] This article also reveals RNAi agents containing a 21-nucleotide sense strand, wherein each nucleotide of the sense strand is a modified nucleotide, and the modified nucleotide containing the 2'-fluorine modification is located at one of the following positions: a. Positions 9, 10, and 11 from the 5' end; b. Positions 7, 9, and 11 from the 5' end; c. Positions 7, 9, and 10 from the 5' end; and d. Positions 7, 10, and 11 from the 5' end, Furthermore, the 2'O-methyl modification is located at all other positions of the sense oligonucleotide.

[0020] This article also discloses a method for increasing the selectivity of RNAi agents, which includes replacing the sugar portion with butylene glycol. The RNAi agents of this invention may include, as needed, a delivery portion conjugated to the RNA duplex via a linker. In some embodiments, the delivery portion may be a GalNAc portion. In some embodiments, the delivery portion is a sense strand conjugated to the RNA duplex. Implementation

[0021] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 560,890, filed March 4, 2024, pursuant to 35 USC §119(e), the disclosure of which is incorporated herein by reference. sequence list

[0022] This application is submitted together with a sequence list in ST.26 XML format. The sequence list is provided as a file titled "30407_WO" created on February 13, 2025, and is 1,351 kilobytes in size. The ST.26 XML format sequence list information is incorporated herein by reference in its entirety.

[0023] Therefore, this paper reveals novel destabilizing molecules, modified nucleotides incorporating such destabilizing molecules, and oligonucleotides and RNAi agents containing such modified nucleotides. These modified nucleotides improve the selectivity of attenuated target gene expression or reduce off-target effects, while maintaining or improving attenuated target gene expression.

[0024] It has been surprisingly found that incorporating the revealed destabilizing molecules into nucleotides, oligonucleotides and / or RNAi agents can improve the selectivity of attenuated target gene expression relative to unmodified nucleotides, oligonucleotides and / or RNAi agents and relative to other modified RNAi agents (such as GNA). definition

[0025] The RNAi agents described in this article contain sense and antisense molecules, each of which is an oligonucleotide.

[0026] As used herein, "antisense" refers to a single-stranded oligonucleotide that is complementary to a region of the target sequence. Similarly, as used herein, "sense" refers to a single-stranded oligonucleotide that is complementary to a region of the antisense.

[0027] As used herein, "oligonucleotide" means a polymer of linked nucleotides, each of which may be modified or unmodified. A typical oligonucleotide is less than about 100 nucleotides in length. Oligonucleotides can be single-stranded (ss) or double-stranded (ds). Oligonucleotides may or may not have double-stranded regions. As a set of non-limiting examples, oligonucleotides may be (but are not limited to) small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer saturated interfering RNA (DsiRNA), or antisense oligonucleotides (ASO).

[0028] As used herein, "nucleotide" means an organic compound having a nucleoside (nucleobase, such as, for example, adenine, cytosine, guanine, thymine, or uracil); a pentose, such as, for example, ribose or 2'-deoxyribose; and a phosphate ester group. "Nucleotides" can serve as monomeric units of nucleic acid polymers, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0029] As used herein, "modified nucleotide linkage" means a nucleotide linkage that has one or more chemical modifications when compared to a reference nucleotide linkage having a phosphodiester bond. Modified nucleotide linkages may be non-naturally occurring linkages. In some embodiments, modified nucleotide linkages are phosphate thioester linkages.

[0030] As used herein, "modified nucleotide" means a nucleotide that has one or more chemical modifications when compared to the corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. Modified nucleotides may be non-naturally occurring nucleotides. Modified nucleotides may have, for example, one or more chemical modifications in their sugar, nucleotide, and / or phosphate groups. Additionally or alternatively, modified nucleotides may have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0031] As used herein, "phosphate ester analog" means a chemical part that mimics the electrostatic and / or stereochemical properties of a phosphate ester group. In some embodiments, the phosphate ester analog is a 5'-terminal nucleotide of an oligonucleotide in place of a 5'-phosphate ester. The 5'-phosphate ester analog may include a phosphatase-resistant linker. Examples of phosphate ester analogs include (but are not limited to) 5'-phosphonates, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). The oligonucleotide may have a phosphate ester analog (referred to as "4'-phosphate ester analog") at the 4'-carbon position of the sugar in the 5'-terminal nucleotide. One example of a 4'-phosphate ester analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon) or an analog thereof. In one embodiment of the butanediol-modified RNAi agent described herein, the 5'-terminal nucleotide of the antisense strand of the RNA duplex may contain an OH group, a phosphate ester group, or a phosphate ester analog. In one embodiment, the 5' terminal nucleotide of the antisense strand comprises a vinylphosphonate, a phosphate, or a hydroxyl group.

[0032] As used herein, the term "double strand" in relation to nucleic acids or oligonucleotides (such as sense strands or antisense strands) means a structure formed by hydrogen bonds between complementary bases of two antiparallel sequences of nucleotides under conditions suitable for promoting this structure. Double strands can form even when the two strands are not perfectly complementary, or when a baseless nucleotide is present.

[0033] As used in this article, "base-free" means purine-free or pyrimidine-free nucleotides, in which the N-glycosyl bond is cleaved between the nitrogenous base and the deoxyribose, leaving the complete phosphodiester backbone.

[0034] As used herein, "iRNA," "iRNA agent," "RNAi," "RNAi agent," and "RNA interfering agent" mean an agent containing RNA and mediating targeted cleavage of RNA transcripts via RNA interference, for example, through the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense oligonucleotide and an antisense oligonucleotide, and the sense oligonucleotide and antisense oligonucleotide form a double strand. In some embodiments, the sense and antisense oligonucleotides of the RNAi agent are 21 to 23 nucleotides in length. In other embodiments, the sense and antisense oligonucleotides may be longer, for example, 25 to 30 nucleotides in length, in which case the longer RNAi sequence is first treated with the Dicer enzyme. In some embodiments, the sense or antisense oligonucleotide is conjugated to the delivery site to facilitate intracellular entry into various tissues. In some embodiments, the sense or antisense oligonucleotide is directly conjugated to the delivery site. In other embodiments, the sense or antisense oligonucleotide is indirectly conjugated to the delivery site via a linker.

[0035] As used herein, “reduced expression” or “reduced performance”, such as in relation to a gene, means a decrease in the amount or concentration of RNA transcripts or proteins encoded by the gene and / or a decrease in the amount or concentration of the gene’s activity in the cell, cell population, sample, or individual compared to a suitable reference (e.g., a reference cell, cell population, sample, or individual).

[0036] As used herein, a "strand" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds or thiophosphate bonds). A strand may have two free ends (e.g., a 5' end and a 3' end).

[0037] As used herein, "HPRT" refers to inosine-guanine phosphoribosyltransferase 1 (HPRT) mRNA, protein, or polypeptide. The nucleic acid sequence of the mouse HPRT mRNA transcript can be found at NM_013556.2:

[0038] The amino acid sequence of mouse HPRT protein can be found at NP_038584.2: MPTRSPSVVISDDEPGYDLDLFCIPNHYAEDLEKVFIPHGLIMDRTERLARDVMKEMGGHHIVALCVLKGGYKFFADLLDYIKALNRNSDRSIPMTVDFIRLKSYCNDQ STGDIKVIGGDDLSTLTGKNVLIVEDIIDTGKTMQTLLSLVKQYSPKMVKVASLLVKRTSRSVGYRPDFVGFEIPDKFVVGYALDYNEYFRDLNHVCVISETGKAKYKA (SEQ ID: 96)

[0039] As used herein, "Fas" refers to the Fas cell surface death receptor (Fas) mRNA, protein, or polypeptide. The nucleic acid sequence of the human Fas mRNA transcript can be found at NM_000043.6: (SEQ ID: 97)

[0040] The amino acid sequence of the human Fas protein can be found at NP_000034.1: MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKE EGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID: 98)

[0041] As used herein, "Nav1.8" refers to sodium voltage-gated channel alpha subunit 10 (SCN10A) (Nav1.8) mRNA, protein, or polypeptide. The nucleic acid sequence of the human Nav1.8 mRNA transcript can be found at NM_001293306.2.

[0042] The amino acid sequence of the human Nav1.8 protein can be found at NP_001280235.2:

[0043] As used herein, "Ang8" refers to angiopoietin-like 8 (Ang8) mRNA, protein, or polypeptide. The nucleic acid sequence of the human Ang8 mRNA transcript can be found in NM_018687.7: ATACCTTAGACCCTCAGTCATGCCAGTGCCTGCTCTGTGCCTGCTCTGGGCCCTGGCAATGGTGACCCGGCCTGCCTCAGCGGCCCCCATGGGCGGCCCAGAACTGGCACAGCATGAGGAGCTGACCCTGCTCTTCCATGGGACCCTGCAGCTGGGCCAGGCCCTCAACGGTGTGTACAGGACCACGGAGGGACGGCTGACAAAGGCCAGGAACAGCCTGGGTCTCTATGGCCGCACAATAGAACTCCTGGGGCAGGAGGTCAGCCGGGGCCGGGATGCAGCCCAGGAACTTCGGGCAAGCCTGTTGGAGACTCAGATGGAGGAGGATATTCTGCAGCTGCAGGCAGAGGCCACAGCTGAGGTGCTGGGGGAGGTGGCCCAGGCACAGAAGGTGCTACGGGACAGCGTGCAGCGGCTAGAAGTCCAGCTGAGGAGCGCCTGGCTGGGCCCTGCCTACCGAGAATTTGAGGTCTTAAAGGCTCACGCTGACAAGCAGAGCCACATCCTATGGGCCCTCACAGGCCACGTGCAGCGGCAGAGGCGGGAGATGGTGGCACAGCAGCATCGGCTGCGACAGATCCAGGAGAGACTCCACACAGCGGCGCTCCCAGCCTGAATCTGCCTGGATGGAACTGAGGACCAATCATGCTGCAAGGAACACTTCCACGCCCCGTGAGGCCCCTGTGCAGGGAGGAGCTGCCTGTTCACTGGGATCAGCCAGGGCGCCGGGCCCCACTTCTGAGCACAGAGCAGAGACAGACGCAGGCGGGGACAAAGGCAGAGGATGTAGCCCCATTGGGGAGGGGTGGAGGAAGGACATGTACCCTTTCATGCCTACACACCCCTCATTAAAGCAGAGTCGTGGCATCTCA (SEQ ID: 101)

[0044] The amino acid sequence of the human Ang8 protein can be found at NP_061157.3: MPVPALCLLWALAMVTRPASAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA (SEQ ID: 102)

[0045] As used herein, "HMGCR" refers to 3-hydroxy-3-methylpentanediol-CoA reductase mRNA, protein, or polypeptide. The nucleic acid sequence of the human HMGCR mRNA transcript can be found at NM_000859.3:

[0046] The amino acid sequence of human HMGCR protein can be found in NP_000850.1: MLSRLFRMHGLFVASHPWEVIVGTVTLTICMMSMNMFTGNNKICGWNYECPKFEEDVLSSDIIILTITRCIAILYIYFQFQNLRQLGSKYILGIAGLFTIFSSFVFSTVVIHFLDKELTGLNEALPFFLLLIDLSRASTLAKFALSSNSQDEVRENIARGMAILGPTFTLDALVECLVIGVGTMSGVRQLEIMCCFGCMSVLANYFVFMTFFPACVSLVLELSRESREGRPIWQLSHFARVLEEEENKPNPVTQRVKMIMSLGLVLVHAHSRWIADPSPQNSTADTSKVSLGLDENVSKRIEPSVSLWQFYLSKMISMDIEQVITLSLALLLAVKYIFFEQTETESTLSLKNPITSPVVTQKKVPDNCCRREPMLVRNNQKCDSVEEETGINRERKVEVIKPLVAETDTPNRATFVVGNSSLLDTSSVLVTQEPEIELPREPRPNEECLQILGNAEKGAKFLSDAEIIQLVNAKHIPAYKLETLMETHERGVSIRRQLLSKKLSEPSSLQYLPYRDYNYSLVMGACCENVIGYMPIPVGVAGPLCLDEKEFQVPMATTEGCLVASTNRGCRAIGLGGGASSRVLADGMTRGPVVRLPRACDSAEVKAWLETSEGFAVIKEAFDSTSRFARLQKLHTSIAGRNLYIRFQSRSGDAMGMNMISKGTEKALSKLHEYFPEMQILAVSGNYCTDKKPAAINWIEGRGKSVVCEAVIPAKVVREVLKTTTEAMIEVNINKNLVGSAMAGSIGGYNAHAANIVTAIYIACGQDAAQNVGSSNCITLMEASGPTNEDLYISCTMPSIEIGTVGGGTNLLPQQACLQMLGVQGACKDNPGENARQLARIVCGTVMAGELSLMAALAAGHLVKSHMIHNRSKINLQDLQGACTKKTA (SEQ ID: 104) Butanediol

[0047] This article reveals novel butanediol moieties that can be incorporated into one or more nucleosides and / or nucleotides by substituting one or more sugar moieties. The modified nucleotides can be incorporated into oligonucleotides (such as RNAi agents) as destabilizing agents in RNAi agents. The butanediol moieties can be represented by compounds of formula A, A', and / or A''. Formula A. Butanediol, where B is a nucleobase. Formula A'. (R,S)-Butanediol, where B is a nucleobase. Formula A''. (S,S)-Butanediol, where B is a nucleobase.

[0048] The butanediol moiety may comprise a mixture of (R,S) and (S,S) non-mirror image isomers (Formula A), (R,S) non-mirror image isomers (Formula A'), or (S,S) non-mirror image isomers (Formula A''). In some embodiments, the butanediol moiety may comprise (R,R), (R,S), (S,S), and / or (S,R) non-mirror image isomers. Surprisingly, it has been found that introducing (S,S)-butanediol into the nucleotide at position 8 of the oligonucleotide improves the targeting selectivity of the oligonucleotide.

