Conjugates of n-acetyl-galactosamine (galnac) and oligonucleotides and uses thereof

Abstract

The present disclosure provides, in part, conjugates of N-actylgalactosamine (GalNAc) and oligonucleotides are their methods of use in inhibiting gene expression and the treatment of various disorders.

Description

CONJUGATES OF N-ACETYL-GALACTOSAMINE (GALNAC) AND OLIGONUCLEOTIDES AND USES THEREOF CROSS-REFERENCE

[0001] This application claims priority to US Provisional Application No.63 / 516,762 filed July 31, 2023 and International Application No. PCT / US2024 / 019424 filed March 11, 2024, the contents of each of which are incorporated herein by reference. FIELD OF THE INVENTION

[0002] The disclosure relates to conjugates of N-acetylgalactosamine (GalNAc) and an oligonucleotide therapeutic, and methods using the conjugates to treat various disorders in patients in need thereof. SUMMARY

[0003] The present disclosure is based, in part, on conjugates comprising N- acetylgalactosamine (GalNAc) moieties as targeting ligands and oligonucleotides as therapeutic payloads, and methods of use thereof. The conjugates described herein, in certain embodiments, are contemplated as useful for the targeted delivery of oligonucleotide therapeutics to inhibit the expression of a gene (e.g., expression of a gene described herein, e.g., ANGPLT3) for the treatment of various disorders.

[0004] In one aspect, disclosed herein is a compound represented by Formula (I):or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene, wherein 5-membered heterocyclyl is optionally substituted with one or more – (CH2CH2O)q-, wherein q is an integer from 1 to 20, and alkylene is optionally substituted with one or more -OH; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

[0005] In one aspect, disclosed herein is a compound represented by Formula (I):or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

[0006] In another aspect, provided herein is a compound represented by Formula (II):Formula (II) or a pharmaceutically acceptable salt thereof, wherein: A2is an oligonucleotide; X is selected from hydroxy and thiol; each occurrence of L and L1is a linker; and LAis absent or a linker.

[0007] In another aspect, provided herein is a compound represented by Formula (III):ormu a ( ) or a pharmaceutically acceptable salt thereof, wherein: each occurrence of L1and L is a linker; n is an integer from 1 to 3; A1is an oligonucleotide; X is selected from hydroxy and thiol; and LAis absent or a linker.

[0008] In another aspect, provided herein is a compound represented by Formula (IV):

[0009] or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl substituted with –(CH2CH2O)q-, wherein q is an integer from 1 to 20; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

[0010] In another aspect, provided herein is a pharmaceutical composition comprising a compound described herein (e.g., a compound of Formula (I), Formula (II), or Formula (III)) or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0011] In another aspect, provided herein is a method of treating a disorder (e.g., a disorder described herein) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I), Formula (II), or Formula (III)) or pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

[0012] The present disclosure is also based, in part, upon the development of double-stranded ribonucleic acid (dsRNA) targeting AGT genes, pharmaceutical compositions comprising thedsRNAs targeting AGT genes and methods of using the dsRNA to inhibit expression of AGT in a cell.

[0013] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of AGT comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 613; (b) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 614; or (c) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 618, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 615.

[0014] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of AGT, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 622, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 619; (b) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 623, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 620; or (c) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 624, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 621.

[0015] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of AGT comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 616, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 613; (b) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 617, and the sense strand comprises at least 15 contiguous nucleotides of asense strand sequence comprising the sequence of SEQ ID NO: 614; or (c) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 618, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 615.

[0016] In some aspects, the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of AGT, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 622, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 619; (b) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 623, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 620; or (c) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 624, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 621.

[0017] In some embodiments, the AGT gene is human AGT. In some embodiments, the AGT is human AGT comprising the sequence shown in SEQ ID NO: 441 (NM_001384479.1).

[0018] In some embodiments, the sense strand is 70%, 80%, 90%, 95% or more identical to the sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a sense strand sequence shown in Table 1 and Table 2. In some embodiments, the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in Table 1 and Table 2. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621. In some embodiments, the antisense strand comprises: (a) 21 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619,SEQ ID NO: 620, or SEQ ID NO: 621; (b) 22 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621; and / or (c) 23 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 615, SEQ ID NO: 619, or SEQ ID NO: 621. In some embodiments, the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621, and the antisense strand is selected from an antisense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO: 624. In some embodiments, the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621. In some embodiments, the antisense strand is selected from an antisense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO: 624. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 616 and the sense strand comprises the sequence of SEQ ID NO: 613. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 617 and the sense strand comprises the sequence of SEQ ID NO: 614. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 618 and the sense strand comprises the sequence of SEQ ID NO: 615. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 622 and the sense strand comprises the sequence of SEQ ID NO: 619. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 623 and the sense strand comprises the sequence of SEQ ID NO: 620. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 624 and the sense strand comprises the sequence of SEQ ID NO: 621. In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide selected from the group consisting of: a 5’-vinyl phosphonate nucleotide, a 2'-O-methyl modified nucleotide, an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’-5’ linked nucleotide), a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide, and a nucleotide comprising a modified nucleotide component represented by Formula (I):wherein: B1is a nucleobase; and R1is selected from the group consisting of hydrogen and C1–6alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide.

[0019] In some embodiments, the antisense strand has a 3’ end nucleotide overhang compared to the sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides. In some embodiments, the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of AGT mRNA. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the AGT mRNA. In some embodiments, the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the AGT mRNA.

[0020] In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide. In some embodiments, the modified nucleotide is at least one of a modified nucleotide selected from the group consisting of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’-5’ linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-Isomer, and a nucleotide comprising a modified nucleotide component represented by Formula (I):wherein: B1is a nucleobase; and R1is selected from the group consisting of hydrogen and C1–6alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide. In some embodiments, B1is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof. In some embodiments, B1is independently selected from uracil, cytosine, and modified analogs thereof. In some embodiments, R1is C1–6alkyl. In some embodiments, R1is -CH3. In some embodiments, B1is uracil. In some embodiments, R1is -CH3and B1is uracil. In some embodiments, the sense strand comprises an inverted deoxyribonucleotide at the 5’ end; optionally wherein the inverted deoxyribonucleotide is a 5'-5' linked deoxythymidine. In some embodiments, the sense strand comprises an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide is a 3'-3' linked deoxythymidine. In some embodiments, the sense strand comprises an inverted deoxyribonucleotide at the 5’ end and an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide at the 5’ end is a 5'-5' linked deoxythymidine and the inverted deoxyribonucleotide at the 3’ end is a 3'-3' linked deoxythymidine. In some embodiments, the sense strand comprises a nucleotide comprising the modified nucleotide component represented by Formula (I) at the 3’ end; optionally wherein R1is -CH3and B1is uracil.

[0021] In some embodiments, the modified nucleotide is at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide. In some embodiments, the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

[0022] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond.

[0023] In some embodiments, the dsRNA further comprises a ligand or targeting moiety. In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end of the sense strand of the dsRNA. In some embodiments, ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc). In some embodiments, the ligand or targeting moiety is represented by represented by Formula (I):ormu a or a pharmaceutically acceptable salt thereof, wherein: A1is the point of attachment to the dsRNA; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6. In some embodiments, each occurrence of T1and T2is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C1–6alkylene. In some embodiments, the compound is represented by Formula (I-A):Formula (I-A) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (I-A-A):Formu a (I-A-A) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (I-A-I):Formula (I-A-I) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (I-A-I-A):Formula (I-A-I-A) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (I-A-II):Formula (I-A-II) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (I-A-II- A):Formula (I-A-II-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (I-A-III):Formula (I-A-III) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (I-A-III- A):Formula (I-A-III-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the ligand or targeting moiety is tri-GalNAc6.

[0024] In some embodiments, a cell comprising a dsRNA of the disclosure is provided. In some embodiments, a vector encoding at least one unmodified strand a dsRNA of the disclosure is provided, optionally both strands. of the disclosure is provided a cell comprising the vector is provided.

[0025] In some embodiments, a pharmaceutical composition for inhibiting expression of AGT comprising the dsRNA and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof of the disclosure is provided.

[0026] In some embodiments, a method of inhibiting AGT expression in a cell is provided, the method comprising (a) contacting the cell with the dsRNA of the disclosure or the pharmaceutical composition of the disclosure; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an AGT gene,thereby inhibiting expression of the AGT gene in the cell, optionally wherein the method is in vivo. In some embodiments, the AGT expression is inhibited by at least 30% relative to a control.

[0027] In some embodiments, a method of treating a disorder mediated by or associated with AGT is provided, comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA of the disclosure, or a pharmaceutical composition of the disclosure. In some embodiments, the disorder is a cardiovascular disorder. In some embodiments, the disorder is cardiovascular disease. BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG.1 depicts an exemplary comparison of AGT mRNA reduction in a mouse HDI in vivo model for GalNAc conjugates of a certain oligonucleotide sequence.

[0029] FIG.2. depicts an exemplary comparison of AGT mRNA reduction in a mouse HDI in vivo model for GalNAc conjugates of a certain oligonucleotide sequence.

[0030] FIG.3. depicts an exemplary comparison of ANGPTL3 reduction in wild-type mice for GalNAc conjugates dosed at 1 mg / kg as compared to a control conjugate containing L96 GalNAc.

[0031] FIG.4. depicts an exemplary comparison of ANGPTL3 reduction in wild-type mice for GalNAc conjugates dosed at 1 mg / kg as compared to a control conjugate containing L96 GalNAc.

[0032] FIG.5. depicts an exemplary comparison of ANGPTL3 reduction in wild-type mice for GalNAc conjugates dosed at 1 mg / kg as compared to a control conjugate containing L96 GalNAc.

[0033] FIG.6. depicts an exemplary comparison of ANGPTL3 reduction in wild-type mice for GalNAc conjugates dosed at 3 mg / kg as compared to a control conjugate containing L96 GalNAc.

[0034] FIG.7. depicts an exemplary comparison of ANGPTL3 reduction in wild-type mice for GalNAc conjugates dosed at 3 mg / kg as compared to a control conjugate containing L96 GalNAc.

[0035] FIG.8. depicts an exemplary comparison of ANGPTL3 reduction in wild-type mice for GalNAc conjugates dosed at 3 mg / kg as compared to a control conjugate containing L96 GalNAc.

[0036] FIG.9 depicts a protocol for the ANGPTL3 mice knockdown study of Example 8.

[0037] FIGs.10A-10B show line graphs depicting the ratio of human AGT mRNA level in transgenic mice injected with a DNA plasmid encoding the full-length human AGT transcript that were subcutaneously injected with PBS or one of 13 indicated modified AGT siRNA compounds at 2 mg / kg. AGT mRNA levels were measured by ELISA and normalized to individual animal at day -1 and PBS control at given time points throughout the experiment. FIG.10A depicts the line graph for Duplexes 100329 – 100341. FIG.10B depicts the line graph for Duplexes 100329 – 100336. FIG.10C depicts the line graph for Duplexes 100329 and 100337 – 100341. DETAILED DESCRIPTION

[0038] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, the drawings, and from the claims. Definitions

[0039] As used herein, all numerical values or numerical ranges comprise whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, comprises 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold comprises 1-, 2-, 3-, 4- , 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold, etc., as well as 1.1-, 1.2-, 1.3-, 1.4-, or 1.5-fold, etc., 2.1-, 2.2-, 2.3-, 2.4-, or 2.5-fold, etc., and so forth.

[0040] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

[0041] The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0042] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range

[0043] The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

[0044] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.

[0045] "G," "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising anucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the disclosure by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the disclosure.

[0046] “ANGPTL3” refers to the Angiopoietin-like 3 gene. According to the NCBI NLM website, this gene encodes a secreted protein that functions in angiogenesis. The encoded protein, which is expressed predominantly in the liver, is further processed into an N-terminal coiled-coil domain-containing chain and a C-terminal fibrinogen chain. The N-terminal chain is important for lipid metabolism, while the C-terminal chain may be involved in angiogenesis. Mutations in this gene cause familial hypobetalipoproteinemia type 2. Diseases associated with ANGPTL3 include Hypobetalipoproteinemia, Familial, 2 and Hypobetalipoproteinemia, Familial, 1. A human ANGPTL3 mRNA sequence is GenBank accession number NM_014495.4. A rhesus monkey (Macaca mulatta) ANGPTL3 mRNA sequence is GenBank accession number XM_015141187.2; a dog (Canis familiaris) ANGPTL3 mRNA sequence is GenBank accession number XM_038666015.1. A mouse (Mus musculus) mRNA sequence is GenBank accession number NM_013913.4.

[0047] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene (e.g., a gene described herein), including mRNA that is a product of RNA processing of a primary transcription product.

[0048] As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0049] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.

[0050] For example, a first nucleotide sequence can be described as complementary to a second nucleotide sequence when the two sequences hybridize (e.g., anneal) under stringent hybridization conditions. Hybridization conditions include temperature, ionic strength, pH, and organic solvent concentration for the annealing and / or washing steps. The term stringent hybridization conditions refers to conditions under which a first nucleotide sequence will hybridize preferentially to its target sequence, e.g., a second nucleotide sequence, and to a lesser extent to, or not at all to, other sequences. Stringent hybridization conditions are sequence dependent, and are different under different environmental parameters. Generally, stringent hybridization conditions are selected to be about 5 ^C lower than the thermal melting point (Tm) for the nucleotide sequence at a defined ionic strength and pH. The Tmis the temperature (under defined ionic strength and pH) at which 50% of the first nucleotide sequences hybridize to a perfectly matched target sequence. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes part I, chap. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).

[0051] Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0052] This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

[0053] “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogsteen base pairing.

[0054] The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

[0055] As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a gene described herein) including a 5’ UTR, an open reading frame (ORF), or a 3’ UTR. For example, a polynucleotide is complementary to at least a part of an a gene described herein mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a gene described herein.

[0056] In one embodiment, the antisense strand of the dsRNA is sufficiently complementary to a target mRNA so as to cause cleavage of the target mRNA.

[0057] The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and / or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

[0058] The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a dsRNA as described above.

[0059] As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3'-end of one strand of the dsRNA extends beyond the 5'-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

[0060] The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5’ and / or 3’ terminus.

[0061] The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0062] “Introducing into a cell,” when referring to a compound of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)), means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake ofdsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be "introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art.

[0063] The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to a gene described herein, herein refer to the at least partial suppression of the expression of a gene described herein, as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a gene described herein is transcribed and which has or have been treated such that the expression of a gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of (mRNAin control cells) - (mRNA in treated cells)^100 % (mRNAin control cells)

[0064] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to gene expression, e.g., the amount of protein encoded by a gene described herein which is secreted by a cell, the level of plasma lipid levels or the number of cells displaying a certain phenotype. In principle, gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a gene by a certain degree and therefore is encompassed by of the disclosure, the assays provided in the Examples below shall serve as such reference.

[0065] For example, in certain instances, expression of a gene described herein is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the disclosure. In some embodiments, a gene described herein is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the disclosure. In some embodiments, a gene describedherein is suppressed by at least about 85%, 90%, or 95% by administration of the double- stranded of the disclosure.

[0066] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle–aged adult or senior adult)) and / or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms “patient,” “individual,” and “subject” are used interchangeably herein.

[0067] As used herein, and unless otherwise specified, the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition. In an alternative embodiment, the present disclosure contemplates administration of the compounds described herein as a prophylactic before a subject begins to suffer from the specified disease, disorder or condition.

[0068] In general, the “effective amount” of a compound as used herein refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the present disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and condition of the subject.

[0069] As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0070] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a compound described herein and a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

[0071] The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Chemical Definitions

[0072] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0073] When a range of values is listed, it is intended to encompass each value and sub– range within the range. For example “ C1–6alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1–6, C1–5, C1–4, C1–3, C1–2, C2–6, C2–5, C2–4, C2–3, C3–6, C3–5, C3–4, C4–6, C4–5, and C5–6alkyl.

[0074] The term “alkyl” as used herein refers to a radical of a straight–chain or branched saturated hydrocarbon group. In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1–12alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1–10alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1–9alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1–8alkyl”). In someembodiments, an alkyl group has 1 to 7 carbon atoms (“C1–7alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1–6alkyl”, also referred to herein as “lower alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1–5alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1–4alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1–3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1–2alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2–6alkyl”). Examples of C1–6alkyl groups include methyl (C1), ethyl (C2), n–propyl (C3), isopropyl (C3), n–butyl (C4), tert–butyl (C4), sec–butyl (C4), iso–butyl (C4), n–pentyl (C5), 3– pentanyl (C5), amyl (C5), neopentyl (C5), 3–methyl–2–butanyl (C5), tertiary amyl (C5), and n– hexyl (C6). Additional examples of alkyl groups include n–heptyl (C7), n–octyl (C8) and the like. Common alkyl abbreviations include Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (- CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0075] The term “alkenyl” as used herein refers to a radical of a straight–chain or branched hydrocarbon group having , one or more carbon–carbon double bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2–10alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2–9alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2–8alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2–7alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2–6alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2–5alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2–4alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2–3alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2alkenyl”). The one or more carbon–carbon double bonds can be internal (such as in 2–butenyl) or terminal (such as in 1–butenyl). Examples of C2–4alkenyl groups include ethenyl (C2), 1– propenyl (C3), 2–propenyl (C3), 1–butenyl (C4), 2–butenyl (C4), butadienyl (C4), and the like. Examples of C2–6alkenyl groups include the aforementioned C2–4alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.

[0076] The term “alkynyl” as used herein refers to a radical of a straight–chain or branched hydrocarbon group having one or more carbon–carbon triple bonds (e.g., 1, 2, 3, or 4 carbon– carbon triple bonds). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2–10alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2–9alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2–8alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2–7alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2–6alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2–5alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2–4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2–3alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon– carbon triple bonds can be internal (such as in 2–butynyl) or terminal (such as in 1–butynyl). Examples of C2–4alkynyl groups include, without limitation, ethynyl (C2), 1–propynyl (C3), 2–propynyl (C3), 1–butynyl (C4), 2–butynyl (C4), and the like. Examples of C2–6alkenyl groups include the aforementioned C2–4alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like.

[0077] The term “alkylene” as used herein refers to a divalent radical of a straight–chain or branched saturated hydrocarbon group. In some embodiments, an alkylene group has 1 to 12 carbon atoms (“C1–12alkylene”). In some embodiments, an alkylene group has 1 to 10 carbon atoms (“C1–10alkylene”). In some embodiments, an alkylene group has 1 to 9 carbon atoms (“C1–9alkylene”). In some embodiments, an alkylene group has 1 to 8 carbon atoms (“C1–8alkylene”). In some embodiments, an alkylene group has 1 to 7 carbon atoms (“C1–7alkylene”). In some embodiments, an alkylene group has 1 to 6 carbon atoms (“C1–6alkylene”, also referred to herein as “lower alkylene”). In some embodiments, an alkylene group has 1 to 5 carbon atoms (“C1–5alkylene”). In some embodiments, an alkylene group has 1 to 4 carbon atoms (“C1–4alkylene”). In some embodiments, an alkylene group has 1 to 3 carbon atoms (“C1–3alkylene”). In some embodiments, an alkylene group has 1 to 2 carbon atoms (“C1–2alkylene”). In some embodiments, an alkyl group has 1 carbon atom (“C1alkylene”).

[0078] The term “cycloalkyl” as used herein refers to a radical of a saturated or partially unsaturated cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3–10 cycloalkyl”) and zero heteroatoms in the ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3–8cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3–6cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3–6cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5–10cycloalkyl”). Exemplary C3–6cycloalkyl groupsinclude, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3–8cycloalkyl groups include, without limitation, the aforementioned C3–6cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3–10cycloalkyl groups include, without limitation, the aforementioned C3–8cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro–1H–indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”). “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the cycloalkyl ring or the one or more aryl or heteroaryl groups, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system.

[0079] The term “heterocyclyl” as used herein refers to a radical of a saturated or partially unsaturated 3 to 10 membered ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3 to 10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”). Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring or the one or more aryl or heteroaryl groups, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.

[0080] In some embodiments, a heterocyclyl group is a 5 to 10 membered saturated or partially unsaturated ring system having ring carbon atoms and 1 to 4 ring heteroatoms,wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5 to 10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5 to 8 membered saturated or partially unsaturated ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 to 8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5 to 6 membered saturated or partially unsaturated ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 to 6 membered heterocyclyl”). In some embodiments, the 5 to 6 membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5 to 6 membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5 to 6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0081] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4–membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5–membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl–2,5–dione. Exemplary 5– membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5–membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6–membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6–membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6– membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7–membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8–membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0082] The term “aryl” as used herein refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6–14aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10aryl”; e.g., naphthyl such as 1–naphthyl and 2–naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14aryl”; e.g., anthracyl). Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene. Particularly aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.

[0083] The term “heteroaryl” as used herein refers to a radical of a 5 to 10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic array) having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5 to 10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2– indolyl) or the ring that does not contain a heteroatom (e.g., 5–indolyl).

[0084] In some embodiments, a heteroaryl group is a 5 to 10 membered aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system,wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 to 10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5 to 8 membered aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 to 8 membered heteroaryl”). In some embodiments, a heteroaryl group is a monocyclic 5 to 6 membered aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 to 6 membered heteroaryl”). In some embodiments, the 5 to 6 membered heteroaryl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5 to 6 membered heteroaryl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5 to 6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, a heteroaryl group is a monocyclic 5 membered aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- membered heteroaryl”). In some embodiments, a heteroaryl group is a monocyclic 6 membered aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“6-membered heteroaryl”).

[0085] Exemplary 5–membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5–membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6–membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6–membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6–membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7–membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6–bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6– bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0086] The term “alkoxy” as used herein refers to the group –OR100where R100is alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n- pentoxy, n-hexoxy, and 1,2-dimethylbutoxy. Other exemplary alkoxy groups are lower alkoxy, i.e. with between 1 and 6 carbon atoms. In other examples, alkoxy groups have between 1 and 4 carbon atoms.

[0087] The term “hydroxy” as used herein refers to the radical -OH.

[0088] The term “thiol” as used herein refers to the radical -SH.

[0089] The term “cyano” as used herein refers to the radical -CN. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2–hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2–naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3–phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p–toluenesulfonate,undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1–4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. GalNAc Conjugates

[0090] In one aspect, disclosed herein is a compound represented by Formula (I):Formula (I) or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene, wherein 5-membered heterocyclyl is optionally substituted with one or more – (CH2CH2O)q-, wherein q is an integer from 1 to 20, and alkylene is optionally substituted with one or more -OH; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

[0091] In one aspect, disclosed herein is a compound represented by Formula (I):

[0092] or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

[0093] In some embodiments, each occurrence of T1and T2is independently selected from 5- membered heterocyclyl having at least one ring oxygen and C1–6alkylene.

[0094] In some embodiments, the compound is represented by Formula (I-A):or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, the compound is represented by Formula (I-A-A):Formula (I-A-A) or a pharmaceutically acceptable salt thereof.

[0096] In some embodiments, the compound is represented by Formula (I-A-I):or a pharmaceutically acceptable salt thereof.

[0097] In some embodiments, the compound is represented by Formula (I-A-I-A): HOOHHOOHOH OH HFormula (I-A-I-A) or a pharmaceutically acceptable salt thereof.

[0098] In some embodiments, the compound is represented by Formula (I-A-II):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

[0099] In some embodiments, the compound is represented by Formula (I-A-II-A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

[0100] In some embodiments, the compound is represented by Formula (I-A-III):

[0101] or a ph, each occurrence of a and b is an integer from 1-20.

[0102] In some embodiments, the compound is represented by Formula (I-A-III-A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

[0103] In some embodiments, the compound is represented by Formula (I-A-IV): HO OH OHOHHOOHA1or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

[0104] In some embodiments, the compound is represented by Formula (I-A-IV-A):ormua ( - - V- ) or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

[0105] In some embodiments, the compound is represented by Formula (I-A-V):or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

[0106] In some embodiments, the compound is represented by Formula (I-A-V-A):or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

[0107] In some embodiments, n is 2.

[0108] In some embodiments, each occurrence of a and b is 3.

[0109] In some embodiments, each occurrence of d and e is 1.

[0110] In some embodiments, each occurrence of X is independently selected from - OH and -SH, or a pharmaceutically acceptable salt thereof.

[0111] In some embodiments, the compound is selected from a compound of Formula (I-A-VI) and a compound of Formula (I-A-VII):ndFormula (I-A-VII) or a pharmaceutically acceptable salt thereof.

[0112] In some embodiments, the compound is selected from a compound of Formula (I-A-VI-A) and a compound of Formula (I-A-VII-A): ndFormula (I-A-VI-A)or a pharmaceutically acceptable salt thereof.

[0113] In some embodiments, the compound is represented by Formula (A):Formula (A) or a pharmaceutically acceptable salt thereof.

[0114] In some embodiments, the compound is represented by Formula (A-A):ormu a - or a pharmaceutically acceptable salt thereof.

[0115] In some embodiments, the compound is represented by Formula (A-1):or a pharmaceutically acceptable salt thereof.

[0116] In some embodiments, the compound is represented by Formula (A-1-A):or a pharmaceutically acceptable salt thereof.

[0117] In some embodiments, the compound is represented by Formula (A-2):or a pharmaceutically acceptable salt thereof.

[0118] In some embodiments, the compound is represented by Formula (A-2-A):LAA1O O O P X NH H OHor a pharmaceutically acceptable salt thereof.

[0119] In some embodiments, the compound is represented by Formula (A-3):or a pharmaceutically acceptable salt thereof, wherein each of a, b, and c is an integer from 1- 20.

[0120] In some embodiments, the compound is represented by Formula (A-3-A):or a pharmaceutically acceptable salt thereof, wherein each of a, b, and c is an integer from 1- 20.

[0121] In some embodiments, the compound is represented by Formula (A-4):or a pharmaceutically acceptable salt thereof, wherein each of a, b, and c is an integer from 1- 20.

[0122] In some embodiments, the compound is represented by Formula (A-4-A):LAA1O O O P X NH H OH Hor a pharmaceutically acceptable salt thereof, wherein each of a, b, and c is an integer from 1- 20.

[0123] In some embodiments, the compound is represented by Formula (A-5):Formula (A-5) or a pharmaceutically acceptable salt thereof, wherein each of d, e, and f is an integer from 1- 20.

[0124] In some embodiments, the compound is represented by Formula (A-5-A):ormu a - - or a pharmaceutically acceptable salt thereof, wherein each of d, e, and f is an integer from 1- 20.

[0125] In some embodiments, the compound is represented by Formula (A-6):or a pharmaceutically acceptable salt thereof, wherein each of d, e, and f is an integer from 1- 20.

[0126] In some embodiments, the compound is represented by Formula (A-6-A): LAA1O O H OH HFormula (A-6-A) or a pharmaceutically acceptable salt thereof, wherein each of d, e, and f is an integer from 1- 20.

[0127] In some embodiments, the compound is represented by Formula (I-B):o u a - or a pharmaceutically acceptable salt thereof.

[0128] In some embodiments, the compound is represented by Formula (I-B-A):or a pharmaceutically acceptable salt thereof.

[0129] In some embodiments, the compound is represented by Formula (I-B-I):or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the compound is represented by Formula (I-B-I-A):o ua - -- or a pharmaceutically acceptable salt thereof.