[0049] In formulas A, A', and A'', nucleobase (B) can be any nucleobase. Nucleobases can be naturally occurring nucleobases, non-naturally occurring artificial or modified nucleobases, or combinations thereof. Suitable modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouridine, 5-methylcytidine, and / or pseudouridine. Suitable naturally occurring nucleobases may include adenine, guanine, cytosine, thymine, and / or uracil. In one state, the nucleobase group is selected from the following: adenine, cytosine, thymine, uracil, and guanine. In one state, the nucleobase group is selected from the following: adenine, cytosine, and uracil. In another state, the nucleobase system is selected from the following groups: cytosine and uracil. In another state, the nucleobase system is uracil. nucleosides

[0050] This paper discloses a novel nucleoside comprising a butanediol moiety. As disclosed herein, the butanediol moiety can be incorporated into one or more nucleosides by substituting the sugar moiety in the nucleoside. The novel nucleoside may be represented by compounds of formula B, B', and / or B''. Formula B. Novel nucleosides, wherein the A group is independently selected from H, phosphate esters, C1 to C6 alkyl groups and organic protecting groups, and the B group is a nucleobase. Formula B'. Novel nucleosides, wherein the A group is independently selected from H, phosphate esters, C1 to C6 alkyl groups and organic protecting groups, and the B group is a nucleobase. Formula B''. Novel nucleosides, wherein the A group is independently selected from H, phosphate esters, C1 to C6 alkyl groups and organic protecting groups, and the B group is a nucleobase.

[0051] In formulas B, B', and B'', A can be independently selected from H, phosphate esters, C1 to C6 alkyl groups, and organic protecting groups. Suitable organic protecting groups may include methoxymethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytriphenylmethyl (DMT), methoxytriphenylmethyl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), trimethylacetyl (Piv), tetrahydropiperanyl (THP), tetrahydrofuran (THF), triphenylmethyl (Tr), and / or trimethylsilyl (TMS). The organic protecting group may include dimethoxytriphenylmethyl (DMT).

[0052] In formulas B, B', and B'', the nucleobase (B) can be any nucleobase. The nucleobase can be a naturally occurring nucleobase, a non-naturally occurring artificial or modified nucleobase, or a combination thereof. Suitable modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouridine, 5-methylcytidine, and / or pseudouridine. Suitable naturally occurring nucleobases may include adenine, guanine, cytosine, thymine, and / or uracil. In one state, the nucleobase group is selected from the following: adenine, cytosine, thymine, uracil, and guanine. In one state, the nucleobase group is selected from the following: adenine, cytosine, and uracil. In another state, the nucleobase system is selected from the following groups: cytosine and uracil. In another state, the nucleobase system is uracil.

[0053] Novel nucleosides may also be represented by compounds of formula C, C' and / or C''. Formula C. Novel nucleosides, wherein B-series nucleobases and Y-series organic protecting groups. Formula C'. Novel nucleoside, wherein B-series nucleobases and Y-series organic protecting groups. Formula C''. Novel nucleoside, wherein B-series nucleobases and Y-series organic protecting groups.

[0054] Novel nucleosides may also be represented by compounds of formula D, D' and / or D''. Formula D. Novel nucleosides, in which B-series nucleobases. Formula D'. Novel nucleoside, wherein B is a nucleobase. Formula D''. Novel nucleoside, in which B is a nucleobase.

[0055] In formulas C, C', and C'', the organic protecting group may include methoxymethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytriphenylmethyl (DMT), methoxytriphenylmethyl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), trimethylacetyl (Piv), tetrahydropiperanyl (THP), tetrahydrofuran (THF), triphenylmethyl (Tr), and / or trimethylsilyl (TMS). The organic protecting group may include dimethoxytriphenylmethyl (DMT).

[0056] In formulas C, C', C'', D, D', and / or D'', the nucleobase (B) can be any nucleobase. The nucleobase can be a naturally occurring nucleobase, a non-naturally occurring artificial or modified nucleobase, or a combination thereof. Suitable modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouridine, 5-methylcytidine, and / or pseudouridine. Suitable naturally occurring nucleobases may include adenine, guanine, cytosine, thymine, and / or uracil. In one state, the nucleobase group is selected from the group consisting of: adenine, cytosine, thymine, uracil, and guanine. In one state, the nucleobase group is selected from the group consisting of: adenine, cytosine, and uracil. In another state, the nucleobase system is selected from the following groups: cytosine and uracil. In another state, the nucleobase system is uracil. Nucleotides

[0057] This article discloses a novel nucleotide comprising a butanediol moiety, a nucleobase, and a phosphate ester or phosphorylamine to allow selective incorporation into RNAi agents (such as oligonucleotides). As disclosed herein, the butanediol moiety can be incorporated into one or more nucleotides by substituting the sugar moiety in the nucleotide. The novel nucleotide may be represented by compounds of formula E, E', and / or E''. Formula E. A novel nucleotide, wherein B is a nucleobase, Y is an organic protecting group, and Z is a phosphate ester or a phosphorylated amine represented by Formula F. Formula E'. A novel nucleotide, wherein B is a nucleobase, Y is an organic protecting group, and Z is a phosphate ester or a phosphorylated amine represented by Formula F. Formula E''. Novel nucleotide, wherein B is a nucleobase, Y is an organic protecting group, and Z is a phosphate ester or a phosphorylated amine represented by Formula F. Formula F. phosphorous amine

[0058] In formulas E, E', and E'', the organic protecting group may include methoxymethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytriphenylmethyl (DMT), methoxytriphenylmethyl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), trimethylacetyl (Piv), tetrahydropiperanyl (THP), tetrahydrofuran (THF), triphenylmethyl (Tr), and / or trimethylsilyl (TMS). The organic protecting group may include dimethoxytriphenylmethyl (DMT).

[0059] Novel nucleotides may also be represented by compounds of the formula G, G' and / or G''. Formula G. Modified nucleotides, in which B is a nucleobase. Formula G'. is a modified nucleotide in which B is a nucleobase. Formula G''. Modified nucleotides, in which B is a nucleobase.

[0060] In formulas E, E', E'', G, G', and / or G'', the nucleobase (B) can be any nucleobase. The nucleobase can be a naturally occurring nucleobase, a non-naturally occurring artificial or modified nucleobase, or a combination thereof. Suitable modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouridine, 5-methylcytidine, and / or pseudouridine. Suitable naturally occurring nucleobases may include adenine, guanine, cytosine, thymine, and / or uracil. In one state, the nucleobase group is selected from the group consisting of: adenine, cytosine, thymine, uracil, and guanine. In one state, the nucleobase group is selected from the group consisting of: adenine, cytosine, and uracil. In another state, the nucleobase system is selected from the following groups: cytosine and uracil. In another state, the nucleobase system is uracil. Oligonucleotides

[0061] This paper discloses a novel oligonucleotide containing a butanediol moiety. As disclosed herein, the butanediol moiety can be incorporated into one or more nucleotides by replacing the sugar portion of the nucleotide.

[0062] Novel oligonucleotides can be represented by compounds of Formula 1. Formula H. Oligonucleotide, wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are one or more independently selected nucleotides, X4 is the 3' portion of the oligonucleotide, m and n are independently selected from any integer from 0 to 40, and XB is a butanediol-modified nucleotide.

[0063] In formula H, m and n can be independently selected from any integer from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, m can be from 0 to 7, n can be from 0 to 20, and m+n can be from 0 to 40.

[0064] Novel oligonucleotides may also be represented by compounds of formula I, I' and / or I''. Formula I. Oligonucleotide, wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is the 3' portion of the oligonucleotide, and m and n are any integers independently selected from 0 to 40. Formula I'. Oligonucleotide, wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is the 3' portion of the oligonucleotide, and m and n are any integers independently selected from 0 to 40. Formula I''. Oligonucleotide, wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is the 3' portion of the oligonucleotide, and m and n are any integers independently selected from 0 to 40.

[0065] The 5' portion of the oligonucleotide (such as X1 in formula I, I', and / or I'') is the 5' end of the oligonucleotide. The 5' portion of the oligonucleotide may include nucleotides, nucleosides, nucleobases, pentose sugars, phosphate esters, vinylphosphonates, halogens, alcohols, C1 to C20 alkyl groups, C1 to C20 alkyl-O-C1 to C20 alkyl groups, and / or combinations thereof. The 5' end of the oligonucleotide can be readily modified using methods known to those skilled in the art.

[0066] The 3' portion of the oligonucleotide (such as X4 in formula I, I', and / or I'') is the 3' end of the oligonucleotide. The 5' portion of the oligonucleotide may include nucleotides, nucleosides, nucleobases, pentose sugars, phosphate esters, vinylphosphonates, halogens, alcohols, C1 to C20 alkyl groups, C1 to C20 alkyl-O-C1 to C20 alkyl groups, and / or combinations thereof. The 3' end of the oligonucleotide can be readily modified using methods known to those skilled in the art.

[0067] Oligonucleotides may also include one or more nucleotides that do not contain a butanediol moiety, such as X2 and X3 in formulas I, I' and / or I''. Suitable nucleotides may include pentose sugars, phosphate esters, nucleobases and / or other phosphate ester linkers.

[0068] In formulas I, I', and / or I'', m and n can be independently selected from any integer from 0 to 20. In one embodiment, m can be any integer from 0 to 7, n can be any integer from 0 to 40, and m+n can be any integer from 0 to 40.

[0069] Novel oligonucleotides can be represented by compounds of formula J. Formula J. Oligonucleotides and / or RNAi agents, wherein B is a nucleobase, m and n are independently selected from any integer from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

[0070] In formula J, m and n can be independently selected from any integer from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0071] In Formula J, the nucleobases may be selected from the group consisting of: adenine, cytosine, thymine, uracil, and guanine. In one embodiment, the nucleobases are selected from the group consisting of: adenine, cytosine, and uracil. In another embodiment, the nucleobases are selected from the group consisting of: cytosine and uracil. In another embodiment, the nucleobase is uracil. In one embodiment, the modified nucleotide is located at position 3, 4, 5, 6, 7, or 8 counting from the 5' end. In one embodiment, the modified nucleotide is located at position 5, 6, 7, or 8 counting from the 5' end. In one embodiment, the modified nucleotide is located at position 5, 6, or 8 counting from the 5' end. In one embodiment, the modified nucleotide is located at position 5 counting from the 5' end.

[0072] The modified nucleotide may be present in the antisense and / or sense strands. Preferably, the modified nucleotide is present in the antisense strand.

[0073] Novel oligonucleotides can be represented by compounds of formula K. Formula K. Oligonucleotides and / or RNAi agents, wherein B is a nucleobase, m and n are independently selected from any integer from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

[0074] In formula K, m and n can be independently selected from any integer from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0075] In formula K, the nucleobases can be selected from the group consisting of: adenine, cytosine, thymine, uracil, and guanine. In one embodiment, the nucleobases are selected from the group consisting of: adenine, cytosine, and uracil. In another embodiment, the nucleobases are selected from the group consisting of: cytosine and uracil. In another embodiment, the nucleobase is uracil. In another embodiment, the modified nucleotide is located at position 3, 4, 5, 6, 7, or 8 counting from the 5' end. In another embodiment, the modified nucleotide is located at position 5, 6, 7, or 8 counting from the 5' end. In another embodiment, the modified nucleotide is located at position 5, 6, or 8 counting from the 5' end. In another embodiment, the modified nucleotide is located at position 5 counting from the 5' end.

[0076] Novel oligonucleotides can be represented by compounds of formula L. Formula L. oligonucleotides and / or RNAi agents, wherein B is a nucleobase, m and n are independently selected from any integer from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

[0077] In formula L, m and n can be independently selected from any integer from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0078] In Formula L, the nucleobases may be selected from the group consisting of: adenine, cytosine, thymine, uracil, and guanine. In one embodiment, the nucleobases are selected from the group consisting of: adenine, cytosine, and uracil. In another embodiment, the nucleobases are selected from the group consisting of: cytosine and uracil. In another embodiment, the nucleobase is uracil. In another embodiment, the modified nucleotide is located at position 3, 4, 5, 6, 7, or 8 counting from the 5' end. In another embodiment, the modified nucleotide is located at position 5, 6, 7, or 8 counting from the 5' end. In another embodiment, the modified nucleotide is located at position 5, 6, or 8 counting from the 5' end. In another embodiment, the modified nucleotide is located at position 5 counting from the 5' end.

[0079] One or more butanediol moieties may be incorporated at any position in the RNAi agent. For example, the butanediol moieties may be incorporated at position 22, position 23, or both positions 22 and 23 to achieve a potential increase in persistence, particularly with respect to the antisense strand of the RNAi agent. Butanediol may also be incorporated at positions 1, 2, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the oligonucleotide.

[0080] The oligonucleotide may contain a sense term and / or an antisense term. In other embodiments, the oligonucleotide is an antisense oligonucleotide. In another embodiment, the antisense oligonucleotide is 15 to 30 nucleotides in length. In still other embodiments, the antisense oligonucleotide is 18 to 30 nucleotides in length. In some embodiments, the antisense oligonucleotide is 23 nucleotides in length. RNAi drugs

[0081] This paper discloses an RNAi agent containing a butanediol moiety. As disclosed herein, the butanediol moiety can be incorporated into one or more nucleotides by replacing the sugar portion in the nucleotide. Modified nucleotides can be incorporated into the RNAi agent.

[0082] RNAi agents may comprise sense oligonucleotides and antisense oligonucleotides as described herein. In another embodiment, the RNAi agent is siRNA.

[0083] In another embodiment, an RNAi agent is described herein comprising sense oligonucleotides and antisense oligonucleotides, each independently being 15 to 30 nucleotides in length, wherein the sense oligonucleotides and the antisense oligonucleotides form a double strand, wherein, if desired, one or more sense nucleotides and the antisense nucleotides are independently modified nucleotides, and wherein, if desired, the linkage between one or more sense oligonucleotides and the antisense oligonucleotides is a modified nucleotide linkage, and wherein the antisense oligonucleotide is represented by formula I, II or III.

[0084] In yet another embodiment of the RNAi agent disclosed herein, the antisense oligonucleotide of formula J, K, or L comprises a 3' overhang of two nucleotides. In another embodiment, the antisense oligonucleotide of formula J, K, or L is 23 nucleotides in length. In yet another embodiment, the sense oligonucleotide is 21 nucleotides in length.

[0085] In other embodiments, each nucleotide of the sense oligonucleotide and antisense oligonucleotide of formula J, K or L is modified or further modified and independently contains a 2-fluorine modified nucleotide or a 2'O-methyl nucleotide.

[0086] In one embodiment of the RNAi agent described herein, the antisense oligonucleotide of formula J, K, or L is 23 nucleotides in length, wherein each nucleotide of the antisense is a modified nucleotide, and wherein the 2'-fluorine modified nucleotide is modified to be present at a position selected from one of the following: a. Positions 2, 3, 7, 14, and 16 from the 5' end of the antonymous stock; b. Positions 2, 5, 7, 14, and 16 from the 5' end of the antonymous stock; c. Positions 2, 3, 8, 14, and 16 from the 5' end of the antonymous stock; d. Positions 2, 5, 8, 14, and 16 from the 5' end of the antonymous stock; e. Positions 2, 6, 14, and 16 from the 5' end of the opposite stock; and f. Positions 2, 14, and 16 from the 5' end of the opposite stock.