[0131] In some embodiments, the compound is represented by Formula (I-B-II):or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

[0132] In some embodiments, the compound is represented by Formula (I-B-II-A):Formula (I-B-II-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

[0133] In some embodiments, the compound is represented by Formula (I-B-III):o u a - - or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

[0134] In some embodiments, the compound is represented by Formula (I-B-III-A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

[0135] In some embodiments, the compound is represented by Formula (I-B-IV):or a pharmaceutically acceptable salt thereof.

[0136] In some embodiments, the compound is represented by Formula (I-B-IV-A):or a pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the compound is represented by Formula (B-1):Formula (B-1) or a pharmaceutically acceptable salt thereof.

[0138] In some embodiments, the compound is represented by Formula (B-1-A):or a pharmaceutically acceptable salt thereof.

[0139] In some embodiments, the compound is represented by Formula (B-2):Formula (B-2) or a pharmaceutically acceptable salt thereof.

[0140] In some embodiments, the compound is represented by Formula (B-2-A):or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the compound is represented by Formula (B-3): HOHN OH OHFormula (B-3) or a pharmaceutically acceptable salt thereof, wherein each of g, h, and j is an integer from 1- 20.

[0142] In some embodiments, the compound is represented by Formula (B-3-A):or a pharmnteger from 1- 20.

[0143] In some embodiments, the compound is represented by Formula (B-4): HOHN OH OHFormula (B-4) or a pharmaceutically acceptable salt thereof, wherein each of g, h, and j is an integer from 1- 20.

[0144] In some embodiments, the compound is represented by Formula (B-4-A):or a phareger from 1- 20.

[0145] In some embodiments, LAis absent.

[0146] In some embodiments, LAis a cleavable linker.

[0147] In some embodiments, LAis a non- cleavable linker.

[0148] In some embodiments, A1is a double-stranded RNA (dsRNA) molecule, wherein LAis attached to only one strand of the dsRNA.

[0149] In another aspect, provided herein is a compound represented by Formula (II):HO O HO O L H N A2or a pharmaceutically acceptable salt thereof, wherein: A2is an oligonucleotide; X is selected from hydroxy and thiol; each occurrence of L and L1is a linker; and LAis absent or a linker.

[0150] In some embodiments, the compound is represented by Formula (II-A-I): .Formula (II-A-I) or a pharmaceutically acceptable salt thereof.

[0151] In some embodiments, each occurrence of L is independently selected from the group consisting of alkylene, heteroalkylene, and –(CH2)j-C(O)NH-(CH2)k-, wherein each of j and k is independently 1 to 10.

[0152] In some embodiments, the compound is represented by Formula (II-A-II):o u a - - or a pharmaceutically acceptable salt thereof, wherein each occurrence of n is an integer from 1-20.

[0153] In some embodiments, the compound is represented by Formula (II-A-III):or a pharmaceutically acceptable salt thereof, wherein each occurrence of n is an integer from 1-20.

[0154] In some embodiments, L1is alkylene.

[0155] In some embodiments, the compound is represented by Formula (II-A-IV):Formula (II-A-IV) or a pharmaceutically acceptable salt thereof.

[0156] In some aspects, provided herein is a compound represented by Formula (III):ormu a or a pharmaceutically acceptable salt thereof, wherein: each occurrence of L1and L is a linker; n is an integer from 1 to 3; A1is an oligonucleotide; X is selected from hydroxy and thiol; and LAis absent or a linker. In some embodiments, the compound is represented by Formula (III-A):Formula (III-A) or a pharmaceutically acceptable salt thereof. In some embodiments, L1is -CH2C(H)m-, wherein m is an integer from 0 to 2, provided that the sum of m and n is 3. In some embodiments, each occurrence of L is:wherein p is an i

[0157] In another aspect, provided is a compound represented by Formula (IV):Formula (IV) or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl substituted with –(CH2CH2O)q-, wherein q is an integer from 1 to 20; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

[0158] In some embodiments, each occurrence of T1and T2is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C1–6alkylene. In some embodiments, the compound is represented by Formula (IV-A-A):ormu a ( V- - ) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (IV-A):or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (IV-A-I-A): HOOHHOOHOH OH HFormula (IV-A-I-A) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (IV-A-I): HOOHOH HOOHOHA1Formula (IV-A-I) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (IV-A-II-A):Formula (IV-A-II-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A- II):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A- III-A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A- III):ormu a - - or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A- IV-A):ormua (V- -V- ) or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A- IV):Formula (IV-A-IV) or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A-V- A):Formula (IV-A-V-A) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20. In some embodiments, the compound is represented by Formula (IV-A-V):HO OHOHHO OHOHHN HN A1o u a - - or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20. In some embodiments, n is 2. In some embodiments, each occurrence of a and b is 3. In some embodiments, each occurrence of d and e is 1. In some embodiments, each occurrence of X is independently selected from - OH and -SH, or a pharmaceutically acceptable salt thereof. In some embodiments, q is from 2 to 6. In some embodiments, q is 2. In some embodiments, the compound is A1-LA-D, wherein D is selected from:nd,or a pharmaceutically acceptable salt thereof.

[0159] In some embodiments, LAis absent.

[0160] In some embodiments, LAis a cleavable linker.

[0161] In some embodiments, LAis a non- cleavable linker.

[0162] In some embodiments, A2is a double-stranded RNA (dsRNA) molecule, wherein LAis attached to only one strand of the dsRNA.

[0163] In some embodiments, LAis attached to A2at the 5’ end, the 3’ end, or both ends of A2.

[0164] In some embodiments, LAis attached to A2at the 5’ end, the 3’ end, or both ends of A2.

[0165] Exemplary compounds of the disclosure also include, for example, those compounds disclosed in Tables 1-4 and 7, such as BCR-0000739, BCR-0000740, BCR- 0000741, BCR-0000742, BCR-0000743, BCR-0000744, BCR-0000745, BCR-0000746, BCR-0000747, BCR-0000748, BCR-0000749, BCR-0000750, BCR-0000751, BCR- 0000752, BCR-0000753, BCR-0000754, BCR-0001075, BCR-0001076, BCR-0001077, and BCR-0001078, or a pharmaceutically acceptable salt thereof. In some embodiments the compound is disclosed in Tables 1-4, such as BCR-0000739, BCR-0000740, BCR-0000741, BCR-0000742, BCR-0000743, BCR-0000744, BCR-0000745, BCR-0000746, BCR- 0000747, BCR-0000748, BCR-0000749, BCR-0000750, BCR-0000751, BCR-0000752, BCR-0000753, and BCR-0000754, or a pharmaceutically acceptable salt thereof.

[0166] The compounds provided herein can be administered as the sole active agent, or they can be administered in combination with other active agents. In some embodiments, the present invention provides a combination of a compound of the present invention and another pharmacologically active agent. Administration in combination can proceed by any technique apparent to those of skill in the art including, for example, separate, sequential, concurrent, and alternating administration.

[0167] The present disclosure also embraces isotopically labeled compounds which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36Cl, respectively. For example, a compound of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) may have one or more H atom replaced with deuterium. Double-stranded ribonucleic acids (dsRNA)

[0168] The present disclosure provides, in certain embodiments, GalNAc compounds comprising a double stranded ribonucleic acid (dsRNA) molecule. In one aspect of the disclosure, provided herein are compounds comprising double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a gene e.g., in a cell within a subject,such as a mammal (for example a human). In some embodiments, the gene is a gene of the disclosure, e.g., AGT or ANGPTL3. The use of these dsRNA oligonucleotides enables the targeted degradation of mRNAs of the corresponding gene (e.g., a gene described herein, such as AGT or ANGPTL3) in mammals.

[0169] In certain embodiments, the dsRNA comprises an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA or an mRNA fragment formed in the expression of a gene, e.g., a gene of the disclosure. In some embodiments, the dsRNA comprises at least 70% complementarity to the mRNA or the fragment mRNA of human mRNA.

[0170] In certain embodiments, the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in Table 1, 2, 3, or 4.

[0171] In some embodiments, the sense strand is 70 - 80% or more identical to the sense strands listed in Table 1, 2, 3, or 4. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence shown in Table 1, 2, 3, or 4. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a sense strand sequence shown in Table 1, 2, or 3. In some embodiments, the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in Table 1, 2, or 3. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in Table 1, 2, 3, or 4.

[0001] In some embodiments, the antisense strand is 70 - 80% or more identical to the antisense strands listed in Table 1, 2, 3, or 4. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of an antisense sense strand sequence shown in Table 1, 2, 3, or 4. In some embodiments, the antisense strand comprises 21 contiguous nucleotides of an antisense sense strand sequence shown in Table 1, 2, 3, or 4. In some embodiments, the antisense strand is selected from an antisense strand sequence shown in Table 1, 2, 3, or 4.

[0002] In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in Table 1, 2, 3, or 4 and the antisense strand is selected from an antisense strand sequence shown in Table 1, 2, 3, or 4.

[0003] In some embodiments, the dsRNA has a mismatch to a fragment of an mRNA (e.g., an mRNA for a gene such as AGT or ANGPTL3). In some embodiments, the dsRNA comprises one or two mismatches to the mRNA or fragment of a human mRNA (e.g., a human mRNA for a gene described herein). In some embodiments, the dsRNA is more than 70% identical to the mRNA or fragment of a human mRNA (e.g., a human mRNA for a gene described herein). In some embodiments, the dsRNA is more than 70%, 75%, 80%, 85%, 90%, or 95 % identical to the mRNA or fragment of a human mRNA (e.g., a human mRNA for a gene described herein). In some embodiments, the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of an mRNA of the disclosure. In some embodiments, the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the mRNA of the disclosure. In some embodiments, the mismatch is in the sense strand. In some embodiments, the mismatch is in the antisense strand. In some embodiments, the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the mRNA of the disclosure. In some embodiments, the mismatch is located in the middle of the dsRNA. In some embodiments, the mismatch is in the 5’ or 3’ region of the dsRNA. In some embodiments, the mismatch is no more than 5 nucleotides from the 5’ or 3’ end of the dsRNA.

[0004] In some embodiments, at least one strand of the dsRNA comprises a 3’ or 5’ overhang of at least 1 nucleotide. In some embodiments, the overhang is at least 2 or a at least 3 nucleotides. In some embodiments, in the dsRNA at least one strand comprises a 3’ overhang. In some embodiments, in the dsRNA at least one strand comprises a 5’ overhang.

[0005] In some embodiments, the antisense strand has a 3’ end nucleotide overhang compared to the sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.

[0006] The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The dsRNA includes twoRNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is complementary to a target sequence, derived from the sequence of an mRNA formed during the expression of a gene of the disclosure, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.

[0007] In some embodiments, the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 20 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different single stranded RNAs (ssRNA) are used in combination, the duplex lengths can be identical or can differ.

[0008] In some embodiments, each strand of the dsRNA of the disclosure is between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is about 25-30 nucleotides in length. In some embodiments, each strand of the duplex is the same length or of different lengths. When two different ssRNAs are used in combination, the lengths of each strand of each ssRNA can be identical or can differ.

[0009] In some embodiments, the dsRNA includes dsRNA that is longer than 21-23 nucleotides, e.g., dsRNA that is long enough to be processed by the RNase III enzyme Dicer into 21-23 base pair siRNA which is then incorporated into a RNA-induced silencing complex (RISC). Accordingly, a dsRNA of the disclosure is at least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100 base pairs in length.

[0010] Inhibition of the expression of a gene of the disclosure can be assayed by, for example, a nucleic acid based assay, such as by quantitative PCR, or by a protein-based method, such as by Western blot. Expression of a gene of the disclosure can be reduced by at least 50% when measured by an assay as described in the Examples below. For example, expression of a gene of the disclosure in cell culture, such as in Huh-7 cells, can be assayed by measuring mRNA levels for a gene of the disclosure, such as by quantitative PCR assay, or by measuring protein levels, such as by ELISA assay.

[0011] In another aspect, the disclosure provides a single-stranded antisense oligonucleotide RNAi. An antisense oligonucleotide is a single-stranded oligonucleotide thatis complementary to a sequence within the target mRNA. Antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther.1:347- 355. Antisense oligonucleotides can also inhibit target protein expression by binding to the mRNA target and promoting mRNA target destruction via RNase-H. The single-stranded antisense RNA molecule can be about 13 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule can comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Table 1, 2, 3, or 4. Modifications

[0012] In certain embodiments, the dsRNA is chemically modified to enhance stability of the dsRNA. The nucleic acids featured in the disclosure may be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this disclosure include dsRNAs containing modified backbones or non- natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0013] In some embodiments, a modified dsRNA backbone includes at least one of: a 2'- O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'- fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide), and a thymidine-glycol nucleic acid (GNA) S-Isomer.

[0014] In some embodiments, the modification includes one or more phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0015] In some embodiments, the modified nucleotide includes at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide. In some embodiments, the modified nucleotide antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O- phosphonate.

[0016] In some embodiments, the modified nucleotide comprises at least one of: a 2'- deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

[0017] In some embodiments, the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage. In some embodiments, the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond. Conjugates

[0018] Another modification of the dsRNAs of the disclosure involves chemically linking to the dsRNA one or more ligand or targeting moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA (e.g., a GalNAc of the present disclosure, e e.g., GalNAc3, tri-GalNAc4, tri-GalNAc5, and tri-GalNAc6). Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533- 538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl- ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969- 973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0019] In some embodiments, the ligand or targeting moiety (e.g., a GalNAc described herein, e.g., GalNAc3, tri-GalNAc4, tri-GalNAc5, and tri-GalNAc6) is conjugated to the 5’ end, 3’ end or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety (e.g., a GalNAc described herein, e.g., GalNAc3, tri-GalNAc4, tri-GalNAc5, and tri- GalNAc6) is conjugated to the 3’ end of the sense strand of the dsRNA. In some embodiments, the ligand or targeting moiety (e.g., a GalNAc described herein, e.g., GalNAc3, tri-GalNAc4, tri-GalNAc5, and tri-GalNAc6) is conjugated to the 3’ end of the antisense strand of the modified dsRNA.

[0020] In some embodiments, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the oligonucleotide sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. The dsRNA conjugate can be purified for example by HPLC methods.

[0172] Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate- receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Linkers

[0173] The term "linker" or “linking group” as used herein means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2- 24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0174] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In an exemplary embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0175] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze ordegrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0176] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a selected pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

[0177] A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[0178] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

[0179] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In certain embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0180] Redox cleavable linking groups: In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulfide linking group (-S-S-). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0181] Phosphate-based cleavable linking groups: In other embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O- P(S)(ORk)- O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O- P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S- P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)( Rk)-S-, wherein Rk at each occurrence can be, independently, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include -O- P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S- , -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O- P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, - S-P(O)(H)-S-, and -O-P(S)(H)-S-. In certain embodiments a phosphate-based linking group is -O- P(O)(OH)-O-. These candidates can be evaluated using methods analogous to those described above.

[0182] Acid cleavable linking groups: In other embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In certain embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). An exemplary embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

[0183] Ester-based linking groups: In other embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O-, or - OC(O)-. These candidates can be evaluated using methods analogous to those described above.

[0184] Peptide-based cleaving groups: In yet other embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RAand RBare the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.Pharmaceutical Compositions Excipients

[0185] Described herein in certain embodiments is a pharmaceutical composition comprising a compound of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) and a pharmaceutically acceptable excipient. In contrast to a carrier compound, a “pharmaceutical carrier” or “pharmaceutically acceptable excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0186] Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0187] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0188] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0189] The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art- established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0190] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. Administration

[0191] Also disclosed herein are methods of administration for the pharmaceutical compositions and formulations which include the compounds and pharmaceutical compositions of the disclosure. In some embodiments, the compound or pharmaceutical composition of the disclosure is administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

[0192] In certain embodiments, administration of the pharmaceutical composition is topical (including buccal and sublingual), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. In some embodiments, parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.

[0193] Pharmaceutical compositions containing a compound of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)), can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0194] Pharmaceutical formulations, for example, are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0195] In certain embodiments, the compound of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) is delivered in a manner to target a particular tissue, for example the liver.

[0196] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound (e.g., a compound of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)), e.g., a compound of Formula (I), Formula (II), or Formula (III)) which produces a therapeutic effect.

[0197] In certain embodiments, a formulation of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present disclosure. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)).

[0198] Formulations of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose andacacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) as an active ingredient. A compound of the present disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) may also be administered as a bolus, electuary or paste.

[0199] Liquid dosage forms for oral administration of the compounds of the disclosure (e.g., a compound of Formula (I), Formula (II), or Formula (III)) include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liposomal Formulations

[0200] In certain embodiments, pharmaceutical compositions disclosed herein comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

[0201] In some embodiments, the GalNAc compounds of the disclosure (e.g., a compound of Formula (I) of Formula (II)) is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain embodiments, dsRNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue. Methods of Use

[0202] In an embodiment, provided herein are methods of inhibiting the expression of a gene (e.g., a gene described herein) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., acompound of Formula (I), Formula (II), or Formula (III)) or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof.

[0203] The instantly disclosed compounds (e.g., a compound of Formula (I), Formula (II), or Formula (III)) are useful in the treatment of various disorders. In an embodiment, provided herein is a method of treating a disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I), Formula (II), or Formula (III)) or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof. In some embodiments, the disorder is a liver disorder. In some embodiments, the disorder is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), obesity, hypertension, and hypercholesteremia. In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). In another embodiment, provided herein is a method of treating a disorder mediated by the expression of ANGPTL3 or AGT in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I), Formula (II), or Formula (III)) or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof. In some embodiments, the method further comprises administering to the patient one or more additional therapeutic agents. EXAMPLES

[0204] The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization. Abbreviations ACN acetonitrile AGT angiotensinogen ANGPTL3 Angiopoietin-like 3 genearaF arabino fluoro CPG controlled pore glass DCE 1,2-Dichloroethane DCI 4,5-Dicyanoimidazole DCM dichloromethane DIPEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMSO dimethyl sulfoxide DMTr 4,4'-Dimethoxytrityl DMTrCl / DMT-Cl 4,4'-Dimethoxytrityl chloride DNA deoxyribonucleic acid dsRNA double-stranded RNA DTT dithiothreitol EA ethyl acetate EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide ELISA enzyme-linked immunosorbent assay ESI electrospray ionization GalNAc N-acetylgalactosamine GNA thymidine-glycol nucleic acid h hour(s) HBTU hexafluorophosphate benzotriazole tetramethyl uronium HDI hydrodynamic injection HOBT hydroxybenzotriazole HPLC high-performance liquid chromatography iRNA informational RNA KD knockdown LCMS liquid chromatography mass spectrometry LNA locked nucleic acid min minute(s) MOE methoxyethyl mRNA messenger RNA MTBE methyl tert-butyl ether NAFLD non-alcoholic fatty liver diseaseNASH non-alcoholic steatohepatitis NeoR neorhodopsin NMR nuclear magnetic resonance ORF open reading frame PBS phosphate-buffered saline PCR polymerase chain reaction PE petroleum ether PS phosphorothioate Py pyridine RISC RNA-induced silencing complex RNA ribonucleic acid r.t. room temperature siRNA small interfering RNA ssRNA single stranded RNA TBSCl tert-Butyldimethylsilyl chloride TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin-layer chromatography TMS trifluoromethyltrimethylsilane TMSOTf trimethylsilyl trifluoromethanesulfonate UTR untranslated region UV ultra-violetEXAMPLE 1. Preparation of GalNAc-3-CPG HO 1d

[0205] Step 1: To a solution of 1c (30.0 g, 130.0 mmol) in DCM (300.0 mL) was added at r.t. in N2 atmosphere. Next the 1d (18.0 g, 170.0 mmol) was added at r.t. in the mixture. Then EDCI (27.0 g, 140.0 mmol) and DMAP (1.6 g, 13.0 mmol) were added at 0 °C in the mixture. The reaction mixture was stirred at r.t. for 1 h. The LCMS showed 1c was consumed. Quenched by water (500.0 mL). The product was extracted with DCM (300.0 mL *3). The organic layer was washed with brine and dry over Na2SO4. After filtration, the organic solvent was removed in vacuo and the crude residue was purified by flash column chromatography (SiO2, PE / EA = 6 / 1) to afford the compound 2c (37.0 g, 115.1 mmol, 95.0% purity) as a pale-yellow oil with 88.0% yield. ESI-LCMS: m / z 322.1 [M+H]+.1H-NMR (400 MHz, CDCl3) δ = 7.38 – 7.26 (m, 5H), 5.11 (s, 2H), 4.51 (s, 1H), 3.08 (s, 2H), 2.37 – 2.33 (m, 2H), 1.69 – 1.62 (m, 2H), 1.51 – 1.43 (m, 11H), 1.37 – 1.29 (m, 2H).

[0206] Step 2: To a solution of 2c (13.0 g, 40.5 mmol) in DCM (130.0 mL) was added TFA (4.6 g, 40.5 mmol, 26.0 mL). The reaction was stirred at 25 °C for 1 h. LCMS showed the reaction completion. The reaction mixture was concentrated under reducedpressure to afford crude product benzyl 6-aminohexanoate (10.0 g, crude). The crude product was used to next step without purification. ESI-LCMS: m / z 222.1 [M+H]+.

[0207] Step 3: To the solution of 1 (100.0 g, 262.9 mmol) in THF (1 L) was added DIPEA (44.2 g 347.7 mmol) at -15oC under N2. The mixture was stirred at -4oC for 30 min. Then added TMSCHN2(250.0 mL) and the reaction was stirred at 20oC for 16 h. LCMS showed the 1 was consumed. The mixture was quenched by saturated NaHCO3, extracted with DCM, washed by brine and dried by Na2SO4. The organic phase was concentrated in vacuum and purified by column chromatography (silica gel, PE: EA= 1:1) to get product 2 (28.0 g, 69.2 mmol). ESI-LCMS: 377.1 [M+H]+.

[0208] Step 4: To the solution of 2 (28.0 g, 69.2 mmol) in THF (432.0 mL) and H2O (72.0 mL) was added silver; benzoate (3.5 g, 15.2 mmol) and TEA (17.5 g, 173.1 mmol, 24.1 mL) under N2. The reaction was stirred at 25 °C for 16 h and protect from light. LCMS showed the reaction completion. The mixture was quenched with NaHCO3, extract with EA. Aqueous phase was acidified with AcOH to pH=6, and extract with EA again. The organic layer was dried over by Na2SO4, concentrated under reduced pressure and crude product was purified by Flash-Prep-HPLC [with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5% NH4HCO3) = 1 / 0 within 30 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 10 / 1; Detector, UV 254 nm.] to get 3 (13.0 g, 33.0 mmol, 47.6% yield). ESI-LCMS: m / z 395.2 [M+H]+.1H-NMR (400 MHz, CD3OD): δ = 7.40~7.25 (m, 5H), 5.06 (s, 2H), 3.98~3.93 (m, 1H), 3.02~2.94 (m, 2H), 2.51~2.41 (m, 2H), 1.57~1.25 (m, 17H).

[0209] Step 5: To the solution of 3 (13.0 g, 33.0 mmol) in THF (130.0 mL) was added Pd / C (2.6 g, 6.6 mmol). The reaction was stirred at 25°C for 2 h under H2. LCMS showed the reaction completion. The reaction mixture was filtered and the filtered cake was washed with CH3OH and the filtrate was concentrated under reduced pressure to afford crude product. The crude product was triturated with ACN and filtered to get product 4 (7.8 g, 30.0 mmol, 90.9% yield) as white solid. ESI-LCMS: m / z 261.2 [M+H]+.1H-NMR (400 MHz, CD3OD): δ = 3.36~3.34 (m, 2H), 3.06~3.03 (m, 2H), 2.52~2.25 (m, 2H), 1.65~1.60 (m, 2H), 1.52~1.43 (m, 13H).

[0210] Step 6: To the solution of 4 (7.8 g, 30.0 mmol) in THF (60.0 mL) and H2O (20.0 mL) was added Boc2O (13.1 g, 59.9 mmol) and NaOH (1.8 g, 44.9 mmol). The reaction was stirred at 25°C for 2h. LCMS showed the reaction completion. The mixture was extracted with EA and washed with aqueous NH4Cl and brine, dried over by Na2SO4. Concentrated under reduced pressure and the mixture was purified by column chromatography on silica gel (SiO2, CH3OH: DCM =0~10%) to get 5 (9.4 g, 26.1 mmol, 87.0% yield) as white solid. ESI -LCMS: m / z 361.3 [M+H]+.1H-NMR (400 MHz, CD3OD): δ = 3.89~3.78 (m, 1H), 3.31~3.30 (m, 1H), 3.03~3.90 (m, 2H), 2.44~2.38 (m, 2H), 1.58~1.26 (m, 25H).

[0211] Step 7: To the solution of 6 (12.0 g, 46.1 mmol) in DCM (100.0 mL) was added benzyl 6-aminohexanoate (12.2 g, 55.3 mmol), Then added EDCI (13.0 g, 92.2 mmol), HOBT (12.5 g, 92.2 mmol) and DIPEA (11.9 g, 92.2 mmol, 16.1 mL). The reaction was stirred at 25 °C for 2 h. LCMS showed the reaction completion. The mixture was extracted with DCM and washed with H2O and brine, dried over by Na2SO4. The organic layer was concentrated under reduced pressure and crude product was triturated with ACN and filtered to get 7 (15.4 g, 26.6 mmol, 57.6% yield) as white solid. ESI-LCMS: m / z 686.3 [M+H]+.1H- NMR (400 MHz, DMSO-d6): δ = 7.90~7.86 (m, 2H), 7.77~7.67 (m, 3H), 7.43~7.30 (m, 9H), 7.10~7.78 (m, 1H), 6.80~6.70 (m, 1H), 5.10~5.05 (s, 2H), 4.27~4.19 (m, 3H), 3.77~3.72 (m, 1H), 3.01~2.85 (m, 4H), 2.33~2.12 (m, 4H), 1.56~1.45 (m, 2H), 1.41~1.15 (m, 20H).

[0212] Step 8: To the solution of 7 (15.0 g, 21.4 mmol) in DMF (150.0 mL) was added piperidine (1.8 g, 21.4 mmol). The reaction was stirred at 25°C for 2h. LCMS showed the reaction completion. The mixture was extracted with EA and washed with H2O and brine, dried over by Na2SO4. The organic layer was concentrated under reduced pressure and crude product was triturated with ACN and filtered to get 8 (10.0 g, 20.9 mmol, 97.6% yield) as white solid. ESI-LCMS: m / z 464.3 [M+H]+.1H-NMR (400 MHz, DMSO-d6): δ = 7.93~7.90 (m, 1H), 7.40~7.31 (m, 5H), 6.77~6.74 (m, 1H), 5.08 (s, 2H), 3.03~2.86 (m, 5H), 2.36~2.30 (m, 2H), 2.16~1.96 (m, 2H), 1.58~1.50 (m, 2H), 1.41~1.21 (m, 19H).