[0087] In another embodiment of the RNAi agent described herein, the sense oligonucleotide is 21 nucleotides in length, wherein each nucleotide of the sense strand is a modified nucleotide, and wherein one or more of the modified nucleotides contain a 2' fluorine modification present at a position selected from one of the following: a. Positions 9, 10, and 11 from the 5' end of the share capital; b. Positions 7, 9, 10, and 11 from the 5' end of the share; c. Positions 7, 9, and 10 from the 5' end of the share; or d. Positions 7, 10 and 11 from the 5' end of the right stock.

[0088] In another embodiment of the RNAi agent described herein, the modified nucleotide comprises a 2' fluorine modification in an antisense oligonucleotide of formula J, K, or L located at one of the following positions: a. Positions 2, 3, 7, 14, and 16 from the 5' end of the antonymous stock; b. Positions 2, 5, 7, 14, and 16 from the 5' end of the antonymous stock; c. Positions 2, 3, 8, 14, and 16 from the 5' end of the antonymous stock; d. Positions 2, 5, 8, 14, and 16 from the 5' end of the antonymous stock; e. Positions 2, 6, 14, and 16 from the 5' end of the opposite stock; and These modified nucleotides contain 2' fluorine modification in the sense strand at one of the following positions: a. Positions 9, 10, and 11 from the 5' end of the share capital; b. Positions 7, 9, 10, and 11 from the 5' end of the share; c. Positions 7, 9, and 10 from the 5' end of the share; or d. Positions 7, 10 and 11 from the 5' end of the right stock.

[0089] In other embodiments of the RNAi agents described herein, the nucleotides contain modifications or further modifications, which are inter-nucleotide or inter-nucleoside linkages. In one embodiment, the sense oligonucleotide has four or five phosphate thioester linkages. In another embodiment, the antisense oligonucleotide of formula J, K, or L has four or five phosphate thioester linkages.

[0090] In another embodiment, the RNAi agent of this article comprises at least four internucleotide links, which are equivalent to phosphate thioester links at the two terminal nucleotides at the 5' and 3' ends of each of the sense and antisense strands.

[0091] In some embodiments, the sense or antisense further comprises a baseless portion or a reverse baseless portion, for example, as shown in Table A. In some embodiments, the sense comprises a reverse baseless portion. Table A. Baseless and Reverse Baseless (iAb) Section structure 1 (No bases) 2 (iAb) "5'" and "3'" indicate the 5' to 3' direction of the sequence.

[0092] In other embodiments, the RNAi agents described herein include a modified nucleotide at the 5' end of an antisense oligonucleotide of formula J, K, or L, which further modifies the phosphate ester group to a phosphate ester analog. In another embodiment, this further modification to a phosphate ester analog is a 5'-(E)-vinylphosphonate.

[0093] In other embodiments of the RNAi agents disclosed herein, the sense or antisense strands are further modified to be baseless.

[0094] RNAi agents may further include a delivery moiety that enables the RNAi agent to target specific cells, tissues, and / or organs of a patient or individual. By delivering oligonucleotides to the desired cells or tissues of the patient, gene expression can be regulated at its most advantageous location. In one morphology, the delivery moiety may be a lipophilic delivery moiety.

[0095] In one embodiment, the delivery portion may be a GalNAc delivery portion; that is, a portion comprising at least one N-acetylglucosamine (GalNAc). This portion can target desialylate glycoprotein receptors on hepatocytes, thereby increasing delivery to the liver. In one embodiment, the delivery portion may include one, two, three, or four GalNAc portions. Suitable delivery portions are described in WO 2022 / 271806.

[0096] In one particular embodiment, the delivery portion may also be described by a compound of formula M. Formula M. Delivery Part

[0097] In the part of formula M, the L-line delivers a partial linker or bond (i.e., there is no additional linker), and the Z-line delivers RNA molecules.

[0098] In another state, the delivery portion can be described by a compound of formula M': Formula M'. Delivery part

[0099] For formulas M' and L, L can be a linker or bond, and Z-series RNA molecules.

[0100] The delivery moiety can be attached to the RNAi agent at either the 3' or 5' end. The delivery moiety can be attached to the RNAi agent via a delivery moiety linker. This delivery moiety linker can be any suitable organic linker. The delivery moiety linker can be described by a compound of formula N. Formula N. Delivery part connector

[0101] In another embodiment, the connector can be described by equation N': Formula N'

[0102] In another embodiment, the connector can be described by equation N'': Formula N'', In some embodiments, it may have formula N''-a or formula N''-b: Formula N''-a Formula N''-b.

[0103] In one embodiment, the RNAi agent as described herein may have the following structure: Among them, Z-series RNA molecules.

[0104] In another embodiment, the RNAi agent as described herein may have the following structure: Among them, Z-series RNA molecules.

[0105] In some embodiments, the RNAi agent comprises any of the dsRNAs in Tables 1a to 2d, conjugated to the delivery portion at 3' or 5' as needed. In some embodiments, the delivery portion is GalNAc of formulation 29. In some embodiments, GalNAc has formula M. In some embodiments, the dsRNA and GalNAc are conjugated via a linker, such as a delivery portion linker of formula N.

[0106] RNAi agents may also contain other suitable delivery portions. Other suitable delivery portions may contain cholesterol, such as 5' or 3'-attached cholesterol-tetraethylene CE-phosphorusamine (CholTEG). Suitable phosphorusamines that can attach to cholesterol molecules may include the phosphorusamines listed in Table B. Table B: Phosphine phosphorus amine CAS DMT-2'-FA(Bz)-CEphosphosamide 136834-22-5 DMT-2'-FC(Ac)-CE phosphorusamine 159414-99-0 DMT-2'-FG(iBu)-CE phosphorusamine 144089-97-4 DMT-2'-FU-CEphosphosamide 146954-75-8 DMT-2'-O-Me-A(Bz)-CEphosphosamide 110782-31-5 DMT-2'-O-Me-C(Ac)-CEphosphosamide 199593-09-4 DMT-2'-O-Me-G(iBu)-CEphosphosamide 150780-67-9 DMT-2'-O-Me-U-CE phosphorus amine 110764-79-9 5'-bis(POM)vinyl phosphate-2'-Ome-U3'CE amino phosphate Lilly Reverse non-basic amino phosphate 401813-16-9 Example

[0107] Some abbreviations are defined as follows: "AS" refers to the antisense term; "BD" and "bd" are used interchangeably to refer to butanediol; "t-BuOOH" refers to tert-butyl hydroperoxide; "CT" refers to circulation threshold; "DCM" refers to dichloromethane; "DIPT" refers to diisopropyl tartrate; "DMF" refers to dimethylformamide; "DMT" refers to dimethoxytriphenylmethyl; "dsRNA" refers to double-stranded ribonucleic acid; "EtOAc" refers to ethyl acetate; "GNA" refers to ethylene glycol nucleic acid; "hiPSC" refers to human induced pluripotent stem cells; "MeCN" refers to acetonitrile; "MeOH" refers to methanol and methyl alcohol. alcohol); PBS (phosphate-buffered saline); PCR (polymerase chain reaction); RBF (round-bottom flask); RT-PCR (reverse transcription polymerase chain reaction); siRNA (small interfering RNA); SS (sense RNA); TEA (triethylamine); and Ti(Oi-Pr)4 (titanium isopropoxide). Option 1

[0108] Scheme 1, Step A illustrates the synthesis of compound (2) using the Sharpless asymmetric epoxidation protocol, the conditions of which will be known to those skilled in the art. Step B shows the conversion of compound (2) to compound (3) using 4-nitrobenzoic acid under Mitsunobu reaction conditions, the conditions of which will be known to those skilled in the art. Step C shows the hydrolysis of compound (3) in a solvent (such as dichloromethane) using a base (such as cesium carbonate) to provide compound (4), which is then protected with DMT in step E to provide compound (6). Step D is carried out in a manner substantially similar to that of step E to provide compound (5). Option 2

[0109] Scheme 2, Step A, compound (5) is treated with compound (7) and with a base (such as NaH) in a suitable solvent (such as DMF) to provide compound (8). In Step B, the secondary alcohol of compound (8) is phosphite-esterified with 3-((bis(diisopropylamino)phosphino)oxy)propionitrile in a suitable solvent (such as DCM) to provide compound (9).

[0110] The scheme was repeated starting with compound (6) to provide other non-mirror isomers. Option 3 []

[0111] In scheme 3, step A is performed in a manner substantially similar to step A of scheme 2 to produce compound (11). Step B is performed in a manner substantially similar to step B of scheme 2 to produce compound (12). The scheme is repeated starting with compound (6) to provide other non-mirror image isomers. [] Option 4

[0112] In scheme 4, step A is carried out in a manner substantially similar to that in step A of scheme 2 to produce compound (14). For step B, compound (14) is treated with 1,1-dimethoxy-N,N-dimethylmethylamine in a suitable solvent (such as DMF) and heated to provide compound (15). Step C is carried out in a manner substantially similar to that in step B of scheme 2 to produce compound (16). This scheme is repeated starting with compound (6) to provide other non-mirror image isomers. Option 5

[0113] In Scheme 5, step A is performed in a manner substantially similar to that in step A of Scheme 2 to produce compound (18). In step B, the benzyl protecting group is removed by catalytic hydrogenation to Pd / C in a suitable solvent (such as EtOAc) to provide compound (19). Step C is performed in a manner substantially similar to that in step B of Scheme 4 to produce compound (20). Step D is performed in a manner substantially similar to that in step B of Scheme 2 to produce compound (21). The scheme is repeated starting with compound (6) to provide other non-mirror image isomers. Formulation 1 But-3-en-2-ol

[0114] The starting material was purchased from Combi-Blocks, CAS No. 598-32-3. Formulation 2 (R)-1-((S)-Ethyleneoxy-2-yl)ethanol-1-ol

[0115] Under nitrogen, 20 g of 4 Å molecular sieve was added to a dry three-necked RBF. DCM (160 mL) was added, and the suspension was cooled to -23 °C. (-)-DIPT (1.00 mL, 4.88 mmol) was added, followed by Ti(Oi-Pr)₄ (1.20 mL, 4.2 mmol). t-BuOOH (18.2 mL, 100 mmol) was added slowly. The reaction mixture was stirred for 30 min using a mechanical stirrer. 3-Buten-2-ol (6.00 g, 83.2 mmol) in solution dissolved in DCM (20 mL) was added dropwise via a feeding funnel. The reaction mixture was allowed to stir at -23 °C for 64 h. The reaction mixture was quenched by adding 7 mL of dimethyl sulfide. The reaction flask was removed from the cold bath and allowed to warm to ambient temperature. A solution of acetone / water (100 mL / 40 mL) was added, and the suspension was stirred for 1 h. The reactants were filtered through a diatomaceous earth mat and washed with DCM. The filtrate was dried over MgSO4 and filtered. The crude product solution was carefully concentrated under vacuum. The resulting solution was purified by rapid silica gel chromatography and precipitated with 0 to 10% MeOH in DCM to produce the title compound (3.15 g, 43%) as a colorless oil. ¹H NMR (CDCl₃) δ 4.01 (m, 1H), 3.02 (m, 1H), 2.79 (dd, J = 5.0, 3.0 Hz, 1H), 2.73 (dd, J = 4.8, 4.4 Hz, 1H), 1.85 (m, 1H), 1.26 (d, J = 6.4 Hz, 3H). Formulation 3 (S)-4-nitrobenzoic acid 1-((S)-ethylene oxide-2-yl)ethyl ester

[0116] Under nitrogen, (R)-1-((S)-ethyleneoxy-2-yl)ethylene-1-ol (3.00 g, 34 mmol) was added to dry RBF. DCM (60 mL) was added, followed by 4-nitrobenzoic acid (2.85 g, 17.0 mmol) and triphenylphosphine (4.47 g, 17.0 mmol). The solution was cooled to 0 °C, and DIAD (2.65 mL, 13.6 mmol) was slowly added as a solution in DCM (5 mL). After stirring for 10 minutes, the cold bath was removed, and the reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture and the resulting solution were concentrated under vacuum and purified by rapid silica gel chromatography, precipitated with 0 to 30% EtOAc in hexane, yielding the title compound (1.72 g, 64%) as a white solid. ¹H NMR (CDCl₃) δ 8.30 (d, J = 9.2 Hz, 2H), 8.23 ​​(d, J = 9.2 Hz, 2H), 5.02 (quintet, J = 6.4 Hz, 1H), 3.25 (m, 1H), 2.91 (dd, J = 4.4, 4.4 Hz, 1H), 2.72 (dd, J = 4.8, 2.8 Hz, 1H), 1.48 (d, J = 6.4 Hz, 3H). Formulation 4 (S)-1-((S)-epoxyethylene-2-yl)ethanol-1-ol

[0117] Under nitrogen, 1-((S)-4-nitrobenzoic acid 1-((S)-ethylene oxide-2-yl)propyl ester (1.72 g, 7.25 mmol) was added to dry RBF. DCM (10 mL) was added, followed by cesium carbonate (2.60 g, 7.98 mmol). MeOH (1.5 mL, 36.3 mmol) was added, and the reaction mixture was stirred for 2 hours. The suspension was filtered and carefully concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography, precipitated with 0 to 10% MeOH in hexane from DCM, yielding the title compound (0.614 g, 96%) as a colorless oil. 1H NMR (CDCl3) δ 3.63 (m, 1H), 2.97 (m, 1H), 2.82 (dd, J = 4.8, 4.0 Hz, 1H), 2.71 (dd, J = 4.8, 2.4 Hz, 1H), 1.88 (br s, 1H), 1.35 (d, J = 6.4 Hz, 3H). Formulation 5 (S)-2-((S)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)epoxide