[0213] Step 9: To the solution of 8 (10.0 g, 20.9 mmol) in DCM (200.0 mL) was added 5 (12.2 g, 25.0 mmol), EDCI (7.9 g, 41.7 mmol), HOBT (5.6 g, 41.7 mmol) and DIPEA (5.4 g, 41.7 mmol, 7.3 mL). The reaction was stirred at 25°C for 2h under N2. LCMS showed the reaction completion. The mixture was extracted with DCM and washed with H2O and brine, dried over by Na2SO4. The organic layer was concentrated under reducedpressure and crude product was triturated with ACN to get 9 (15.0 g, 15.8 mmol, 75.6% yield) as white solid. ESI-LCMS: m / z 804.6 [M-H] -.1H-NMR (400 MHz, DMSO-d6): δ = 7.76~7.73 (m, 1H), 7.61~7.59 (m, 1H), 7.39~7.32 (m, 5H), 6.75~6.69 (m, 2H), 6.57~6.55 (d, 1H, J= 4 Hz), 5.08 (s, 2H), 4.09~3.95 (m, 1H), 3.72~3.68 (m, 1H), 3.19~3.16 (m, 1H), 3.01~2.96 (m, 2H), 2.88~2.84 (m, 4H), 2.37~2.29 (m, 2H), 2.20~2.07 (m, 4H), 1.57~1.49 (m, 3H)，1.41~1.25 (m, 45H).

[0214] Step 10: To the solution of 9 (10.0 g, 10.5 mmol) in DCM (100.0 mL) was added TFA (50.0 mL). The reaction was stirred at 25 °C for 0.5 h. LCMS showed the reaction completion. The reaction mixture was concentrated under reduced pressure to afford crude product 10 (16.2 g, crude). The crude product was used at next step without purification. ESI- LCMS: m / z 506.2 [M+H]+.

[0215] Step 11: To the solution of 10 (6.9 g, 10.6 mmol) in DCM (200.0 mL) was added 6a (18.8 g, 36.9 mmol), EDCI (8.1 g, 42.2 mmol), HOBT (5.7 g, 42.2 mmol), DIPEA (10.9 g, 84.4 mmol, 14.7 mL). The reaction was stirred at 25°C for 2h. LCMS showed the reaction completion. The mixture was extracted with DCM and washed with H2O and brine, dried over by Na2SO4. The organic layer was concentrated under reduced pressure and crude product was purified by column chromatography on silica gel (SiO2, CH3OH: DCM =0~10%) to get 11 (12.0 g, 5.7 mmol, 53.6% yield) as a white solid. ESI-LCMS: m / z 987.9 [M+H]+.1H-NMR (400 MHz, DMSO-d6): δ = 7.82~7.75 (m, 6H), 7.64~7.60 (m, 2H), 7.39~7.32 (m, 5H), 5.22~5.21 (d, 3H, J=4.00 Hz), 5.08 (s, 2H), 5.00~4.96 (m, 3H), 4.56~4.54 (d, 3H, J=8.00 Hz), 4.05~4.01 (m, 11H), 3.92~3.84 (m, 3H), 3.79~3.75 (m, 3H), 3.61~3.45 (m, 27H), 2.99~2.98 (m, 6H), 2.35~2.27 (m, 8H), 2.19~2.10 (m, 12H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77(s, 9H), 1.55~1.51 (m, 2H), 1.39~1.19 (m, 17H).

[0216] Step 12: To the solution of 11 (11.6 g, 5.5 mmol) in THF: CH3OH=2:1 (110.0 mL) was added Pd / C (132.8 mg, 1.1 mmol) under H2. The reaction was stirred at 25 °C for 2 h. LCMS showed the reaction completion. The reaction mixture was filtered through celite pad and concentrated under reduced pressure to afford crude product 12 (11.0 g, crude). The crude product was used at next step without purification. ESI-LCMS: m / z 1884.1 [M+H]+.

[0217] Step 13: To the solution of 12 (11.0 g, 5.4 mmol) in DCM (200.0 mL) was added EDCI (2.1 g, 10.8 mmol), HOBT (1.5 g, 10.8 mmol), 4b (3.2 g, 8.1 mmol) and DIPEA (2.8 g, 21.7 mmol). The reaction was stirred at 25°C for 2h. LCMS showed the reactioncompletion. The mixture was extracted with DCM and washed with H2O and brine, dried over by Na2SO4. The organic layer was concentrated under reduced pressure and crude product was purified by column chromatography on silica gel (SiO2, CH3OH: DCM =0~10%) and Flash-Prep-HPLC [with the following conditions (IntelFlash-1): Column, C18silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5% NH4HCO3) = 1 / 0 within 30 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 10 / 1; Detector, UV 254 nm.] to get 13 (10 g, 4.2 mmol, 76.7% yield). ESI-LCMS: m / z 979.2 [M+H]+.1H-NMR (400 MHz, DMSO-d6): 7.81~7.79 (m, 6H), 7.62~7.60 (m, 4H), 7.38~7.36 (m, 3H), 7.30~7.20 (m, 9H), 6.88~6.85 (m, 5H), 5.22~5.21 (m, 3H), 4.99~4.59 (m, 3H), 4.56~4.54 (m, 3H), 4.05~3.98 (m, 12H), 3.91~3.86 (m, 3H), 3.79~3.73 (m, 11H), 3.59~3.43 (m, 29H), 3.01~2.94 (m, 9H), 2.33~2.27 (m, 7H), 2.19~2.10 (m, 16H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77(s, 9H), 1.51~1.15 (m, 21H).

[0218] Step 14: To the solution of 13 (10.0 g, 4.2 mmol) in DCM (100.0 mL) was added DMAP (507.0 mg, 4.2 mmol), TEA (1.3 g, 12.5 mmol, 1.7 mL) and succinic anhydride (1.0 g, 10.4 mmol). The reaction was stirred at 25 °C for 2 h under N2. LCMS showed the reaction completion. The mixture was extracted with DCM and washed with H2O and brine, dried over by Na2SO4. The organic layer was concentrated under reduced pressure to get product cmp2 (5.0 g, 4.0 mmol, 96.6% yield). ESI-LCMS: m / z 1029.4 [M+H]+.1H-NMR (400 MHz, DMSO-d6): δ = 8.23~7.52 (m, 9H), 7.41~7.05 (m, 9H), 6.90~6.81 (m, 2H), 5.21~5.20 (m, 3H), 4.99~4.59 (m, 3H), 4.57~4.54 (m, 3H), 4.18~4.16 (m, 2H), 4.08~3.96 (m, 12H), 3.92~3.88 (m, 3H), 3.81~7.32 (m, 9H), 3.62~3.44 (m, 29H), 3.02~2.90 (m, 10H), 2.67~2.64 (m, 2H), 2.41~2.39 (m, 5H), 2.33~2.27 (m, 9H), 2.19~2.04 (m, 12H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.47~1.15 (m, 21H).

[0219] Step 15: To a solution of compound cmp2 (5.0 g, 2.0 mmol) in THF (240.0 mL) was added HBTU (3.8 g, 10.0 mmol,), DMAP (244.8 mg, 2.0 mmol) and 115DEA (2.1 g, 16.0 mmol). Then added CPG Resin (30.0 g). The mixture was stirred by mechanical agitation at 40oC for 16 h. The reaction solution was filtered and the filter cake rinsed successively with DCM (240.0 mL x 4) to give a white solid. The suspension of resin was added to the mixture of pyridine, Ac2O (5 / 1, 180.0 mL). The suspension was agitated by mechanism stir at 40oC for 4 h. The reaction solution was filtered and the filter cake rinsed successively with DCM (240.0 mL x 4) and dried at 30oC under reduced pressure to get the solid to a fine powder. Corresponding GalNAc-3-CPG was obtained, and loading value ofthe corresponding GalNAc-3-CPG calculated by UV-Vis (Total volume of DCA added in mL = 40 mL; Abs value at 500 nm = 0.567; Mg of CPG taken = 5.74 mg; loading in μmol / g = ((40)*(0.567)*1000) / 76*(5.74) = 53 μmol / g).EXAMPLE 2. Preparation of GalNAc-4-amidite and GalNAc-4-CPG

[0220] Step 1: To a solution of 1a (255.0 g, 2.4 mol) in DMF (2.6 L), was added imidazole (196.3 g, 2.9 mol) and TBSCl (245.4 g, 3.6 mol) at 0oC under N2 atmosphere, and the mixture was stirred at 25oC for 3 h. LCMS showed starting material was consumed. The reaction mixture was quenched by water (100.0 mL) and added EA (10.0 L), the mixture was washed by water (1.0 L *3), the organic was concentrated and then purified by column chromatography (SiO2, EA / PE = 50:1 to 10:1) to give 2 (340.0 g, 90.0% purity, 67% yield) as yellow solid l. ESI-LCMS: m / z 212.2 [M+H]+.

[0221] Step 2: To a solution of 2a (340.0 g, 1.5 mol) in I (3.5 L), was added Cs2CO3 (551.5 g, 1.7 mol) and 2b (198.7 g, 2.3 mol) at 0oC under N2atmosphere, the mixture was stirred at 25oC for 1 h. LCMS showed starting material was consumed. The solution was filtrated and organic concentrated to give crude 3a (350.5 g) as yellow oil. ESI-LCMS: m / z 324.2 [M+NH3]+.

[0222] Step 3: To a solution of 3a (350.5 g, 1.5 mol) in MeOH (3.5 L) was added LiOH (94.5 g, 2.3 mol) at 25oC under N2 atmosphere, the mixture was stirred at 25oC for 2h. LCMS showed starting material was consumed. The solution was concentrated and extracted by EA (1.0 L *3) and then adjust PH=5 extracted by EA (1.0 L *3) again and the organic was concentrated to give crude 4a (174.0 g) as yellow oil. ESI-LCMS: m / z 293.1 [M+H]+.

[0223] Step 4: Compound 4a (174.0 g) was added in DCM (200.0 mL), followed by EDCI (125.9 g, 0.6 mol) and DMAP (7.3 g, 59.6 mmol), BnOH (106.7 g, 1.0 mol) was added in the mixture at r.t. Then the reaction mixture was stirred at r.t. for 3 h. The LCMS showed compound 4a was consumed. The reaction mixture extracted with DCM (200.0 mL *4). The organic layer was washed with brine and dried over Na2SO4and concentrated to give crude 5a (270.0 g) as yellow oil. ESI-LCMS: m / z 400.1 [M+NH3]+.

[0224] Step 5: To a solution of 5a (270.0 g) in THF (2.7 L) was added 3HF.TFA (227.6 g, 1.4 mol) at 25oC under N2 atmosphere, the mixture was stirred at 50oC for 2 h. LCMS showed 5a was consumed. The solution was concentrated and extracted by EA (2.0 L *3) and the organic was washed by brine, dried and concentrated and then purified by column chromatography (SiO2, EA / PE = 20:1 to 5:1) to give 6a (106.0 g, 96.0% purity, 55.9% yield) as yellow oil. ESI-LCMS: m / z 286.1 [M+NH3]+.1H-NMR (600 MHz, DMSO-d6) δ = 7.39- 7.31 (m, 5H), 5.11 (s, 2H), 4.57-4.55 (t, J = 5.5 Hz 1H), 3.67-3.65 (t, J = 6.2 Hz 1H), 3.50- 3.46 (m, 6H), 3.41-3.39 (t, J = 5.3 Hz 1H), 2.61-2.59 (t, J = 6.2 Hz 1H).

[0225] Step 6: To a solution of 6a (106.0 g, 0.4 mol) and 9a (100.0 g, 0.3 mol) in DCE (1.0 L) was added TMSOTf (6.8 g, 30.4 mmol) at 0oC under N2atmosphere, the mixture was stirred at 25oC for night. LCMS showed starting material was consumed. Mixture was quenched by water (100.0 mL) and extracted by EA (200.0 mL *4), the organic was washed by brine, dried and concentrated. The residue was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5% NH4HCO3) = 1 / 0 within 30 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 10 / 1; Detector, UV 254 nm. This resulted in to give 7a (140.0 g, 95% purity, 77.1% yield) as yellow oil. ESI-LCMS: m / z 598.5 [M+H]+.1H-NMR (400 MHz, CDCl3) δ = 7.37-7.32 (m, 5H), 6.39-6.37 (d, J = 9.3 Hz 1H), 5.32-5.30 (m, 2H), 5.14 (s, 2H), 5.08-5.05 (m, 1H), 4.76- 4.74 (d, J = 8.6 Hz 1H),4.27-4.09 (m, 3H), 3.91-3.79 (m, 5H), 3.69-3.57 (m, 6H), 2.65-2.67 (d, J = 6.2 Hz 1H), 2.15 (s, 3H), 2.04 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H),

[0226] Step 7: To a solution of 7a (140.0 g, 234.5 mmol) was dissolved in THF (150.0 mL). To this mixture was added Pd / C (14.0 g). The mixture was warmed at 50 °C for 2 hours at H2 atmosphere. The LCMS showed 7a was consumed. The solution was filtrated and organic concentrated purified by column chromatography (SiO2, DCM / MeOH = 100:1 to 10:1) to give 8a (80.0 g) as yellow oil. ESI-LCMS: m / z 508.0 [M+H]+.1H-NMR (400 MHz, CDCl3) δ = 6.49-6.47 (d, J = 9.3 Hz 1H), 5.33-5.32 (d, J = 2.8 Hz 1H), 5.12-5.08 (m, 1H), 4.76-4.74 I, 4.29-4.11 (m, 3H), 3.97-3.54 (m, 15H), 2.16 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H), 1.88-1.84 (m, 2H).

[0227] Step 8: To a solution of 1 (100.0 g, 260.0 mmol) in DCM (800.0 mL) was added TMSCN (38.3 g, 390.0 mmol) at 25oC under N2 atmosphere, the mixture was stirred at -78oC for 30 minutes and added BF3.Et2O (10.9 g, 77.2 mmol). LCMS showed 1 was consumed. Mixture was quenched by NaHCO3 to pH=7 and extracted by DCM (200.0 mL *4), the organic was washed by brine, dried and concentrated. The residue was purified by column chromatography (SiO2, EA / PE = 8:1 to 1:1) to give 2 (32.7 g, 96.0% purity, 40.0% yield) as yellow oil. ESI-LCMS: m / z 379.2 [M+NH3]+.1H-NMR (400 MHz, CDCl3) δ = 7.98-7.96 (m, 2H), 7.90-7.88 (m, 2H), 7.26-7.24 (m, 4H), 5.60-5.59 (m, 1H), 4.93-4.90 (m, 1H), 4.62-4.49 (m, 3H), 2.78-2.61 (m, 2H), 2.42-2.41 (d, J = 3.7 Hz 6H).