[0118] Under nitrogen, (S)-1-((S)-ethylene-2-yl)ethanol-1-ol (0.614 g, 6.97 mmol) was added to dry RBF. DCM (35 mL) was added and the solution was cooled to 0 °C. 2,4,6-Trimethylpyridine (5.53 mL, 41.8 mmol) was added, followed by 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (3.31 g, 9.76 mmol). AgNO3 (1.54 g, 9.06 mmol) was added slowly in five portions over 10 minutes each. The cold bath was removed and the reaction mixture was allowed to stir at ambient temperature for 3 hours. The reaction mixture was diluted with EtOAc (15 mL) and quenched with water (3 mL). The suspension was stirred for 10 minutes and then filtered through a diatomaceous earth mat and washed with DCM. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting solution was purified by rapid silica gel chromatography and precipitated with 0 to 30% EtOAc in hexane (the precipitate contained 1% TEA) to produce the title compound (2.40 g, 88%) as a colorless oil. 1H NMR (CDCl3) δ 7.52 (d, J = 8.0 Hz, 2H), 7.41 (m, 4H), 7.30-7.19 (m, 3H), 6.82 (d, J = 8.8 Hz, 4H), 3.79 (s, 6H), 3.62 (quint, J = 6.4 Hz, 1H), 2.85 (m, 1H), 2.61 (dd, J = 4.8, 4.4 Hz, 1H), 2.51 (dd, J = 4.8, 2.8 Hz, 1H), 0.72 (d, J = 6.4 Hz, 3H). Table 1: Formulation 6 synthesized in a manner substantially similar to that of Formulation 5 preparation structure Preparation Name 1H NMR (CDCl3) 6 (S)-2-((R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)epoxide δ 7.54 (d, J = 7.2 Hz, 2H), 7.43 (m, 4H), 7.32-7.21 (m, 3H), 6.84 (d, J = 9.2 Hz, 4H), 3.82 (s, 6H), 3.50 (quint, J = 6.0 Hz, 1H), 2.81 (m, 1H), 2.54 (dd, J = 4.8, 4.4 Hz, 1H), 2.19 (dd, J = 5.2, 2.8 Hz, 1H), 1.26 (d, J = 6.4 Hz, 3H) Formulation 7 1-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)pyrimidine-2,4(1H,3H)-dione

[0119] Under nitrogen, pyrimidine-2,4(1H,3H)-dione (0.517 g, 4.61 mmol) was added to dry RBF. DMF (5 mL) was added, followed by NaH (46 mg, 1.15 mmol, 60% dispersion in mineral oil). The suspension was stirred for 1.5 hours. (S)-2-((R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)ethylene oxide (1.50 g, 3.84 mmol) was added in a solution of DMF (5 mL). The reaction mixture was heated to 110 °C and stirred for 48 hours. The reaction mixture was cooled to ambient temperature and diluted with EtOAc (20 mL) and washed with 5% NaCl in water (2 x 50 mL). The organic layer was separated and concentrated under vacuum. The resulting solution was purified by rapid silica gel chromatography, followed by precipitation in hexane with 0 to 100% EtOAc (the precipitate contained 1% TEA), yielding the title compound (1.32 g, 68%) as a white foam. ES / MS (m / z): 501.3 (MH). Table 2: The following formulations are synthesized in a manner substantially similar to that of formulation 7. preparation structure Preparation Name ES / MS (m / z) 8 1-((2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)pyrimidine-2,4(1H,3H)-dione 501.4 (MH). 9 N-(1-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-2-sideoxy-1,2-dihydropyrimidin-4-yl)benzamide 606.2 (M+H). 10 N-(1-((2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-2-sideoxy-1,2-dihydropyrimidin-4-yl)benzamide 606.2 (M+H). 11 (2S,3R)-1-(6-amino-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-ol) 526.3 (M+H). 12 (2S,3S)-1-(6-amino-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-ol) 526.3 (M+H). 13 (2S,3R)-1-(2-amino-6-(benzyloxy)-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-ol) 630.4 (MH). 14 (2S,3S)-1-(2-amino-6-(benzyloxy)-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-ol) 630.4 (MH). Formulation 15 (2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-(2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)but-2-yl(2-cyanoethyl)diisopropylphosphoniumamine

[0120] Under nitrogen, 1-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)pyrimidine-2,4(1H,3H)-dione (1.32 g, 2.63 mmol) was added to dry RBF. DCM (13 mL) was added, followed by 2H-tetrazole (4.09 mL, 1.84 mmol, 0.45 M in MeCN). 3-((bis(diisopropylamino)phosphino)oxy)propionitrile (0.918 mL, 2.89 mmol) was added. The reaction mixture was stirred for 20 hours. The reaction mixture was concentrated under vacuum. The resulting solution was purified by rapid silica gel chromatography and precipitated with 0 to 100% EtOAc in hexane (the precipitate contained 1% TEA), yielding the title compound (1.32 g, 71%) as a colorless foam. 31P NMR (CDCl3) δ 149.87, 149.43; ES / MS (m / z): 701.4 (MH). Table 3: The following formulations are synthesized in a manner substantially similar to that of formulation 15. preparation structure Preparation Name 16 (2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-(2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)but-2-yl(2-cyanoethyl)diisopropylphosphonium 31P NMR (CDCl3) δ 149.96, 148.85; ES / MS (m / z): 701.4 (MH). 17 (2S,3R)-1-(4-benzopyrimidin-2-sideoxypyrimidin-1(2H)-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-yl(2-cyanoethyl)diisopropylphosphonium 31P NMR (CDCl3) δ 149.61, 149.52; ES / MS (m / z): 804.4 (MH). 18 (2S,3S)-1-(4-benzopyrimidin-2-sideoxypyrimidin-1(2H)-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-yl(2-cyanoethyl)diisopropylphosphonium 31P NMR (CDCl3) δ 150.14, 149.12; ES / MS (m / z): 804.3 (MH) 19 (2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-(6-(((E)-(dimethylamino)methylene)amino)-9H-purine-9-yl)but-2-yl(2-cyanoethyl)diisopropylphosphonium 31P NMR (CDCl3) δ 149.63, 149.50; ES / MS (m / z): 781.4 (M+H). 20 (2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-(6-(((E)-(dimethylamino)methylene)amino)-9H-purine-9-yl)but-2-yl(2-cyanoethyl)diisopropylphosphonium 31P NMR (CDCl3) δ 149.95, 148.56; ES / MS (m / z): 781.4 (M+H). twenty one (2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-(2-(((E)-(dimethylamino)methylene)amino)-6-sideoxy-1,6-dihydro-9H-purine-9-yl)but-2-yl(2-cyanoethyl)diisopropylphosphonium 31P NMR (CDCl3) δ 150.12, 149.00; ES / MS (m / z): 797.4 (M+H). twenty two (2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-(2-(((E)-(dimethylamino)methylene)amino)-6-sideoxy-1,6-dihydro-9H-purine-9-yl)but-2-yl(2-cyanoethyl)diisopropylphosphatidylamine 31P NMR (CDCl3) δ 149.75, 149.57; ES / MS (m / z): 797.4 (M+H). Formulation 23 (E)-N'-(9-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-9H-purine-6-yl)-N,N-dimethylformamidin

[0121] Under nitrogen, (2S,3R)-1-(6-amino-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-ol (2.04 g, 3.88 mmol) was added to dry RBF. DMF (15 mL) was added, followed by 1,1-dimethoxy-N,N-dimethylmethylamine (2.75 mL, 20.6 mmol). The suspension was heated to 60 °C and stirred for 1.5 h. The reaction mixture was cooled to ambient temperature and diluted with EtOAc (20 mL) and washed with a solution of 5% NaCl in water (2 x 70 mL). The organic layer was separated and concentrated under vacuum. The resulting solution was purified by rapid silica gel chromatography, precipitated with 0–20% MeOH in EtOAc (the precipitate contained 1% TEA), yielding the title compound (1.58 g, 70%) as a pale yellow foam. ES / MS (m / z): 581.4 (M+H). Table 4: The following formulations are synthesized in a manner substantially similar to that of formulation 23. preparation structure Preparation Name ES / MS (m / z): twenty four (E)-N'-(9-((2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-9H-purine-6-yl)-N,N-dimethylformamidin 581.4 (M+H). 25 (E)-N'-(9-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-6-sideoxy-6,9-dihydro-1H-purine-2-yl)-N,N-dimethylformamidin 597.4 (M+H). 26 (E)-N'-(9-((2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-6-sideoxy-6,9-dihydro-1H-purine-2-yl)-N,N-dimethylformamidin 597.4 (M+H). Formulation 27 2-Amino-9-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-1,9-dihydro-6H-purine-6-one

[0122] (2S,3R)-1-(2-amino-6-(benzyloxy)-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)but-2-ol (1.82 g, 2.88 mmol) and Pd / C (0.92 g, 10% carbon-supported) were suspended in EtOAc (72 mL) in dry RBF. The reaction flask was purged with nitrogen, followed by hydrogen. The reaction mixture was stirred for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered through diatomaceous earth and washed with 5:1 DCM / MeOH. The solution was concentrated under vacuum to give the title compound (1.32 g, 85%) as a white solid. ES / MS (m / z): 542.2 (M+H). Table 5: The following formulations are synthesized in a manner substantially similar to that of formulation 27. preparation structure Preparation Name ES / MS (mz) (M+H) 28 2-Amino-9-((2S,3S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-1,9-dihydro-6H-purine-6-one 542.9 Formulation 29 Resin loaded with 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropiperan-2-yl]oxypentaamino]-5-[bis[2-[5-[3-acetamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropiperan-2-yl]oxypentaamino]ethyl]amino]-5-sideoxy-pentaamino]amino]hexylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propionic]-4-piperidinyl]methoxy]-4-sideoxy-butyric acid []

[0123] The title compound was prepared in a manner similar to that of the formulation found in WO2022 / 271806. Table 6a: HPRT Unmodified Sequences dsRNA No. Stocks with positive connotations (5' to 3') SEQ ID NO Antonyms (5' to 3') SEQ ID NO Origin of the target region on mouse HPRT transcript NM_013556.2 1a UCCUAUGACUGUAGAUUUUAA 1 UUAAAAUCUACAGUCAUAGGAAU 2 427 Table 6b: Unmodified Fas sequences dsRNA No. Stocks with positive connotations (5' to 3') SEQ ID NO Antonyms (5' to 3') SEQ ID NO Human FAS transcripts The starting position of the target area on NM_000043.6 1b GUUGACUUGAGUAAAUAUAUA 3 UAUAUAUUUACUCAAGUCAACAU 4 760 Table 6c: Nav1.8 Unmodified Sequences dsRNA No. Stocks with positive connotations (5' to 3') SEQ ID NO Antonyms (5' to 3') SEQ ID NO Human Nav1.8 transcript The starting position of the target area on NM_001293306.2 1c CCUUUGAAGCCUUGAUAAAGA 5 UCUUUAUCAAGGCUUCAAAGGUG 6 496 Table 6d: Unmodified Ang8 sequences dsRNA No. Stocks with positive connotations (5' to 3') SEQ ID NO Antonyms (5' to 3') SEQ ID NO Human Ang8 transcripts The starting position of the target area on NM_018687.7 1d AUGGAGGAGGAUAUUCUGCAA 7 UUGCAGAAUAUCCUCCUCCAUCU 8 319 Table 6e: Unmodified HMGCR sequences dsRNA No. Stocks with positive connotations (5' to 3') SEQ ID NO Antonyms (5' to 3') SEQ ID NO Origin of the target region on the human HMGCR transcript NM_000859.3 1e GUAGUUCAAACUGUAUUACUA 105 UAGUAAUACAGUUUGAACUACCA 106 2e CUGUAUUACUUUAAUGGAAGA 107 UCUUCCAUUAAAGUAAUACAGUU 108 Table 7a: HPRT modified sequences dsRNA No. share Oligomeric sequence 5' to 3' SEQ ID NO 2a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmAmAmAmUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 10 3a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmU*mA*mA 9 AS mU*fU*mA(sgA)mAmUmCMUMAmCmAmGfUmCfAmUmAmGmGMA*mA*mU 11 4a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmU*mA*mA 9 AS mU*fU*mA(ssbdA)mAmUmCMUMAmCmAmGfUmCfAmGmGmA*mA*mU 12 5a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmU*mA*mA 9 AS mU*fU*mA(rsbdA)mAmUmCmUmAmCmAmGfUmCfAmUmGmGMA*mA*mU 13 6a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmU*mA*mA 9 AS mU*fU*mA(sgA)mAmUmCmUmAmCmAmGfUmCfAmUmAmGmGMA*mA*mU 14 7a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmU*mA*mA 9 AS mU*fU*mAmA(ssbdA)mAmUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 15 8a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmA(rsbdA)mAmUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 16 9a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmAmA(sgA)mUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 17 10a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmAmA(ssbdA)mUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 18 11a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmAmA(rsbdA)mUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 19 12a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmAmA(sgU)MCMUMAmCmGfUmCfUmAmGmGMA*mA*mU 20 13a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmU*mA*mA 9 AS mU*fU*mAmAmA(ssbdU)MCMUMAmCmGfUmCfUmGmGMA*mA*mU 21 14a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmU*mA*mA 9 AS mU*fU*mAmAmA(rsbdU)MCMUMAmCmGfUmCfUmAmGmGMA*mA*mU 22 15a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmU*mA*mA 9 AS mU*fU*mAmAmAmU(sgC)MUMAmCmAmGfUmCfAmGmGMA*mA*mU 23 16a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmU*mA*mA 9 AS mU*fU*mAmAmAmU(ssbdC)MUMAmCmAmGfUmCfAmGmGMA*mA*mU 24 17a S mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 9 AS mU*fU*mAmAmAmAmU(rsbdC)mUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 25 Abbreviations: "m" indicates 2'-OMe; "f" indicates 2'-fluorine; "*" indicates thiophosphate linkage; "sg" indicates GNA; "rsbd" indicates R,S-butanediol; and "ssbd" indicates S,S-butanediol. Table 7b: Fas modified sequences dsRNA No. share Oligomeric sequence 5' to 3' SEQ ID NO 2b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAfUmAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 27 3b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAfU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 28 4b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAfUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 29 5b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS mU*fA*fUmAmUmAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 30 6b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*fUmAmUmAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 31 7b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*fUmA(ssbdU)mAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 32 8b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*fUmAmU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 33 9b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*fUmAmUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 34 10b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAfUmAmUfUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 35 11b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAfU(ssbdA)mUfUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 36 12b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAfUmA(ssbdU)fUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 37 13b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAmUmAmUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 38 14b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAmUmA(ssbdU)mUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 39 15b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS PmU*fA*mUmAmUfAmUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 40 16b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS mU*fA*mUmAfU(rsbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 41 17b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS mU*fA*mUmAfU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 42 18b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS mU*fA*fUmAmU(rsbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 43 19b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmAmUmA*mU*mA 26 AS mU*fA*fUmAmU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 44 20b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmAmUmA*mU*mA 26 AS mU*fA*mUmAfUmA(rsbdU)fUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 45 21b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmAmUmA*mU*mA 26 AS mU*fA*mUmAfUmA(ssbdU)fUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 46 22b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmAmUmA*mU*mA 26 AS mU*fA*mUmAfUmAfU(rsbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 47 23b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmAmUmA*mU*mA 26 AS mU*fA*mUmAfUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 48 24b S mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 26 AS mU*fA*fUmAmUmAfU(rsbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 49 25b S mG*mU*mUmGmAmCfUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 50 AS mU*fA*fUmAmUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 51 Abbreviations: "m" indicates 2'-OMe; "f" indicates 2'-fluorine; "*" indicates thiophosphate linkage; "sg" indicates GNA; "rsbd" indicates R,S-butanediol; and "ssbd" indicates S,S-butanediol. Table 7c: Modified sequences of Nav1.8 dsRNA No. share Oligomeric sequence 5' to 3' SEQ ID NO 2c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 53 3c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*(sgU)mUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 54 4c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*(rsbdU)mUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 55 5c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*(ssbdU)mUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 56 6c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mU(sgU)mUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 57 7c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mU(rsbdU)mUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 58 8c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mU(ssbdU)mUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 59 9c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmU(sgU)fAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 60 10c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmU(rsbdU)fAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 61 11c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmU(ssbdU)fAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 62 12c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmU(sgA)mUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 63 13c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmU(rsbdA)mUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 64 14c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmU(ssbdA)mUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 65 15c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfA(sgU)mCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 66 16c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfA(rsbdU)mCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 67 17c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfA(ssbdU)mCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 68 18c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfAmU(sgC)mAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 69 19c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfAmU(rsbdC)mAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 70 20c S mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 52 AS PmU*fC*mUmUmUfAmU(ssbdC)mAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 71 Abbreviations: "m" indicates 2'-OMe; "f" indicates 2'-fluorine; "*" indicates thiophosphate bonding; "sg" indicates GNA; "rsbd" indicates R,S-butanediol; and "ssbd" indicates S,S-butanediol. Table 7d: Modified sequence of Ang1.8 dsRNA No. share Oligomeric sequence 5' to 3' SEQ ID NO 2d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS PmU*fU*mGmCmAfGmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 73 3d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS PmU*fU*mGmCfAmGfAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 75 4d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS PmU*fU*fGmCmAmGfAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 76 5d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS PmU*fU*mGmCfAmGmAfAmUmCmCmCfUmCfCmUmCmCmAmU*mC*mU 77 6d S mA*mU*mGmGmAmGfGmAfGfGfAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*mGmCfAmGfAmUmAmCmCfUmCfCmUmCmCmAmU*mC*mU 78 7d S mA*mU*mGmGmAmGfGmAfGfGfAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*fGmC(ssbdA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 79 8d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*fGmC(rsbdA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 80 9d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*mGmCfA(ssbdG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 81 10d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*mGmCfA(rsbdG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 82 11d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*mGmCmAmG(ssbdA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 83 12d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*mGmCmAmG(rsbdA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 84 13d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*fGmCmAmGfA(ssbdA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 85 14d S mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 72 AS mU*fU*fGmCmAmGfA(rsbdA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 86 15d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCfAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 78 16d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmCmAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 87 17d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fG(sgC)mAmGfAmAmUmAmCmCfUmCfCmUmCmCmAmU*mC*mU 88 18d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fG(ssbdC)mAmGfAmAmUmAmCmCfUmCfCmUmCmCmAmU*mC*mU 89 19d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fG(rsbdC)mAmGfAmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 90 20d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmC(sgA)mGfAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 91 21d S mA*mU*mGmGmAmGmGmGmAfGfGfAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmC(ssbdA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 79 22d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmC(rsbdA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 80 23d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCfA(sgG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 92 24d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCfA(ssbdG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 81 25d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCfA(rsbdG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 82 26d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCmAmG(sgA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 93 27d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCmAmG(ssbdA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 83 28d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*mGmCmAmG(rsbdA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 84 29d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmCmAmGfA(sgA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 94 30d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmCmAmGfA(ssbdA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 85 31d S mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 74 AS mU*fU*fGmCmAmGfA(rsbdA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 86 Abbreviations: "m" indicates 2'-OMe; "f" indicates 2'-fluorine; "*" indicates thiophosphate linkage; "sg" indicates GNA; "rsbd" indicates R,S-butanediol; and "ssbd" indicates S,S-butanediol. Table 7e: HMGCR modified sequences dsRNA No. share Oligomeric sequence 5' to 3' SEQ ID NO 3e S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS PmU*fA*mGmUfAmAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 110 4e S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS VPmU*fA*mGmUfAmAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 111 5e S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*mG(rsbdU)fAmAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 112 6th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*mG(ssbdU)fAmAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 113 7th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*fGmU(rsbdA)mAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 114 8th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*fGmU(ssbdA)mAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 115 9th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*mGmUfA(rsbdA)fUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 116 10th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*mGmUfA(ssbdA)fUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 117 11th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*mGmUmAmA(rsbdU)mAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 118 12th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*mGmUmAmA(ssbdU)mAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 119 13th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*fGmUmAmAfU(rsbdA)mCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 120 14th S mG*mU*mAmGmUmUmCmAfAfAfCmUmGmUmAmUmUmAmC*mU*mA 109 AS mU*fA*fGmUmAmAfU(ssbdA)mCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 121 15th S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS PmU*fC*mUmUfCmCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 123 16th S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mU(rsbdU)fCmCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 124 17th S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mU(ssbdU)fCmCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 125 18th S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*fUmU(rsbdC)mCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 126 19th S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*fUmU(ssbdC)mCfAmUmUmAmAmAmAfAmUmAmCmAmG*mU*mU 127 20e S mC*mU*mGmUmAmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mUmUfC(rsbdC)fAmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 128 21e S mC*mU*mGmUmAmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mUmUfC(ssbdC)fAmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 129 22e S mC*mU*mGmUmAmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mUmUmCmC(rsbdA)mUmUmAmAmAmAfAmUmAmCmAmG*mU*mU 130 23e S mC*mU*mGmUmAmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mUmUmCmC(ssbdA)mUmUmAmAmGfUmAfAmUmAmCmAmG*mU*mU 131 24e S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mUmUfCmCfA(rsbdU)mUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 132 25e S mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 122 AS mU*fC*mUmUfCmCfA(ssbdU)mUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 133 Abbreviations: "m" indicates 2'-OMe; "f" indicates 2'-fluorine; "*" indicates thiophosphate linkage; "sg" indicates GNA; "rsbd" indicates R,S-butanediol; and "ssbd" indicates S,S-butanediol.

[0124] The sense and antisense strands of dsRNA can be synthesized using any nucleic acid polymerization method known in this technique, such as solid-phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al., John Wiley & Sons, Inc., New York, NY, USA), H-phosphonates, phosphortriesters, or enzymatic synthesis. Automated commercial synthesizers can be used, such as the MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Sulfidating agents, such as phenacetin disulfide or DDTT (((dimethylaminomethylene)amino)-3H-1,2,4-dithiozoline-3-thionone), can be used to introduce thiophosphate bonds.

[0125] The modified sequences in Tables 7a, 7b, 7c, 7d, and 7e are further modified by introducing a delivery portion (see Table 7f below) at the 3' end of the sense strand of the corresponding dsRNA. GalNAc (GalNAc of Formulation 29) is added to the 3' end of the sense strand of the dsRNA compounds in Tables 7a, 7b, 7d, and 7e. Modified oligonucleotides or conjugated oligonucleotides are known to be synthesized using similar techniques and commercially available modified amides and controlled-pore glass (CPG) products, including the resin-bound GalNAc of Formulation 29. CholTEG (cholesterol triethylene glycol, ChemGenes; N-9166-05) is added to the dsRNA compound in Table 7c (see Table 7f) via a conjugation method well known to those skilled in the art. Each of these delivery-modified dsRNA compounds is used in Examples 1 to 5.

[0126] Purification methods are used to remove unwanted impurities from the final oligonucleotide product. Common purification techniques for single-stranded oligonucleotides include reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion-exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, the oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense and antisense strands can then be annealed to form dsRNA. Table 7f. Delivery Section describe dsRNA No. Delivery section Attachment HPRT modified sequence 2a - 17a Table 7a GalNAc1 3' Righteous Stocks Fas modified sequence 2b - 25b Table 7b GalNAc1 3' Righteous Stocks Nav1.8 modified sequence 2c - 20c Table 7c CholTeg2 3' Righteous Stocks Ang1.8 modified sequence 2d - 31d Table 7d GalNAc1 3' Righteous Stocks HMGCR modified sequence 3e-25e Table 7e GalNAc1 3' Righteous Stocks 1 formulation 29 2ChemGenes, Product No. N-9166-05 Example 1: Using Gal1-conjugated HPRT siRNA with BD and GNA chemical modifications in the seed region to attenuate mouse HPRT in wild-type primary mouse hepatocytes (MPH) in vitro.

[0127] The attenuation of HPRT performance by Gal1-conjugated HPRT siRNA with BD and GNA chemical modifications in the seed region was analyzed using the following procedure: Primary mouse hepatocytes (MPH) were freshly isolated from three-month-old wild-type mice. Three hours after isolation, 15,000 cells were added to each well of a Corning 96-well plate, and the siRNA was added directly to the well. To generate concentration / dose-response curves, the final concentrations of Gal1-conjugated siRNA were 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM.

[0128] The treated cells were directly lysed in 96-well plates, and RNA was isolated using the Quick-RNA 96-pack (Zymo Research). The lysed RNA was used immediately or frozen. cDNA was synthesized using a Fast Advanced RT Master Mix (Invitrogen) in a thermal cycler following the steps below: 37°C for 30 minutes, 95°C for 5 minutes, and held at 4°C. Polymerase chain reaction (PCR) was performed using a TaqMan RT PCR (Life Technologies) system with the following cycling temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds each, and 60°C for 1 minute.

[0129] Mouse HPRT levels were normalized to mouse Rplp0 (Life Technologies) and represent a relative decrease in mouse HPRT mRNA expression compared to mediator-treated control cells. IC50 values ​​were calculated using an XLFit model with a 4-parameter fit.

[0130] Table 8A shows the results of IC50 and maximum attenuation percentage in wild-type mouse primary hepatocytes by freely ingesting indicator Gal1 conjugated HPRT siRNA with BD and GNA chemical modifications in the seed region. Table 8A shows the maintenance of titer and attenuation percentage of introducing destabilized nucleotides containing the butylene glycol moiety relative to the parent compound (2a) without butylene glycol modification in almost all of the compounds tested.

[0131] The second study was conducted in the same manner as the first, except that the cell lines were isolated from two-month-old mice and were inoculated immediately after isolation. The results of this study are shown in Table 8B. Table 8A: dsRNA No. Delivery section Attachment IC50 / nM Maximum KD% 2a GalNAc 3' Righteous Stocks 0.34 98.38 3a GalNAc 3' Righteous Stocks 1.48 97.88 4a GalNAc 3' Righteous Stocks 1.38 97.61 5a GalNAc 3' Righteous Stocks 2.16 94.59 6a GalNAc 3' Righteous Stocks 1.21 97.9 7a GalNAc 3' Righteous Stocks 1.44 97.38 8a GalNAc 3' Righteous Stocks 2.62 94.68 9a GalNAc 3' Righteous Stocks 1.4 98.87 10a GalNAc 3' Righteous Stocks 1.85 98.75 11a GalNAc 3' Righteous Stocks 1.03 96.87 12a GalNAc 3' Righteous Stocks 0.43 97.51 13a GalNAc 3' Righteous Stocks 4.39 97.04 14a GalNAc 3' Righteous Stocks 1.32 96.44 15a GalNAc 3' Righteous Stocks 2.49 94.87 16a GalNAc 3' Righteous Stocks 0.06 91 17a GalNAc 3' Righteous Stocks 770.8 71 Table 8B: dsRNA No. Delivery section Attachment IC50 / nM Maximum KD% 2a GalNAc 3' Righteous Stocks 0.42 97.42 15a GalNAc 3' Righteous Stocks 1.43 83.16 16a GalNAc 3' Righteous Stocks 2.775 86.86 17a GalNAc 3' Righteous Stocks 32.23 78.35 Example 2:

[0132] The attenuation of FAS expression by Gal1 conjugated FAS siRNA modified with butylene glycol (BD) at the seed region was analyzed using the following procedure: RNAiMAX (Life Technologies) transfection reagent was mixed with siRNA at 0.3 µl / well in Corning dishes, followed by the addition of HepG2 (ATCC) cells at 15 kJ / well. To generate concentration / dose-response curves, final concentrations of GalNAc conjugated siRNA of 200, 66.7, 22.2, 7.4, 2.5, 0.82, 0.27, 0.09, 0.03, 0.01, and 0.003 nM were used.

[0133] Cells were lysed after treatment, and RNA was directly isolated into 96-well plates using the Quick-RNA 96 kit (Zymo Research). The lysed RNA was used immediately or frozen. cDNA was synthesized using a Fast Advanced RT Master Mix (Invitrogen) in a thermal cycler following the steps below: 37°C for 30 minutes, 95°C for 5 minutes, and held at 4°C. Polymerase chain reaction (PCR) was performed using a TaqMan RT PCR (Life Technologies) system with the following cycling temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds each, and 60°C for 1 minute.

[0134] Human FAS levels were normalized to human RPLLP0 (Life Technologies) and represent a relative decrease in human FAS mRNA expression compared to control cells treated with the cytokine. IC50 values ​​were calculated using an XLFit model with a 4-parameter fit.