[0228] Step 9: The material 2 (32.7 g, 86.3 mmol) was dissolved in THF (327.0 mL). To this mixture was added ReNi (6.5 g). The mixture was warmed at 50 °C for 2 hours at H2atmosphere. The LCMS showed 2 was consumed. The solution was filtrated and organic concentrated to give crude 3 (40.0 g) as yellow oil. ESI-LCMS: m / z 384.1 [M+H]+.

[0229] Step 10: To the solution of crude 3 (40.0 g, 86.3 mmol) was added CH3NH2 / MeOH (500.0 mL) at 25oC under N2 atmosphere. Reaction was stirred at 50oC for 17 h and then LCMS showed 3 was consumed. Mixture was concentrated and added water (300.0 mL) extracted by EA (100.0 mL *3). The aqueous was freeze-drying to give 4 (7.3 g, 95.0% purity, 57.5% yield) as white solid. ESI-LCMS: m / z 148.1 [M+H]+.1H-NMR (600 MHz, MeOD) δ = 4.25-4.21 (m, 2H), 3.85-3.83 (m, 1H), 3.65-3.62 (m, 1H), 3.58-3.55 (m, 1H), 2.92-2.89 (m, 1H), 2.76-2.72 (m, 1H), 1.93-1.83 (m, 2H).

[0230] Step 11: Compound 8a (24.8 g, 48.9 mmol) was added in DMF (250.0 mL), next EDCI (12.2 g, 63.5 mmol) and HOBT (8.6 g, 63.7 mmol) was added in the mixture at r.t. for the 20 min. Then the DIPEA (12.6 g, 97.7 mmol) and compound 4 (7.2 g, 48.9 mmol) wasadded in the mixture at r.t. Then the reaction mixture was stirred at r.t. for overnight. The LCMS showed compound 4 was consumed. The reaction mixture extracted with DCM (150.0 mL*4). The organic layer was washed with brine and dried over Na2SO4. The residue was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5% NH4HCO3) = 1 / 0 within 30 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 10 / 1; Detector, UV 254 nm. This resulted in to give 5 (21.5 g, 97% purity, 68.3% yield) as an oil. ESI-LCMS: m / z 637.3 [M+H]+.1H-NMR (600 MHz, MeOD) δ = 5.34-5.33 (d, J =3.0 HZ, 1H), 5.09-5.06 (m, 1H), 4.66-4.64 d, J =8.0 HZ, 1H),4.24-4.20 (m, 2H),4.17-4.09 (m, 3H), 4.04-4.02 (m, 1H), 3.93-3.90 (m, 1H), 3.79-3.70 (m, 6H), 3.65-3.58(m, 7H), 3.33 (s, 6H), 3.32-3.30 (m, 2H), 2.52-2.43 (m, 2H), 2.14 (s, 3H), 2.03 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H), 1.90-1.79 (m, 2H).

[0231] Step 12: To a solution of 5 (21.5 g, 33.8 mmol) in pyridine (210.0 mL) was added DMTrCl (13.7 g, 40.4 mmol) at 0oC under N2atmosphere. Reaction was stirred at 25oC for 15h. LCMS showed starting material was consumed. Mixture was quenched by water (100.0 mL) and extracted by EA (200.0 mL *4), the organic was washed by brine, dried and concentrated and then purified by column chromatography (SiO2, DCM / MeOH = 200:1 to 50:1) to give 6 (21.4 g, 96.0% purity, 67.5% yield) as yellow solid. ESI-LCMS: m / z 937.3 [M+H]+;1H-NMR (400 MHz, DMSO-d6) δ = 7.80-7.78 (m, 2H), 7.40-7.20 (m, 10H), 6.90- 6.88 (m, 5H), 5.22-5.21 (d, J = 3.4 Hz 1H), 5.00-4.96 (m, 1H), 4.56-4.54 (d, J = 8.5 Hz 1H), 4.09-4.03 (m, 5H), 3.91-3.84 (m, 1H), 3.79-3.74 (m, 1H), 3.60-3.43 (m, 10H), 3.10-3.24 (m, 2H),2.97-2.92 (m, 2H), 2.31-2.28 (m, 2H), 2.10 (s, 3H), 2.07 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.78 (s, 3H).

[0232] Step 13 (GalNAc-4-amidite): To a solution of 6 (13.0 g, 13.8 mmol) in dry DCM (130.0 mL) were added CEP[N(iPr)2]2(5.0 mL, 16.6 mmol) and DCI (1.5 g, 12.5 mmol) under N2 atmosphere. The reaction mixture was stirred at 25°C for 30 min. LCMS showed 6 was consumed. The residue was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5% NH4HCO3) = 1 / 0 within 30 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 10 / 1; Detector, UV 254 nm. This resulted in to give GalNAc-4-amidite (12.0 g, 99% purity, 76.4% yield) as yellow solid. ESI-LCMS: m / z 1156.4 [M+NH3]+;1H-NMR (600 MHz, DMSO-d6) δ = 7.93-7.90 (m,2H), 7.79-7.78 (m, 10H), 7.40-7.38 (m, 2H), 7.40-7.38 (m, 2H),7.32-7.20 (m, 7H), 6.89-6.87 (m, 4H), 5.22-5.21 (d, J = 3.4 Hz 1H), 4.99-4.97(m, 1H), 4.56-4.55 (d, J = 8.5 Hz 1H), 4.31- 4.30 (m, 1H), 4.09-4.00 (m, 4H), 3.96-3.86 (m, 2H), 3.90-3.44 (m, 20H), 3.30-3.15 (m, 2H), 3.02-2.98 (m, 2H), 2.75-2.73 (m, 2H), 2.65-2.61 (m, 1H) 2.30-2.28 (m, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H).31P-NMR (242 MHz, DMSO-d6) δ = 146.68, 146.53.

[0233] Step 14: To a solution of 6 (8.4 g, 9.0 mmol) in DCM (84 mL), succinic anhydride (2.2 g, 22.0 mmol), DMAP (1.1 g, 9.0 mmol), Et3N (2.7 g, 26.9 mmol) was added. The mixture was stirred for 1 hour at 25 °C. LCMS showed the reaction completion. Mixture was concentrated and the crude product was purified by column chromatography (silica gel / PE / EA; 53% EA) to give A-2 (8.0 g, 7.7 mmol, 83% purity%, 85.6% yield) as a yellow solid. ESI-LCMS: m / z 1056.5 [M+NH3]+;1H-NMR (400 MHz, DMSO-d6) δ = 8.11-7.82 (m, 3H), 7.40-7.14 (m, 11H), 6.90-6.88 (m, 4H), 7.40-7.38 (m, 2H),7.32-7.20 (m, 7H), 6.89-6.87 (m, 4H), 6.59-6.58 (m, 2H), 5.22-5.21 (d, J = 3.4 Hz 1H), 5.08-5.07(m, 1H), 5.00-4.97 (m, 1H), 4.57-4.55 (d, J = 8.5 Hz 1H), 4.08-4.03 (m, 5H) 3.92-3.85 (m, 2H), 3.79-3.75 (m, 8H), 3.59-3.43 (m, 10H), 3.34-3.14 (m, 2H), 3.08-3.01 (m, 2H), 2.55-2.40 (m, 20H) 2.32-2.29 (m, 3H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H).

[0234] Step 15 (GalNAc-4-CPG): NH2-CPG was washed by I, DMF and DCM. To a solution of compound A-2 (5.74 g, 5.0 mmol) in THF (640.0 mL) was added HBTU (9.5 g, 25.2 mmol), DMAP (0.6 g, 5.0 mmol) and DEA (5.2 g, 40.3 mmol). Then added CPG Resin (A-2 / CPG = 1 / 14, mass ratio). The mixture was stirred by mechanical agitation at 40oC for 16 h. Filtered the reaction solution and rinse the filter cake successively with DCM (400.0 mL *4) To give a white solid. The suspension of resin was added to the mixture of (Pyridine: Ac2O = 5:1, 360.0 mL) in total. The suspension was agitated by mechanism stir at 40oC for 4 h. The reaction solution was filtered and the filter cake rinsed successively with DCM (400.0 mL *4) and dried at 30oC under reduced pressure to get the solid to a fine powder. Corresponding GalNAc-4-CPG was obtained and loading value of the corresponding GalNAc-4-CPG calculated by UV-vis (Total volume of DCA added in mL = 40 mL; Abs value at 500 nm = 0.567; Mg of CPG taken = 5.74 mg; Loading in μmol / g = ((40)*(0.567)*1000) / 76*(5.74) = 51.99 μmol / g).EXAMPLE 3. Preparation of GalNAc-5

[0235] Step 1: Compound 1 (65.0 g, 166.9 mmol) was dissolved in DCE (1.0 L) and TMSOTf (40.8 g, 183.6 mmol) was added. The reaction was stirred for 2 hours at 55 °C. After workup, check the reaction via LCMS and TLC (DCM / MeOH=20 / 1). The reaction mixture was cooled down to room temperature and then poured into TEA (25.3 g, 250.4mmol, 34.9 mL) and extraction with DCM. The reaction mixture was dried over and evaporated to dryness. The crude product was purified by flash chromatography (SiO2, DCM / MeOH=40 / 1) to give 2 (52.0 g, 94.8% purity, 94.7 yield). ESI-LCMS: m / z 330.1 [M+H]+;1H-NMR (400 MHz, DMSO-d6) δ = 6.06-6.04 (m, 1H), 5.25-5.23 (m, 1H), 4.90- 4.87 (m, 1H), 4.28-4.24 (m, 1H), 4.27-4.00 (m, 2H), 3.97-3.87 (m, 1H), 2.07-2.00 (m, 9H), 1.95-1.97 (m, 2H).

[0236] Step 2: Compound 2a (40.0 g, 179.9 mmol), EDCI (44.8 g, 233.9 mmol) and HOBT (31.6 g, 233.9 mmol) were dissolved in DMF (400.0 mL). After the mixture was stirred at room temperature for 15 minutes, Compound 2b (20.3 g, 269.9 mmol) and DIPEA (46.5 g, 359.9 mmol) were added and reaction at room temperature for 1 hour. A sample from the reaction mixture was taken by dropping. After workup, the reaction was checked via LCMS and TLC (DCM / MeOH=10 / 1). The reaction mixture was poured into NaHCO3 and extraction with DCM. Then dried over and evaporated to dryness. The crude product was purified by flash chromatography (SiO2 / DCM / MeOH=150 / 1) to give 2c (35.1 g, 98.5% purity, 50.3% yield). ESI-LCMS: m / z 280.2 [M+H]+;1H-NMR (400 MHz, CDCl3) δ = 8.00 (s, 1H), 7.37-7.29 (m, 5H), 6.39 (s, 1H), 5.11 (s, 2H), 3.62-3.59 (m, 2H), 3.43-3.36(m, 2H), 2.43-2.41 (m, 2H),2.25-2.23 (m, 2H), 2.00-1.67 (m, 2H), 1.66-1.64 (m, 2H).

[0237] Step 3: Compound 2 (31.4 g, 95.4 mmol) and 2c (31.9 g, 114.4 mmol) were dissolved in DCE (600.0 mL) and molecular sieve (31.0 g, 95.3 mmol) was added. After the reaction was stirred for 20 minutes, TMSOTf (2.1 g, 9.5 mmol) was added at 0 °C and reaction at room temperature overnight. After workup, check the reaction via LCMS and TLC (DCM / MeOH=20 / 1). The reaction mixture was filtered and the filter was concentrated under reduced pressure. Poured the filtrate into the saturated NaHCO3and extraction with DCM. And then the filtrate was dried over and evaporated to dryness. The crude product was purified by flash chromatography (SiO2, DCM / MeOH, =40 / 1) to give 3 (36.0 g, 96.5% purity, 62.1% yield).ESI-LCMS: m / z 609.3 [M+H]+;1H-NMR (400 MHz, CDCl3) δ = 7.39- 7.30 (m, 5H), 6.49-6.47 (m, 1H), 6.24-6.12 (m, 1H), 5.35-5.30 (m, 1H), 5.16-5.05 (m, 3H), 4.51-4.49 (m, 1H), 4.23-4.10 (m, 3H), 4.02-3.97 (m, 1H), 3.91-3.87 (m, 1H), 3.66-3.60 (m, 1H), 3.47-3.44 (m, 1H), 3.10-3.01 (m, 1H), 2.49-2.39 (m, 2H), 2.31-2.24 (m, 2H), 2.19-2.11 (m, 3H), 2.17-1.97 (m, 12H).

[0238] Step 4: Compound 3 (20.0 g, 32.9 mmol) was dissolved in THF (400.0 mL) and Pd / C (4.0 g, 37.6 mmol) was added. The reaction mixture was stirred at roomtemperature under H2. The reaction was complete detected by LCMS. The organic layer was washed with brine and dried over Na2SO4. Then the solution was concentrated under reduced pressure to give 4 (18.9 g, 90.3% purity, crude) as solid which was used directly for the next step. ESI-LCMS: m / z 519.1[M+H]+.