[0135] Table 9 shows the percentage of potency maintained and reduced by introducing destabilized nucleotides containing the butylene glycol moiety relative to the parent compound (2b) of the nucleotides without butylene glycol modification in almost all of the tested compounds. Table 9: Fas HepG2 dsRNA No Delivery section Attachment IC50 / nM Maximum KD% 2b GalNAc 3' Righteous Stocks 0.094 93.22 3b GalNAc 3' Righteous Stocks 0.052 93.12 4b GalNAc 3' Righteous Stocks 0.153 93.29 5b GalNAc 3' Righteous Stocks 0.148 92.32 6b GalNAc 3' Righteous Stocks 0.053 93.27 7b GalNAc 3' Righteous Stocks 0.094 92.75 8b GalNAc 3' Righteous Stocks 0.093 93.53 9b GalNAc 3' Righteous Stocks 0.091 93.45 10b GalNAc 3' Righteous Stocks 0.111 94.30 11b GalNAc 3' Righteous Stocks 0.221 93.60 12b GalNAc 3' Righteous Stocks 0.911 91.78 13b GalNAc 3' Righteous Stocks 0.074 93.72 14b GalNAc 3' Righteous Stocks 0.364 81.63 15b GalNAc 3' Righteous Stocks 0.014 87.89 16b GalNAc 3' Righteous Stocks 0.039 86.00 17b GalNAc 3' Righteous Stocks 0.052 85.83 18b GalNAc 3' Righteous Stocks 0.033 86.18 19b GalNAc 3' Righteous Stocks 0.058 86.85 20b GalNAc 3' Righteous Stocks 0.19 65.77 21b GalNAc 3' Righteous Stocks 0.182 66.29 22b GalNAc 3' Righteous Stocks 0.174 79.35 23b GalNAc 3' Righteous Stocks 0.043 84.85 24b GalNAc 3' Righteous Stocks 0.094 77.95 25b GalNAc 3' Righteous Stocks 0.032 85.65 Example 3:

[0136] DRG hiPSC-derived sensory neurons (purchased from Anatomic Inc.) were seeded at 20,000 cells / well on 96-well plates coated with polyguanine (P3655; Sigma Aldrich) and human laminin (AMS-892-012; amsbio). These hiPSC sensory neurons were maintained in Senso maturation medium (Anatomic Inc.) for one week. One week after seeding, the hiPSC sensory neurons were treated with siRNA at 4 µM and cultured for another week before harvesting.

[0137] For gene expression analysis, first RNA was purified from siRNA-treated hiPSC-sensory neurons using the RNAdvance v2 kit (A47943; Beckman Coulter) according to the manufacturer's instructions. The total RNA was then used as a template in a one-step universal probe RT-qPCR (E3006E; NEB) analysis, supplemented by TaqMan analysis. SCN1A (Hs00374696_m1;Thermo Fisher), SCN2A (Hs01109871_m1;Thermo Fisher), SCN3A (Hs00366902_m1;Thermo Fisher), SCN4A (Hs01109480_m1;Thermo Fisher), SCN5A (Hs01668074_m1;Thermo Fisher), SCN7A (Hs00161546_m1;Thermo Fisher), SCN8A (Hs00274075_m1;Thermo Fisher), SCN9A (Hs01076699_m1;Thermo Fisher), SCN10A (Hs01045137_m1;Thermo Fisher), SCN11A (Hs00204222_m1;Thermo Fisher) Taqman analysis of the following genes—KCNT2 (Hs01591277_m1; Thermo Fisher), GTF2F1 (Hs00157845_m1; Thermo Fisher), TPCN1 (Hs00330542_m1; Thermo Fisher), NALCN (Hs00291166_m1; Thermo Fisher), TRIP11 (Hs00188542_m1; Thermo Fisher), DCP2 (Hs00968155_m1; Thermo Fisher), NF1 (Hs01035108_m1; Thermo Fisher), and GAPDH (Hs99999905_m1; Thermo Fisher)—was performed using the QuanStudio 6 Pro system to assess gene expression. Quantitative data were obtained from Design & Analysis software 2.6.0, and then GraphPad 9.0 was used for data quantification and visualization. All values ​​in Table 10a below represent the percentage of the specified target transcripts detected by qPCR, normalized to GAPDH. Table 10a: Nav1.8 gene expression analysis maternal body AS3 AS4 AS5 dsRNA No. 2c 3c 4c 5c 6c 7c 8c 9c 10c 11c SCN10A 33 44 57 48 43 56 61 52 47 48 SCN8A 40 84 85 81 76 86 86 96 87 86 SCN9A 56 83 85 86 88 83 88 91 96 94 TPCN1 72 95 99 82 97 94 95 105 92 97 NALCN 47 55 61 51 59 62 57 71 65 69 SCN7A 74 68 75 67 80 78 81 77 81 83 SCN11A 67 65 65 64 61 58 58 68 80 78 SCN1A 64 65 60 62 61 54 66 69 69 70 SCN4A 43 50 50 41 41 45 39 47 56 54 SCN2A 91 103 90 90 94 94 95 105 90 92 SCN5A 87 100 97 113 114 98 97 105 95 112 GTF2F1 79 94 89 83 89 98 77 99 88 102 TRIP11 82 83 75 81 84 89 80 106 95 92 DCP2 79 80 79 79 86 97 90 96 94 93 Off-target average 68 79 78 76 79 80 78 87 84 86 Missing the target / on the target 2.05 1.77 1.36 1.59 1.86 1.43 1.27 1.67 1.80 1.79 maternal body AS6 AS7 AS8 2c 12c 13c 14c 15c 16c 17c 18c 19c 20c SCN10A 33 58 51 51 62 58 55 53 60 47 SCN8A 40 103 98 110 92 97 99 73 88 94 SCN9A 56 109 87 103 107 98 110 95 88 91 TPCN1 72 115 110 111 111 106 113 106 110 106 NALCN 47 100 85 86 86 87 80 97 82 82 SCN7A 74 95 94 93 88 88 104 91 85 89 SCN11A 67 84 81 86 80 79 78 75 87 78 SCN1A 64 83 74 80 72 77 81 67 70 64 SCN4A 43 64 61 49 80 63 59 52 53 42 SCN2A 91 104 90 99 97 94 94 95 99 96 SCN5A 87 110 114 113 109 118 123 106 111 124 GTF2F1 79 111 106 113 111 112 103 112 100 101 TRIP11 82 98 93 104 86 96 99 94 94 91 DCP2 79 99 94 104 105 89 101 94 90 94 Table 10b: Ratio of changes in off-target activity to changes in on-target activity AS3 AS4 AS5 3c 4c 5c 6c 7c 8c 9c 10c 11c SCN10A 11.37 24.03 14.44 9.49 22.54 27.83 19.13 13.53 15.24 SCN8A 3.86 1.86 2.80 3.72 2.00 1.65 2.92 3.44 2.99 SCN9A 2.37 1.83 2.08 3.36 1.20 1.14 1.84 2.96 2.52 TPCN1 2.03 1.12 0.70 2.61 0.95 0.83 1.72 1.50 1.65 NALCN 0.68 -0.48 0.27 1.22 0.65 0.33 1.25 1.30 1.41 SCN7A -0.58 0.03 -0.49 0.56 0.15 0.22 0.14 0.47 0.56 SCN11A -0.19 -0.37 -0.21 -0.72 -0.43 -0.34 0.04 0.95 0.67 SCN1A 0.10 -0.17 -0.09 -0.29 -0.42 0.06 0.29 0.41 0.41 SCN4A 0.59 -0.58 -0.16 -0.26 0.08 -0.15 0.17 0.95 0.70 average value 1.11 0.41 0.61 1.28 0.52 0.47 1.05 1.50 1.36 AS6 AS7 AS8 12c 13c 14c 15c 16c 17c 18c 19c 20c SCN10A 25.24 18.06 17.56 28.97 25.38 22.02 19.65 26.77 14.13 SCN8A 2.50 3.18 3.97 1.77 2.23 2.67 1.68 1.79 3.78 SCN9A 2.11 1.71 2.66 1.77 1.65 2.43 1.96 1.18 2.46 TPCN1 1.68 2.07 2.18 1.34 1.33 1.87 1.73 1.43 2.38 NALCN 2.08 2.07 2.19 1.34 1.57 1.49 2.53 1.29 2.46 SCN7A 0.82 1.09 1.06 0.49 0.55 1.34 0.84 0.40 1.01 SCN11A 0.65 0.77 1.06 0.44 0.45 0.49 0.41 0.73 0.77 SCN1A 0.75 0.59 0.90 0.29 0.52 0.77 0.15 0.23 0.55 SCN4A 0.81 0.98 0.31 1.26 0.76 0.69 0.43 0.34 -0.08 average value 1.42 1.56 1.79 1.09 1.13 1.47 1.22 0.93 1.67 Example 4

[0138] The attenuation of ANGPTL8 expression by Gal1-conjugated ANGPTL8 siRNA modified with butylene glycol (BD) in the seed region was analyzed using the following procedure: Freshly isolated primary mouse hepatocytes (MPH) from AAV-ANGPTL8 humanized mice were added to Corning discs at 15 kJ / well, and siRNA was added directly to the wells. For Hep3B (ATCC) cells, the transfection reagent RNAiMAX (Life Technologies) was mixed with siRNA at 0.3 µl / well in Corning discs, and cells were then added at 8 kJ / well. To generate concentration / dose-response curves, the final concentrations of GalNAc-conjugated siRNA were 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM.

[0139] Cells were lysed after treatment, and RNA was directly isolated into 96-well plates using the Quick-RNA 96 kit (Zymo Research). The lysed RNA was used immediately or frozen. cDNA was synthesized using a Fast Advanced RT Master Mix (Invitrogen) in a thermal cycler following the steps below: 37°C for 30 minutes, 95°C for 5 minutes, and held at 4°C. Polymerase chain reaction (PCR) was performed using a TaqMan RT PCR (Life Technologies) system with the following cycling temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds each, and 60°C for 1 minute.

[0140] Human ANGPTL8 levels were normalized to mouse (for MPH) or human (for Hep3B) Rplp0 (Life Technologies) and represent a relative decrease in human ANGPTL8 mRNA expression compared to mediator-treated control cells. IC50 values ​​were calculated using a 4-parameter fitting model with XLFit, and the results are shown in Table 11.

[0141] Table 11 shows the percentage of potency maintained and reduced by introducing destabilized nucleotides containing the butylene glycol moiety relative to the parent compounds (2d-6d) without butylene glycol modification in almost all of the tested compounds. Table 11: Ang8 AAV-hA8 MPH and Hep3B / RNAiMAX AAV-hA8 MPH Hep3B / RNAiMAX dsRNA No. Delivery section Attachment IC50 / nM Maximum KD% IC50 / nM Maximum KD% 2d GalNAc 3' Righteous Stocks 0.23 96.24 0.08 79.74 3D GalNAc 3' Righteous Stocks 0.329 92.84 0.037 88.62 4d GalNAc 3' Righteous Stocks 0.105 99.01 0.056 84.96 5d GalNAc 3' Righteous Stocks 0.488 91.01 0.104 82.17 6d GalNAc 3' Righteous Stocks 1.204 55 0.137 82.37 7d GalNAc 3' Righteous Stocks 0.215 97.38 0.208 90.17 8d GalNAc 3' Righteous Stocks 0.318 93.6 0.215 84.92 9d GalNAc 3' Righteous Stocks 0.497 91.41 0.134 89.16 10d GalNAc 3' Righteous Stocks 0.704 89.53 0.581 83 11d GalNAc 3' Righteous Stocks 1.637 84.96 0.727 77.52 12d GalNAc 3' Righteous Stocks 1.165 87.76 0.186 81.92 13d GalNAc 3' Righteous Stocks 0.311 94.96 0.129 85.35 14d GalNAc 3' Righteous Stocks 0.544 90.92 0.048 91.28 15d GalNAc 3' Righteous Stocks 2.46 90.88 NA NA 16d GalNAc 3' Righteous Stocks 1.95 94.19 NA NA 17d GalNAc 3' Righteous Stocks 1.09 94.76 NA NA 18d GalNAc 3' Righteous Stocks 2.35 92.43 NA NA 19d GalNAc 3' Righteous Stocks 2.33 94.6 NA NA 20d GalNAc 3' Righteous Stocks 3.29 91.74 NA NA 21d GalNAc 3' Righteous Stocks 3.57 89.74 NA NA 22d GalNAc 3' Righteous Stocks 4.15 92.13 NA NA 23d GalNAc 3' Righteous Stocks 7.82 86.14 NA NA 24d GalNAc 3' Righteous Stocks 5.43 76.25 NA NA 25d GalNAc 3' Righteous Stocks 11.28 77.6 NA NA 26d GalNAc 3' Righteous Stocks 5.42 81.03 NA NA 27d GalNAc 3' Righteous Stocks 12.42 72.59 NA NA 28d GalNAc 3' Righteous Stocks 8.74 84.76 NA NA 29d GalNAc 3' Righteous Stocks 3.71 92.77 NA NA 30d GalNAc 3' Righteous Stocks 3.78 90.42 NA NA 31d GalNAc 3' Righteous Stocks 4.14 85.9 NA NA Example 5: ANGPTL8 siRNA in vivo method In vivo single-dose screening

[0142] GalNAc-siRNA (n=9) was tested in male mice (Taconic farms). The siRNA was tested in a single-dose study. Mice were administered an adeno-associated virus (AAV) vector (Vector BioLabs) containing the plasmid and albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7) via retroorbital injection. Blood was collected from mice via retroorbital puncture 14 days after AAV administration. Serum was prepared from the blood, and triglycerides were measured using a COBAS clinical chemistry analyzer (Roche), while ANGPTL4 / 8 was measured using an internal ELISA (Meso Scale Diagnostics). Mouse body weight was measured 22 days after AAV administration. Mice were assigned to groups (n=5) with similar body weight, serum triglyceride levels, and serum ANGPTL4 / 8. PBS or the test substance GalNac-siRNA was administered subcutaneously to mice at a dose of 5 mg / kg. Seven days after subcutaneous injection, blood was collected from all mice, and serum triglycerides were analyzed as is. Fourteen days after subcutaneous injection, mice were sacrificed under isoflurane anesthesia. Liver samples were collected from these mice and frozen in liquid nitrogen. The livers were homogenized in TriZol (Invitrogen) on FastPrep-24 (MP Bio) using lysis matrix D beads. Chloroform was added, and the aqueous phase was mixed with ethanol to precipitate RNA. RNA was isolated on a PureLink Pro96 total RNA purification kit (Invitrogen) according to the manufacturer's protocol and quantified on NanoDrop (ThermoFisher). An equal volume (1 µg) of RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Life Technologies) on a Mastercycler Nexus (Eppendorf). The thermal cycler was set to 25°C for 10 min, 37°C for 2 hr, and then 85°C for 5 min. Template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probe sets and RT-PCR was performed on a QuantStudio Pro7 (ThermoFisher) with the following parameters: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Fold change (FC) was calculated as follows: delta CT value was obtained by subtracting the CT value of mouse Rplp0 from the CT value of human ANGPTL8. Delta CT value was then calculated by subtracting the delta CT value of the untreated sample (PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the logarithm base 2 of the negative delta CT value.The reduction percentage (KD%) is calculated by subtracting FC from 1 and multiplying by 100. The data is shown in Table 12.

[0143] Table 12 shows the in vivo attenuation of Ang8 in the AAV mouse model. Additionally, Table 12 shows the location of the attenuation and stereochemical variability depending on the position of the modified nucleotide. Table 12. Reduction of ANGPTL8 mRNA at 5 mg / kg dsRNA No. Delivery section Attachment Dosage (mg / kg) FC KD% 2d GalNAc 3' Righteous Stocks 5 0.12 88.3 6d GalNAc 3' Righteous Stocks 5 0.12 88.0 7d GalNAc 3' Righteous Stocks 5 0.21 78.7 8d GalNAc 3' Righteous Stocks 5 0.20 79.7 10d GalNAc 3' Righteous Stocks 5 0.46 53.7 11d GalNAc 3' Righteous Stocks 5 0.31 68.7 12d GalNAc 3' Righteous Stocks 5 0.27 72.8 13d GalNAc 3' Righteous Stocks 5 0.15 84.7 14d GalNAc 3' Righteous Stocks 5 0.25 74.8 Example 6: In vitro attenuation of HMGCR in Hep3B cells by transfection with butylene glycol-modified HMGCR siRNA

[0144] The attenuation of HMGCR expression by butylene glycol-modified HMGCR siRNA was analyzed using the following procedure: siRNA was mixed with RNAiMAX (Life Technologies) transfection reagent at 0.3 µl / well in a Corning dish, and Hep3B (ATCC) cells were added at 20,000 cells per well. To generate concentration / dose-response curves, the final concentrations of butylene glycol-modified siRNA were determined using 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.014, 0.005, 0.002, 0.0005, and 0.0002 nM.

[0145] The treated cells were lysed, and RNA was directly lysed into 96-well plates using the Quick-RNA 96 kit (Zymo Research). The lysed RNA was used immediately or frozen. cDNA was synthesized using a Fast Advanced RT Master Mix (Invitrogen) in a thermal cycler following the steps below: 37°C for 30 minutes, 95°C for 5 minutes, and held at 4°C. Polymerase chain reaction (PCR) was performed using a TaqMan RT PCR (Life Technologies) system with the following cycling temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds each, and 60°C for 1 minute.

[0146] Human HMGCR levels were normalized to human RPLP0 (Life Technologies) and represent a relative decrease in human HMGCR mRNA expression compared to mediator-treated control cells. IC50 values ​​were calculated using an XLFit model with a 4-parameter fit.

[0147] Data confirms the potent potency of RNAi agents containing butylene glycol, especially when the butylene glycol is incorporated at position 6 (counting from the 5' end of the antisense).

[0148] Table 13A: Shows the IC50 and maximum attenuation percentage achieved in Hep3B cells by transfection with RNAiMAX using HMGCR-indicating siRNA. Data are expressed as a percentage attenuation of HMGCR signaling relative to untreated cells. Table 13A: HMGCR IC50 and maximum attenuation percentage in Hep3B cells dsRNA No. Delivery section Attachment IC50 / nM KD% 3e GalNAc 3' Righteous Stocks 0.014 85.87 4e GalNAc 3' Righteous Stocks 5e GalNAc 3' Righteous Stocks 0.509 88.84 6e GalNAc 3' Righteous Stocks 0.25 87.82 7e GalNAc 3' Righteous Stocks 0.142 87.88 8e GalNAc 3' Righteous Stocks 0.19 85.7 9e GalNAc 3' Righteous Stocks 0.086 88.34 10e GalNAc 3' Righteous Stocks 0.043 93.46 11e GalNAc 3' Righteous Stocks 0.327 87.26 12e GalNAc 3' Righteous Stocks 10 79 13e GalNAc 3' Righteous Stocks 0.14 92.45 14e GalNAc 3' Righteous Stocks 0.147 94.51 15e GalNAc 3' Righteous Stocks 0.047 93.11 16e GalNAc 3' Righteous Stocks 0.498 95.57 17e GalNAc 3' Righteous Stocks 0.369 91.53 18e GalNAc 3' Righteous Stocks 0.492 90.82 19e GalNAc 3' Righteous Stocks 0.561 89 20e GalNAc 3' Righteous Stocks 3.643 82 21e GalNAc 3' Righteous Stocks 10 81 22e GalNAc 3' Righteous Stocks 10 36 23e GalNAc 3' Righteous Stocks 1.902 47.46 24e GalNAc 3' Righteous Stocks 10 64 25e GalNAc 3' Righteous Stocks 1.236 97.85 Example 7 In vivo method of HMGCR siRNA in mice

[0149] GalNAc-siRNA was tested in male C57bl / 6 mice (Taconic farms) aged 7–10 weeks. Mice were weighed approximately one week before administration of the test substance or mediator. Mice were assigned to groups with similar body weights. At the start of the study (day 0), mice were subcutaneously administered PBS or the test substance GalNac-siRNA at a dose such as 5 mg / kg. Each group consisted of ten animals. Two weeks after siRNA administration, three mice from each group were sacrificed under isoflurane anesthesia, and their livers were collected and frozen in liquid nitrogen. Eight weeks after siRNA administration, the remaining seven mice were sacrificed under isoflurane anesthesia. Liver samples were collected from these mice. The livers were homogenized in TriZol (Invitrogen) using FastPrep-24 (MP Bio) with lysis matrix D beads. Chloroform was added, and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated from a PureLink Pro96 total RNA purification kit (Invitrogen) column according to the manufacturer's protocol and quantified using NanoDrop (ThermoFisher). An equal volume (1 μg) of RNA was reverse transcribed into cDNA using a Mastercycler Nexus (Eppendorf) using a high-capacity cDNA reverse transcription kit (Life Technologies). The thermal cycler was set to 25°C for 10 min, 37°C for 2 h, and then 85°C for 5 min. The template cDNA was combined with the Taqman Universal Master Mix and Assays on Demand primer / probe set and RT-PCR was performed using a QuantStudio Pro7 (ThermoFisher) with the following parameters: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 seconds each and 60°C for 1 min. The fold change (FC) was calculated by subtracting the CT value of mouse Rplp0 from the CT value of mouse HMGCR to obtain the delta CT value. The delta-CT value was then calculated by subtracting the delta-CT value of the untreated sample (the average of the PBS control) from the delta-CT value of each test sample. The fold change was calculated by taking the logarithm base 2 of the negative delta-CT value. This fold change was then multiplied by 100 to calculate the percentage of remaining mRNA. The data are shown in Table 14. This data confirms that the RNAi agent incorporating butylene glycol at position 6 of the antisense strand exhibits good in vivo attenuation. Table 14: HMGCR mRNA reduction at 5 mg / kg for 2 weeks dsRNA No Multiple change KD% 4e 0.28 72.1 9e 0.18 82.2 10e 0.43 58.3 Sequence Listing SEQ ID NO Sequence 1 UCCUAUGACUGUAGAUUUUAA 2 UUAAAAUCUACAGUCAUAGGAAU 3 GUUGACUUGAGUAAAUAUAUA 4 UAUAUAUUUACUCAAGUCAACAU 5 CCUUUGAAGCCUUGAUAAAGA 6 UCUUUAUCAAGGCUUCAAAGGUG 7 AUGGAGGAGGAUAUUCUGCAA 8 UUGCAGAAUAUCCUCCUCCAUCU 9 mU*mC*mCmUmAmUfGmAfCfUfGmUmAmGmAmUmUmUmU*mA*mA 10 mU*fU*mAmAmAmAmUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 11 mU*fU*mA(sgA)mAmAmUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 12 mU*fU*mA(ssbdA)mAmAmUmCmUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 13 mU*fU*mA(rsbdA)mAmUmCmUmAmCmGfUmCfUmGmGMA*mA*mU 14 mU*fU*mA(sgA)MUMCMUMAmCmGfUmCfAmGmGMA*mA*mU 15 mU*fU*mA(ssbdA)MUMCMUMAmCmGfUmCfUmGmGMA*mA*mU 16 mU*fU*mA(rsbdA)mAmCmUmAmCmGfUmCfAmGmGMA*mA*mU 17 mU*fU*mAmA(sgA)MUMCMUMAmCmGfUmCfAmGmGMA*mA*mU 18 mU*fU*mAmA(ssbdA)MUMCMUMAmCmGfUmCfUmAmGmGMA*mA*mU 19 mU*fU*mAmA(rsbdA)MUMCMUMAmCmGfUmCfAmGmGMA*mA*mU 20 mU*fU*mAmAmA(sgU)MCMUMAmCmGfUmCfUmAmGmGMA*mA*mU 21 mU*fU*mAmAmA(ssbdU)MCMUMAmCmGfUmCfUmGmGMA*mA*mU 22 mU*fU*mAmAmA(rsbdU)MCMUMAmCmGfUmCfUmAmGmGMA*mA*mU 23 mU*fU*mAmAmAmU(sgC)MUMAmCmAmGfUmCfAmGmGMA*mA*mU 24 mU*fU*mAmAmAmU(ssbdC)MUMAmCmAmGfUmCfAmGmGMA*mA*mU 25 mU*fU*mAmAmAmAmU(rsbdC)mUmAmCmAmGfUmCfAmUmAmGmGmA*mA*mU 26 mG*mU*mUmGmAmCmUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 27 PmU*fA*mUmAfUmAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 28 PmU*fA*mUmAfU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 29 PmU*fA*mUmAfUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 30 mU*fA*fUmAmUmAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 31 PmU*fA*fUmAmUmAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 32 PmU*fA*fUmA(ssbdU)mAfUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 33 PmU*fA*fUmAmU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 34 PmU*fA*fUmAmUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 35 PmU*fA*mUmAfUmAmUfUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 36 PmU*fA*mUmAfU(ssbdA)mUfUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 37 PmU*fA*mUmAfUmA(ssbdU)fUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 38 PmU*fA*mUmAmUmAmUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 39 PmU*fA*mUmAmUmA(ssbdU)mUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 40 PmU*fA*mUmAmUfAmUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 41 mU*fA*mUmAfU(rsbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 42 mU*fA*mUmAfU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 43 mU*fA*fUmAmU(rsbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 44 mU*fA*fUmAmU(ssbdA)fUmUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 45 mU*fA*mUmAfUmA(rsbdU)fUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 46 mU*fA*mUmAfUmA(ssbdU)fUmUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 47 mU*fA*mUmAfUmAfU(rsbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 48 mU*fA*mUmAfUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 49 mU*fA*fUmAmUmAfU(rsbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 50 mG*mU*mUmGmAmCfUmUfGfAfGmUmAmAmAmUmAmUmA*mU*mA 51 mU*fA*fUmAmUmAfU(ssbdU)mUmAmCmUmCfAmAfGmUmCmAmAmC*mA*mU 52 mC*mC*mUmUmUmGfAmAfGfCfCmUmUmGmAmUmAmAmA*mG*mA 53 PmU*fC*mUmUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 54 PmU*fC*(sgU)mUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 55 PmU*fC*(rsbdU)mUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 56 PmU*fC*(ssbdU)mUmUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 57 PmU*fC*mU(sgU)mUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 58 PmU*fC*mU(rsbdU)mUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 59 PmU*fC*mU(ssbdU)mUfAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 60 PmU*fC*mUmU(sgU)fAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 61 PmU*fC*mUmU(rsbdU)fAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 62 PmU*fC*mUmU(ssbdU)fAmUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 63 PmU*fC*mUmUmU(sgA)mUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 64 PmU*fC*mUmUmU(rsbdA)mUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 65 PmU*fC*mUmUmU(ssbdA)mUmCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 66 PmU*fC*mUmUmUfA(sgU)mCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 67 PmU*fC*mUmUmUfA(rsbdU)mCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 68 PmU*fC*mUmUmUfA(ssbdU)mCmAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 69 PmU*fC*mUmUmUfAmU(sgC)mAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 70 PmU*fC*mUmUmUfAmU(rsbdC)mAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 71 PmU*fC*mUmUmUfAmU(ssbdC)mAmAmGmGmCfUmUfCmAmAmAmGmG*mU*mG 72 mA*mU*mGmGmAmGfGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 73 PmU*fU*mGmCmAfGmAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 74 mA*mU*mGmGmAmGmGmAfGfGfAmUmAmUmUmCmUmGmC*mA*mA 75 PmU*fU*mGmCfAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 76 PmU*fU*fGmCmAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 77 PmU*fU*mGmCfAmGmAfAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 78 mU*fU*mGmCfAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 79 mU*fU*fGmC(ssbdA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 80 mU*fU*fGmC(rsbdA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 81 mU*fU*mGmCfA(ssbdG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 82 mU*fU*mGmCfA(rsbdG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 83 mU*fU*mGmCmAmG(ssbdA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 84 mU*fU*mGmCmAmG(rsbdA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 85 mU*fU*fGmCmAmGfA(ssbdA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 86 mU*fU*fGmCmAmGfA(rsbdA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 87 mU*fU*fGmCmAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 88 mU*fU*fG(sgC)mAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 89 mU*fU*fG(ssbdC)mAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 90 mU*fU*fG(rsbdC)mAmGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 91 mU*fU*fGmC(sgA)mGfAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 92 mU*fU*mGmCfA(sgG)fAmAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 93 mU*fU*mGmCmAmG(sgA)mAmUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 94 mU*fU*fGmCmAmGfA(sgA)mUmAmUmCmCfUmCfCmUmCmCmAmU*mC*mU 95 96 MPTRSPSVVISDDEPGYDLDLFCIPNHYAEDLEKVFIPHGLIMDRTERLARDVMKEMGGHHIVALCVLKGGYKFFADLLDYIKALNRNSDRSIPMTVDFIRLKSYCNDQSTGDIKVIGGDDLSTLTGKNVLIVEDIIDTGKTMQTLLSLVKQYSPKMVKVASLLVKRTSRSVGYRPDFVGFEIPDKFVVGYALDYNEYFRDLNHVCVISETGKAKYKA 97 98 MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV 99 101 ATACCTTAGACCCTCAGTCATGCCAGTGCCTGCTCTGTGCCTGCTCTGGGCCCTGGCAATGGTGACCCGGCCTGCCTCAGCGGCCCCCATGGGCGGCCCAGAACTGGCACAGCATGAGGAGCTGACCCTGCTCTTCCATGGGACCCTGCAGCTGGGCCAGGCCCTCAACGGTGTGTACAGGACCACGGAGGGACGGCTGACAAAGGCCAGGAACAGCCTGGGTCTCTATGGCCGCACAATAGAACTCCTGGGGCAGGAGGTCAGCCGGGGCCGGGATGCAGCCCAGGAACTTCGGGCAAGCCTGTTGGAGACTCAGATGGAGGAGGATATTCTGCAGCTGCAGGCAGAGGCCACAGCTGAGGTGCTGGGGGAGGTGGCCCAGGCACAGAAGGTGCTACGGGACAGCGTGCAGCGGCTAGAAGTCCAGCTGAGGAGCGCCTGGCTGGGCCCTGCCTACCGAGAATTTGAGGTCTTAAAGGCTCACGCTGACAAGCAGAGCCACATCCTATGGGCCCTCACAGGCCACGTGCAGCGGCAGAGGCGGGAGATGGTGGCACAGCAGCATCGGCTGCGACAGATCCAGGAGAGACTCCACACAGCGGCGCTCCCAGCCTGAATCTGCCTGGATGGAACTGAGGACCAATCATGCTGCAAGGAACACTTCCACGCCCCGTGAGGCCCCTGTGCAGGGAGGAGCTGCCTGTTCACTGGGATCAGCCAGGGCGCCGGGCCCCACTTCTGAGCACAGAGCAGAGACAGACGCAGGCGGGGACAAAGGCAGAGGATGTAGCCCCATTGGGGAGGGGTGGAGGAAGGACATGTACCCTTTCATGCCTACACACCCCTCATTAAAGCAGAGTCGTGGCATCTCA 102 MPVPALCLLWALAMVTRPASAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA 103 104 MLSRLFRMHGLFVASHPWEVIVGTVTLTICMMSMNMFTGNNKICGWNYECPKFEEDVLSSDIIILTITRCIAILYIYFQFQNLRQLGSKYILGIAGLFTIFSSFVFSTVVIHFLDKELTGLNEALPFFLLLIDLSRASTLAKFALSSNSQDEVRENIARGMAILGPTFTLDALVECLVIGVGTMSGVRQLEIMCCFGCMSVLANYFVFMTFFPACVSLVLELSRESREGRPIWQLSHFARVLEEEENKPNPVTQRVKMIMSLGLVLVHAHSRWIADPSPQNSTADTSKVSLGLDENVSKRIEPSVSLWQFYLSKMISMDIEQVITLSLALLLAVKYIFFEQTETESTLSLKNPITSPVVTQKKVPDNCCRREPMLVRNNQKCDSVEEETGINRERKVEVIKPLVAETDTPNRATFVVGNSSLLDTSSVLVTQEPEIELPREPRPNEECLQILGNAEKGAKFLSDAEIIQLVNAKHIPAYKLETLMETHERGVSIRRQLLSKKLSEPSSLQYLPYRDYNYSLVMGACCENVIGYMPIPVGVAGPLCLDEKEFQVPMATTEGCLVASTNRGCRAIGLGGGASSRVLADGMTRGPVVRLPRACDSAEVKAWLETSEGFAVIKEAFDSTSRFARLQKLHTSIAGRNLYIRFQSRSGDAMGMNMISKGTEKALSKLHEYFPEMQILAVSGNYCTDKKPAAINWIEGRGKSVVCEAVIPAKVVREVLKTTTEAMIEVNINKNLVGSAMAGSIGGYNAHAANIVTAIYIACGQDAAQNVGSSNCITLMEASGPTNEDLYISCTMPSIEIGTVGGGTNLLPQQACLQMLGVQGACKDNPGENARQLARIVCGTVMAGELSLMAALAAGHLVKSHMIHNRSKINLQDLQGACTKKTA 105 GUAGUUCAAACUGUAUUACUA 106 WAGUAAUCAGUUUGAWA 107 CUGUAUUACUUUAAUGGAAGA 108 UCUUCCAUAAAAGUAAUCAGUU 109 mG*mU*mAmGmUmCmAfAfCmUmGmUmAmUmAmC*mU*mA 110 PmU*fA*mGmUfAmAfUmAmCmAmGmUmUfUmGfAmCmUmAmC*mC*mA 111 VPmU*fA*mGmUfAmAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 112 mU*fA*mG(rsbdU)fAmAfUmAmCmAmGmUmUfUmGfAmCmUmAmC*mC*mA 113 mU*fA*mG(ssbdU)fAmAfUmAmCmAmGmUmUfUmGfAmCmUmAmC*mC*mA 114 mU*fA*fGmU(rsbdA)mAfUmAmCmAmGmUmUfUmGfAmCmUmAmC*mC*mA 115 mU*fA*fGmU(ssbdA)mAfUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 116 mU*fA*mGmUfA(rsbdA)fUmAmCmAmGmUmUfUmGfAmCmUmAmC*mC*mA 117 mU*fA*mGmUfA(ssbdA)fUmAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 118 mU*fA*mGmUMA(rsbdU)mAmCmAmGmUmUfUmGfAMAmCmUmAmC*mC*mA 119 mU*fA*mGmUmAmA(ssbdU)mAmCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 120 mU*fA*fGmUmAmAfU(rsbdA)mCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 121 mU*fA*fGmUmAmAfU(ssbdA)mCmAmGmUmUfUmGfAmAmCmUmAmC*mC*mA 122 mC*mU*mGmUmAmUmUmAfCfUfUmUmAmAmUmGmGmAmA*mG*mA 123 PmU*fC*mUmUfCmCfAmUmUmAmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 124 mU*fC*mU(rsbdU)fCmCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 125 mU*fC*mU(ssbdU)fCmCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 126 mU*fC*fUmU(rsbdC)mCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 127 mU*fC*fUmU(ssbdC)mCfAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 128 mU*fC*mUmUfC(rsbdC)fAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 129 mU*fC*mUmUfC(ssbdC)fAmUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 130 mU*fC*mUmUmCmC(rsbdA)mUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 131 mU*fC*mUmUmCmC(ssbdA)mUmUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 132 mU*fC*mUmUfCmCfA(rsbdU)mUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU 133 mU*fC*mUmUfCmCfA(ssbdU)mUmAmAmAmGfUmAfAmUmAmCmAmG*mU*mU