[0239] Step 5: Compound 4 (10.0 g, 19.3 mmol) was added in DMF (100.0 L). And then EDCI (4.8 g, 25.1 mmol) and HOBT (3.4 g, 25.1 mmol) was added in the mixture at r.t. for the 20 minutes. Then the DIPEA(4.9 g, 38.6 mmol) and compound 4a (2.8 g, 19.3 mmol) was added in the mixture at r.t. Then the reaction mixture was stirred at r.t. for overnight. A sample from the reaction mixture was taken by dropping. After workup, the reaction was checked via LCMS and TLC (DCM / MeOH=10 / 1). The reaction mixture was extracted with DCM (600.0 mL *5), then dried over and evaporated to dryness. The crude product was purified by flash chromatography (SiO2, DCM / MeOH=20 / 1) to give compound 5 (8.0 g, 82.6% purity, 64.0% yield). ESI-LCMS: m / z 648.2 [M+H]+.

[0240] Step 6: Compound 5 (6.0 g, 9.3 mmol) was dissolved in pyridine (60.0 mL) and DMTrCl (3.5 g, 10.2 mmol) was added. The reaction was stirred for 2 hours at room temperature. A sample from the reaction mixture was taken by dropping. After workup, the reaction was checked via LCMS. The reaction mixture was quenched by H2O and extraction with EA. Then dried over and evaporated to dryness. The crude product was purified by column chromatography to give compound 6 (8.0 g, 97.4% purity, 69.3% yield). ESI-LCMS: m / z 948.2 [M-H]-;1H-NMR (400 MHz, DMSO-d6) δ = 7.80 (s, 1H), 7.68 (s, 1H), 7.41-7.22 (m, 9H), 6.90-6.88 (m, 4H), 5.22-5.21 (m, 1H), 4.97-4.96 (m, 2H), 4.50-4.47 (m, 1H), 4.06- 4.00 (m, 7H), 3.74 (s, 8H), 3.33 (s, 1H), 2.94-2.93 (m, 6H), 2.06-2.05 (m, 3H), 2.03-2.01 (m, 9H), 1.99-1.60 (m, 3H), 1.92-1.16 (m, 9H).

[0241] Step 7: To a solution of 6 (9.0 g, 9.5 mmol) in dichloromethane (90.0 mL) with an inert atmosphere of nitrogen was added CEOP[N(iPr)2]2 (3.4 g, 11.4 mmol) and DCI (1.2 g, 8.5 mmol) in order at room temperature. The resulting solution was stirred for 1.0 h at room temperature and diluted with 50.0 mL dichloromethane and washed with 2 x 50.0 mL of saturated aqueous sodium bicarbonate and 1 x 50.0 mL of saturated aqueous sodium chloride respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated till no residual solvent left under reduced pressure. The residue was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5%NH4HCO3) = 1 / 0 within 20 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 9 / 1; Detector, UV 254 nm. This resulted in to give GalNAc-5 (8.0 g, 99.5% purity, 73.5% yield) as yellow solid. ESI-LCMS: m / z 1095.4 [M-H]-;1H-NMR (400 MHz, DMSO-d6): δ 7.39-7.30 (m, 3H), 7.27-7.24 (m, 9H), 6.90-6.87 (m, 4H), 5.75-5.21 (m, 1H), 4.99-4.91 (m, 1H), 4.50-4.48 (m, 1H), 4.30-4.29 (m, 1H), 4.10-4.01 (m, 4H), 3.96-3.86 (m, 2H), 3.71-3.41 (m, 16H), 3.23-3.18 (m, 2H), 3.08-2.98 (m, 4H), 2.75-2.72 (m, 1H), 2.66-2.63 (m, 1H), 2.51-2.50 (m, 2H), 2.05-1.79 (m, 16H), 1.36-1.1 (m, 12H);31P-NMR (162 MHz, DMSO-d6): δ 146.70, 146.57. EXAMPLE 4. Preparation of GalNAc-6-amidite and GalNAc-6-CPG

[0242] Step 1: Sodium borohydride (31.9 g, 840.0 mmol) was dissolved in THF (460.0 mL). The mixture was warmed at 40 °C for 30 min. To this mixture was added dropwise 1 (100.0 g, 600.0 mmol). The reaction continued for another 5 hours and was then quenched with hydrochloric acid (20.0 mL concentrated HCl in 50.0 mL water) at 0 °C. The mixture was stirred for 1 more hour and was then adjusted to pH=7 with aqueous sodium hydroxide. The solution was extracted with ethyl acetate (500.0 mL) and the organic layer was combined and dried with anhydrous Na2SO4. After filtration, the organic solvent was removed in vacuo and the crude residue was purified by flash column chromatography (SiO2,DCM / MeOH = 100 / 1) to afford the compound 2 (54.0 g, 440.0 mmol, 90% purity) as a pale- yellow oil with 72.6% yield.1H-NMR (400 MHz, CDCl3) δ = 3.99 – 4.05 (m, 1H), 3.82 (m, 2H), 3.59 (m, 1H), 3.50 (m, 2H), 1.72-1.78 (m, 1H).

[0243] Step 2: To a solution of 2 (54.0 g, 440.0 mol) in pyridine (1.1 L) was added at r.t. in N2atmosphere. Next DMT-Cl (140.2 g, 410.0 mmol) was added at 0 °C in the mixture. Then the reaction mixture was stirred at r.t. for 5 hours. The LCMS showed 2 was consumed. Quenched by water (1.5 L). The product was extracted with DCM (500.0 mL *4). The organic layer was washed with brine and dry over Na2SO4. After filtration, the organic solvent was removed in vacuo and the crude residue was purified by flash column chromatography (SiO2,PE / EA= 3 / 1) to afford the compound 3 (170.0 g, 400.0 mmol, 93.0% purity) as a pale-yellow oil with 91.4% yield. ESI-LCMS: m / z 426.3 [M-H]-.1H-NMR (400 MHz, DMSO-d6) δ = 7.38 (d, J =7.3, 2H), 7.24 – 7.29 (m, 7H), 6.80 (d, J =8.8, 4H), 3.97 – 4.00 (m, 1H), 3.75 (s, 6H), 3.43 – 3.52 (m, 2H), 3.34 (m, 1H), 3.25 (m, 1H), 1.79 – 1.82 (m, 2H).

[0244] Step 3: To a solution of 3 (170.0 g, 400.0 mmol) in 1.4-dioxane (1.7 L), 5.0 M sodium hydroxide solution was added dropwise. The solution was stirred overnight at 40 °C and was then diluted with ethyl acetate. The product was extracted with water (500.0 *4mL). The organic layer was washed with brine and dried over Na2SO4. Then the solution was concentrated under reduced pressure to give 4 (176.0 g, 90% purity) as solid which was used directly for the next step. ESI-LCMS: m / z 389.4 [M-H]-.

[0245] Step 4: The product 4 (176.0 g, 450.0 mmol) was dissolved in a saturated solution of ammonium in methanol (7.0 M). The mixture was stirred at r.t. for 24 h and then concentrated in vacuo. The crude residue was purified by flash chromatography (SiO2,DCM / MeOH / TEA = 20 / 1 / 0.1%) to afford 5 (67 g, 0.16 mol) as a colorless viscous oil with a yield of 43%. ESI-LCMS: m / z 406.2 [M+H]+.1H-NMR (400 MHz, CDCl3) δ = 7.43 (d, J =7.4, 2H), 7.18 – 7.32 (m, 3H), 6.83 (d, J =8.8, 4H), 3.78 (s, 6H), 3.72 (m, 1H), 3.30 (m, 1H), 3.22 (m, 1H), 2.75 (m, 1H), 2.58(m, 1H), 1.67 – 1.77 (m, 2H).

[0246] Step 5: Compound 5a (24.0 g, 46.6 mmol) was added in DMF (190.0 mL). Next EDCI (11.6 g, 61.0 mmol) and HOBT (8.2 g, 61.0 mmol) was added in the mixture at r.t. for the 20 min. Then the DIPEA (12 g, 93.0 mmol) and compound 5 (19.0 g, 46.6 mmol) was added in the mixture at r.t. Then the reaction mixture was stirred at r.t. for 17 h. The LCMS showed compound 5 was consumed. The reaction mixture extracted with DCM (600.0 mL *5). The organic layer was washed with brine and dried over Na2SO4. Then the solution was concentrated under reduced pressure to give compound 6 (30.0 g, 33.5 mmol, 96% purity, 71.8% yield) as a yellow solid. ESI-LCMS: m / z 895.3 [M-H]-.1H-NMR (400 MHz, DMSO-d6) δ = 7.81 – 7.78 (d, J =12, 2H), 7.37 – 7.19 (m, 9H), 6.88 – 6.86 (d, J =8, 4H), 5.75 (s, 6H), 5.22 – 5.21 (d, J =4, 1H), 5.00 – 4.96 (m, 1H), 4.59 – 4.54 (m, 2H), 4.05 – 4.00 (m, 1H), 3.91 – 3.84 (m, 1H), 3.79 – 3.73 (m, 1H), 3.63 – 3.54 (m, 4H), 3.52 – 3.43 (m, 6H), 3.09 – 2.95 (m, 4H), 2.35 – 2.32 (m, 2H), 2.12 – 2.10 (s, 3H), 2.00 – 1.99 (s, 3H), 1.92 – 1.86 (s, 3H), 1.72 – 1.67 (m, 1H), 1.55 – 1.50 (s, 1H).

[0247] Step 6 (GalNAc-6-amidite): To a solution of 6 (14.0 g, 15.6 mmol) in dichloromethane (140.0 mL) with an inert of nitrogen were added CEOP[N(iPr)2]2 (5.6 mL, 18.8 mmol) and DCI (1.66 g, 14.1 mmol) in order at room temperature. The resulting solution was stirred for 1 h at room temperature and diluted with 50.0 mL dichloromethane and washed with 50.0 mL *2 of saturated aqueous sodium bicarbonate and 50.0 mL of saturated aqueous sodium chloride respectively. The organic phase was dried over anhydrous sodiumsulfate, filtered and concentrated till no residual solvent left under reduced pressure. The residue was purified by Flash-Prep-HPLC with the following conditions (Intel Flash-1): Column, C18 silica gel; mobile phase, CH3CN / H2O (0.5% NH4HCO3) = 1 / 1 increasing to CH3CN / H2O (0.5% NH4HCO3) = 1 / 0 within 20 min, the eluted product was collected at CH3CN / H2O (0.5% NH4HCO3) = 6 / 1; Detector, UV 254 nm. This resulted to give GalNAc- 6-amidite (12.5 g, 98% purity) as a white solid. ESI-LCMS: m / z 941.2 [M+H]+;1H-NMR (400 MHz, DMSO-d6) δ = 7.83 – 7.70 (m, 2H), 7.38 – 7.18 (m, 9H), 6.88-6.85 (m, 4H), 5.22 – 5.21 (d, J = 4, 1H), 5.00 – 4.96 (m, 1H), 4.56 – 4.54 (d, J = 8, 1H), 4.05 – 4.00 (s, 3H), 3.91 – 3.84 (m, 2H), 3.79 – 3.68 (m, 8H), 3.64 – 3.54 (m, 3H), 3.52 – 3.39 (m, 9H), 3.28 – 3.22 (m, 1H), 3.16 – 3.09 (m, 1H), 3.05 – 2.92 (m, 1H), 2.74 – 2.71 (m, 1H), 2.57 – 2.54 (m, 1H), 2.35 – 2.31 (m, 2H), 2.11 – 2.05 (d, J = 24, 5H), 1.99 (s, 3H), 1.93 – 1.86 (m, 4H), 1.81 – 1.69 (m, 4H), 1.15 – 1.03 (m, 8H), 2.49 (s, 1H), 0.93 – 0.92 (d, J = 4, 3H).31P-NMR (162 MHz, DMSO-d6): δ = 148.01, 147.55.

[0248] Step 7: To a solution of 6 (7.0 g, 7.8 mmol) in DCM (70.0 mL), succinic anhydride (2.0 g, 19.5 mmol), DMAP (953.0 mg, 7.8 mmol) and Et3N (2.4 g, 19.5 mmol) were added. The mixture was stirred for 1 h at 25°C. The reaction was monitored by LCMS. The LCMS showed reaction completion. The crude product was washed with MTBE / EA=50:1 and concentrated under reduced pressure to give Cmp 5 (9.0 g, 9.0 mmol, 91.0% purity) as a yellow solid. ESI-LCMS: m / z 995.2 [M-H]-.

[0249] Step 8 (GalNAc-6-CPG): To a solution of Cmp 5 (4.0 g, 4.0 mmol) in THF (448.0 mL, 8 v / v), HBTU (7.6 g, 20.1 mmol), DMAP (490.0 mg, 4.0 mmol) and DIPEA (4.1 g, 32.1 mmol) were added. The mixture was shook for 10 min at 25oC. Then added CPG Resin (Cmp 5 / CPG=1 / 14, mass ratio). The mixture was stirred by mechanical agitation at 40oC for 16 h. Filtered the reaction solution and rinse the filter cake successively with DCM (8 V * 4). The washed solid is a white solid. The suspension of resin was added to the mixture of pyridine and Ac2O (5 / 1, 6 V in total). The suspension was agitated by mechanism stir at 40oC for 4 h. The reaction solution was filtered and the filter cake rinsed successively with DCM (8 V * 4). The filter cake was dried at 30oC under reduced pressure to get the solid to a fine powder. Corresponding GalNAc-6-CPG was obtained, andloading value of the corresponding GalNAc-6-CPG was calculated by UV-vis (Total volume of DCA added in mL = 40.0 mL; Abs value at 500 nm = 0.623; Mg of CPG taken = 5.92 mg; Loading in μmol / g = ((40)*(0.623)*1000) / (76*(5.92)) = 55.38 μmol / g).EXAMPLE 5. Synthesis of GalNAc3, tri-GalNAc4, tri-GalNAc5, and tri- GalNAc6 conjugates

[0250] Exemplary siRNA conjugates of GalNAc3, tri-GalNAc4, tri-GalNAc5, and tri-GalNAc6 were prepared from GalNAc3-CPG, GalNAc4-CPG, GalNAc5, GalNAc6-CPG, respectively, with the following procedure:

[0251] Sense and antisense strand sequences of siRNAs were synthesized using oligonucleotide synthesizers following a standard solid phase synthesis protocol based on phosphoramidite chemistry. In the case of 3’-conjugation of the ligand, the ligand-CPG succinates were used as solid supports continued by two incorporations of the ligand phosphoramidites. Alternatively, like in the case of tri-GalNAc5, a universal solid support can be used with three consecutive additions of the GalNAc phosphoramidites. In the case of 5’-conjugation three consecutive incorporations of the corresponding GalNAc phosphoramidites were performed. After the final solid phase synthesis step, solid support bound oligomer was cleaved together by treatment with ammonia for 12 hours at 55oC (24 h in the case of tri-GalNAc5). Crude single strand product was isolated by lyophilization and purified by ion pairing reversed phase HPLC (IP-RPHPLC). Purified single strand oligonucleotide product from IP-RP-HPLC was converted to sodium salt by addition of NaOAc followed by desalting. Annealing of equimolar complementary sense stand and antisense strand oligonucleotide was carried out in nuclease-free water to form the double strand siRNAs, followed by a lyophilization procedure.