[0150] TW202600532A_114107737_SEQL.xml

Claims

1. An oligonucleotide comprising a compound of the following formula: , wherein X1 is the 5' portion of the oligonucleotide, X2 and X3 are one or more independently selected nucleotides, X4 is the 3' portion of the oligonucleotide, m and n are independently selected from any integer from 0 to 40, and XB is a butanediol-modified nucleotide of the following formula: .

2. An oligonucleotide as claimed in claim 1, wherein the compound has the following formula: , where B is a nucleobase.

3. An oligonucleotide as claimed in claim 1, wherein the compound has the following formula: , where B is a nucleobase.

4. An oligonucleotide as claimed in claim 1, wherein the compound has the following formula: , where B is a nucleobase.

5. The oligonucleotide of claim 1, wherein the compound has the following formula: , wherein each B is an independently selected nucleobase, m and n are independently selected from any integer from 0 to 20, X is independently O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

6. The oligonucleotide of claim 1, wherein the compound has the following formula: , wherein each B is an independently selected nucleobase, m and n are independently selected from any integer from 0 to 20, X is independently O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

7. The oligonucleotide of claim 1, wherein the compound has the following formula: , wherein each B is an independently selected nucleobase, m and n are independently selected from any integer from 0 to 20, X is independently O or S, R is independently selected from H, O-R1, F, Cl, and R1 is H, C1 to C20 alkyl, or C1 to C20 alkyl-O-C1 to C20 alkyl.

8. An oligonucleotide of any one of claims 1 to 4, wherein each nucleobase system is independently selected from the group consisting of: hypoxanthine, xanthine, 7-methylguanine, inosine, xanthine, 7-methylguanine, 5,6-dihydrouridine, 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouridine, 5-methylcytosine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

9. An oligonucleotide as claimed in any of claims 1 to 4, wherein each nucleobase group is independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

10. An oligonucleotide of any one of claims 1 to 4, wherein the nucleobases in the butanediol-modified nucleotide are independently selected from the group consisting of adenine, cytosine, and uracil.

11. An oligonucleotide of any one of claims 1 to 4, wherein the nucleobases in the butanediol-modified nucleotide are independently selected from the group consisting of cytosine and uracil.

12. An oligonucleotide of any one of claims 1 to 4, wherein the nucleobase of the butanediol-modified nucleotide is uracil.

13. An oligonucleotide of any one of claims 1 to 4, wherein the total length of the oligonucleotide is 15 to 30 nucleotides.

14. An oligonucleotide of any one of claims 1 to 4, wherein the total length of the oligonucleotide is 18 to 30 nucleotides.

15. An oligonucleotide of any one of claims 1 to 4, wherein the total length of the oligonucleotide is 23 nucleotides.

16. An oligonucleotide of any one of claims 1 to 4, wherein the butanediol-modified nucleotide is located at position 3, 4, 5, 6, 7 or 8 from the 5' end.

17. An oligonucleotide of any one of claims 1 to 4, wherein the butanediol-modified nucleotide is located at position 5, 6, 7 or 8, counting from the 5' end.

18. An oligonucleotide of any one of claims 1 to 4, wherein the butanediol-modified nucleotide is located at position 5, 6 or 8, counting from the 5' end.

19. An oligonucleotide of any one of claims 1 to 4, wherein the butanediol-modified nucleotide is located at position 5, counting from the 5' end.

20. An oligonucleotide of any one of claims 5-7, wherein R is 2'-fluorine at one of the following positions: a. 2, 3, 7, 14, and 16 from the 5' end; b. 2, 5, 7, 14, and 16 from the 5' end; c. 2, 3, 8, 14, and 16 from the 5' end; d. 2, 5, 8, 14, and 16 from the 5' end; or e. 2, 6, 14, and 16 from the 5' end, and R is 2'O-methyl at all other positions of the oligonucleotide.

21. An oligonucleotide of any one of claims 5-7, wherein R is 2' fluorine at positions 2, 14 and 16 from the 5' end of the oligonucleotide, and R is 2' O-methyl at all other positions of the oligonucleotide.

22. The oligonucleotide of any one of claims 1 to 4, wherein the butanediol-modified oligonucleotide is located at position 22, position 23 or positions 22 and 23, counting from the 5' end.

23. A compound of the following formula, wherein the B-series nucleobases and the Y-series protecting groups are selected from the group consisting of: methoxymethyl ether (MOM), methoxyethoxymethyl ether (MEM), tributyldimethylsilane (TBDMS), tributyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytriphenylmethyl (DMT), methoxytriphenylmethyl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), trimethylacetyl (Piv), tetrahydropiperanyl (THP), tetrahydrofuran (THF), triphenylmethyl (Tr), and trimethylsilyl (TMS).

24. The compound of claim 23, wherein the compound has the following formula: .

25. The compound of claim 23, wherein the compound has the following formula: .

26. The compound of any one of claims 23 to 25, wherein the organic protecting group is dimethoxytriphenylmethyl (DMT).

27. The compound of claim 23, wherein the compound has the following formula: .

28. The compound of claim 23, wherein the compound has the following formula: .

29. The compound of claim 23, wherein the compound has the following formula: .

30. The compound of any one of claims 23 to 25 and 27 to 29, wherein the nucleobase system is selected from the group consisting of: hypoxanthine, xanthine, 7-methylguanine, inosine, xanthine, 7-methylguanine, 5,6-dihydrouridine, 5-methylcytosine, 5-hydroxymethylcytosine, dihydrouridine, 5-methylcytosine, pseudouridine, adenine, guanine, cytosine, thymine, and uracil.

31. The compound of any one of claims 23 to 25 and 27 to 29, wherein the nucleobase system is selected from the group consisting of adenine, cytosine, thymine, uracil and guanine.

32. The compound of any one of claims 23 to 25 and 27 to 29, wherein the nucleobase system is selected from the group consisting of adenine, cytosine and uracil.

33. The compound of any one of claims 23 to 25 and 27 to 29, wherein the nucleobase system is selected from the group consisting of cytosine and uracil.

34. The compound of any one of claims 23 to 25 and 27 to 29, wherein the nucleobase is uracil.

35. A compound of the following formula, wherein B is a nucleobase, Y is an organic protecting group selected from the group consisting of: methoxymethyl ether (MOM), methoxyethoxymethyl ether (MEM), tributyldimethylsilane (TBDMS), tributyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytriphenylmethyl (DMT), methoxytriphenylmethyl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), trimethylacetyl (Piv), tetrahydropiperanyl (THP), tetrahydrofuran (THF), triphenylmethyl (Tr), and trimethylsilyl (TMS), and Z is a phosphate ester or a phosphoramidene represented by a compound of the following formula: .

36. The compound of claim 35, wherein the compound has the following formula: .

37. The compound of claim 35, wherein the compound has the following formula: .

38. The compound of any one of claims 35 to 37, wherein the organic protecting group is dimethoxytriphenylmethyl (DMT).

39. The compound of claim 35, wherein the compound has the following formula: .

40. The compound of claim 35, wherein the compound has the following formula: .

41. The compound of claim 35, wherein the compound has the following formula: .

42. The compound of any one of claims 35 to 37, 40 and 41, wherein the nucleobase system is selected from the group consisting of adenine, cytosine, thymine, uracil and guanine.

43. The compound of any one of claims 35 to 37, 40 and 41, wherein the nucleobase system is selected from the group consisting of adenine, cytosine and uracil.

44. The compound of any one of claims 35 to 37, 40 and 41, wherein the nucleobase system is selected from the group consisting of cytosine and uracil.

45. The compound of any one of claims 35 to 37, 40 and 41, wherein the nucleobase is uracil.

46. ​​An RNAi agent comprising: (a) a sense oligonucleotide having a length of 15 to 30 nucleotides, and (b) an antisense oligonucleotide comprising any one of claims 1 to 22, wherein the sense oligonucleotide and the antisense oligonucleotide form a double strand.

47. An RNAi agent comprising: (a) a sense oligonucleotide of 15 to 30 nucleotides in length, and (b) an antisense oligonucleotide comprising an oligonucleotide as claimed in claim 21 or claim 22, wherein the sense oligonucleotide and the antisense oligonucleotide form a double strand.

48. An RNAi agent as claimed in claim 46 or claim 47, comprising a delivery portion comprising a GalNAc portion.

49. The RNAi agent of claim 48, wherein the GalNAc portion has the following structure: wherein the L-series bond or the linker selected from the group consisting of: , and; and the Z-series double-stranded RNA therein.

50. The RNAi agent as requested in item 49, wherein the L series is: .

51. The RNAi agent of claim 48, wherein the delivery portion is bound to the sense strand.

52. The RNAi agent of claim 48, wherein the delivery portion is bound to the 3' end of the sense strand.