[0252] Exemplary GalNAc compounds of the disclosure conjugated to modified sense sequences of siRNA molecules were tested, with modified sequences shown in Table 1, 2, 3, and 4. Comparative GalNAc conjugate (L96) sense and antisense sequences are provided in Table 5. Table 1. GalNAc3 conjugate siRNA sequences.GalNAc3 ligand Compound Sense 5’-3’ (modified) SEQ Antisense 5’-3’ (modified) SEQAbbreviation: “*” represents a phosphorothioate (PS) bond; lower case letter represents modification with 2'-OMe; capital letter represents modification with 2'-F; “v” represents vinyl phosphonate; “(L3)” represents GalNAc3 ligand. Table 2. Exemplary tri-GalNAc4 siRNA conjugates.tri-GalNAc4 ligand Compound Sense 5’-3’ (modified) SEQ Antisense 5’-3’ (modified) SEQAbbreviation: “*” represents a phosphorothioate (PS) bond; lower case letter represents modification with 2'-OMe; capital letter represents modification with 2'-F; “v” representsvinyl phosphonate; “invdT” represents inverted Thymidine (5'-5' linked nucleotide); “(L4)” represents tri-GalNAc4 ligand. Table 3. tri-GalNAc5 conjugates SiRNA sense and antisense sequences. tri-GalNAc5 ligandAbbreviation: “*” represents a phosphorothioate (PS) bond; lower case letter represents modification with 2'-OMe; capital letter represents modification with 2'-F; “v” represents vinyl phosphonate; “(L5)” represents tri-GalNAc5 ligand.Table 4. Exemplary tri-GalNAc6 siRNA conjugates. Tri-GalNAc6 ’ ’ ’ ’Abbreviation: “*” represents a phosphorothioate (PS) bond; lower case letter represents modification with 2'-OMe; capital letter represents modification with 2'-F; “v” represents vinyl phosphonate; “(L6)” represents tri-GalNAc6 ligand. Table 5. Exemplary L96 siRNA conjugates.L96 ligandAbbreviation: “*” represents a phosphorothioate (PS) bond; lower case letter represents modification with 2'-OMe; capital letter represents modification with 2'-F; “v” represents vinyl phosphonate; “(L96)” represents L96 ligand. EXAMPLE 6. Protocol for AGT HDI

[0253] Purpose: To evaluate in vivo activity of siRNAs, a hydrodynamic injection mouse model was used for in vivo evaluation of siRNAs conjugated with novel GalNAcs targeting human AGT transcript.

[0254] Protocol: 6–8-week-old male C57 mice are treated subcutaneously with a GalNAc-conjugated AGT siRNA at a dose level of 1 mg / kg. Three days later, the mice arehydrodynamically injected with a DNA plasmid encoding the full-length human AGT transcript. One day after introduction of the plasmid, liver samples are harvested. Total RNA from these mice is subjected to qRT-PCR analysis for AGT mRNA relative to mice only treated with the same volume of PBS. The value is normalized for transfection efficiency using the NeoR gene included in the plasmid.

[0255] Exemplary gene expression results of two siRNA sequences each conjugated to various GalNAc structures of the disclosure, as compared to L96 conjugates, are provided in FIG.1 (Sequence 1) and FIG.2 (Sequence 2). The data show that, in Sequence 1, there is a trend for exemplary GalNAc conjugates of the disclosure to exhibit more potency (downregulated expression of AGT) than a control L96 conjugate.

[0256] Further, in vivo percentage AGT knockdown (KD) at 1 mg / kg dosing in the mice for Sequences 1 and 2 are provided in Tables 5 and 6, respectively, below. Table 5. Sequence 1 KD results Compound ID % KD at 1 mg / kgTable 6. Sequence 2 KD results Compound ID % KD at 1mpkEXAMPLE 7. Protocol for ANGPTL3 In Vivo Study

[0257] Purpose: To test siRNAs conjugated with exemplary GalNAc ligands targeting ANGPTL3 in wildtype mice.

[0258] Protocol: All GalNAc conjugates were administrated subcutaneously at 1 mg / kg or 3 mg / kg, with PBS as a negative control. The expression of mouse ANGPTL3 were measured 7, 14, 21 and 28 days post dosing by enzyme-linked immunosorbent assay (ELISA). Data showed as ANGPTL3 protein expression relative to pretreatment (Day-1).

[0259] FIGS.3, 4, and 5 depict comparison of ANGPTL3 reduction in wild-type mice for exemplary GalNAc conjugates against an L96 control conjugate when dosed at 1 mg / kg. The data demonstrate that, in wild-type mice, ANGPTL3 reduction was significantly higher for the exemplary GalNAc conjugates than for the control L96 conjugate at all data points across three different sequences as identified.

[0260] FIGS.6, 7, and 8 depict comparison of ANGPTL3 reduction in wild-type mice for exemplary GalNAc conjugates against an L96 control conjugate when dosed at 3 mg / kg. The data demonstrate that, in wild-type mice, the difference in ANGPTL3 reduction for the exemplary GalNAc conjugates as compared to the control L96 conjugate was smaller, suggesting dosing saturation.

[0261] In vivo knockdown (KD) Results for GalNAc compounds tested are summarized in Table 7. Table 7. Exemplary data I i % KD d I i % KD d I i % KD d I i % KD dEXAMPLE 8. ANGPTL3 Knockdown Studies of Exemplary siRNA Compounds in Mice

[0262] Exemplary siRNA compounds of the disclosure as shown in Table 8 were tested for their effect on ANGPTL3 expression in naïve mice. The study utilized procedures similar to those described above in Example 7. FIG.9 shows a scheme of the study. Briefly, naïve mice were injected with 1 mg / kg of test compound subcutaneously (n = 4). Mice were treated with 6 TAs under 1 mg / kg. Mouse serum was collected at one day before injection (D-1), 7 days after injection (D7), 14 days after injection (D14), 21 days after injection (D21), and 28 days after injection (D28). ANGPTL3 protein levels in mouse plasma were determined by ELISA.

[0263] Structures of ligands in the compounds of Table 8 are provided below:L96 Table 8. Exemplary siRNA compounds. C d S 5’3’ ( difid) SEQ Ati 5’3’ ( difid) SEQAbbreviation: “*” represents a phosphorothioate (PS) bond; lower case letter represents modification with 2'-OMe; capital letter represents modification with 2'-F; “v” represents vinyl phosphonate; “(LT4)” represents tri-TEG-GalNAc4 ligand; “(LT5)” represents tri- TEG-GalNAc5 ligand; “(LT6)” represents tri-TEG-GalNAc6 ligand; “(L6)” represents tri- GalNAc6 ligand; “(LB6)” represents br-GalNAc6 ligand; “(L96)” represents L96 ligand.

[0264] Table 9 shows percentage of mouse ANGPTL3 knockdown by the siRNA compounds in mice.Table 9. ANGPTL3 Knockdown in naïve mice. % of mouse ANGPTL3 KDEXAMPLE 9. Exemplary syntheses of tri-TEG-GalNAc4, tri-TEG-GalNAc5, tri-TEG- GalNAc6, and br-GalNAc6

[0265] Exemplary siRNA conjugates of tri-TEG-GalNAc4, tri-TEG-GalNAc5, tri- TEG-GalNAc6, and br-GalNAc6 were prepared from GalNAc4, GalNAc5, GalNAc6, and GalNAc6, respectively, with the following procedure:

[0266] Sense and antisense strand sequences of siRNAs were synthesized using oligonucleotide synthesizers following a standard solid phase synthesis protocol based on phosphoramidite chemistry with a universal solid support. In the cases of tri-TEG-GalNAc4, tri-TEG-GalNAc5, and tri-TEG-GalNAc6, conjugation was made as 3’- or 5’- conjugation on oligo using the standard 3’-5’ oligo synthesis with corresponding GalNAc phosphoramidites and Triethylene Glycol (TEG) phosphoramidites in the beginning or end of oligo synthesis, respectively. Alternatively, in the case of br-GalNAc6, 5’- conjugate was made using regularoligo synthesis technology in 3’-5’ direction, and 3’- conjugation product was done using reverse / inverted nucleoside amidites instead of regular amidites. EXAMPLE 10. Evaluation of knockdown of human AGT with modified siRNAs in in vivo mouse hydrodynamic injection (HDI) model

[0267] 13 exemplary modified siRNA compounds (Duplexes 100329 – 100341) were tested for knockdown of human AGT in a mouse model hydrodynamic injected with DNA plasmid encoding the full-length human AGT transcript. Briefly, transgenic mice injected with a DNA plasmid encoding the full-length human AGT transcript were subcutaneously injected with PBS or modified AGT siRNA at 2 mg / kg. After injection with PBS or modified AGT siRNA, plasma samples were drawn weekly from mice. Treatment duration was initially set as 14 days with 4 TAs, and was extended to 28 days with 9 TAs for some compounds. The study design is outlined in Table 10. The unmodified and modified sense and antisense strand sequences of duplexes 100329-100341 are summarized in Tables 11-12, respectively.Table 10. Outline of in vivo study of AGT siRNA treatment of transgenic mice. Group Mice Compound treatment Sample Termination Readout per collectionTable 11. AGT siRNA unmodified sequences Sense 5'-3' SEQ ID Antisense 5'-3' SEQ ID NO NOTable 12. Exemplary tri-GalNAc6 or L96 conjugated, modified siRNA conjugates, wherein the sense strand is conjugated to 5’-triGalNAc6, 3’-triGalNAc6, or 3’-L96 targeting AGT mRNA.Tri-GalNAc6Mo e s nuceot e sugar, weren s te nuceot e ase s urac (nucleotide abbreviation: tmU) or cytosine (nucleotide abbreviation tmC) O B OTNA analog, wherein B is a uracil base (abbreviation: utU), a cytosine base (abbreviation: utC), or an adenine base (abbreviation utA)O NH N O O O Glycol Nucleic Acid (GNA) Com Sense 5'-3' (modified) SEQ Antisense 5'-3' SEQ3 ucagcac Abbreviation: (*) = PS bond; lower case = 2’-OMe; capital = 2'-F; invdN = inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’-5’ linked nucleotide); v = 5'-E-Vinyl- phosphonate; (tmU) = siRNA nucleotide with a modified sugar and a uracil base; (tmC) = siRNA nucleotide with a modified sugar and a cytosine base; (utU) = a TNA analog with a uracil base, i.e., utU from above; (utC) = a TNA analog with a cytosine base, i.e., utC from above; (utA) = a TNA analog with a adenine base, i.e., utA from above; (Tgn) = a thymidine-glycol nucleic acid, i.e., GNA from above; (L6) = tri-GalNAc6 ligand; (L96) = L96 ligand.

[0268] mRNA levels in mice were measured by ELISA and normalized to individual animal at day -1 and PBS control at given time points. Table 13 and FIGs.10A-10C show the results of single dose AGT siRNA injection in AGT C57 mice. The results show that compounds 100331 and 100332 showed improved potency and durability when compared to clinical compound 100329 at day 28, and compound 100348 showed similar potency and durability when compared to clinical compound 100329 at day 28. Table 13 Average % knockdown (KD) for selected modified AGT siRNA compounds (100329 – 100341).% of in vivo KD (days) IDEQUIVALENTS

[0269] While specific embodiments have been discussed, the above specification is illustrative and not restrictive. Many variations of the embodiments will become apparent to those skilled in the art upon review of this specification. The full scope of what is disclosed should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.SEQUENCE LISTING SEQ Description Sequence ID

Claims

CLAIMS What is claimed is:

1. A compound represented by Formula (I):Formula (I) or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene, wherein 5-membered heterocyclyl is optionally substituted with one or more –(CH2CH2O)q-, wherein q is an integer from 1 to 20, and alkylene is optionally substituted with one or more -OH; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; andn is an integer from 1 to 6.

2. The compound of claim 1, wherein each occurrence of T1and T2is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C1–6alkylene.

3. The compound of claim 1, wherein the compound is represented by Formula (I-A-A):Formula (I-A-A) or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein the compound is represented by Formula (I-A):Formula (I-A) or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1 or 2, wherein the compound is represented by Formula (I- A-I-A): HOOHHOOHOH OH HFormula (I-A-I-A) or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein the compound is represented by Formula (I-A-I):or a pharmaceutically acceptable salt thereof.

7. The compound of claim 1, wherein the compound is represented by Formula (I-A-II- A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

8. The compound of claim 1, wherein the compound is represented by Formula (I-A-II):ormua ( - - ) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

9. The compound of claim 1, wherein the compound is represented by Formula (I-A-III- A):o ua - - - or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

10. The compound of claim 1, wherein the compound is represented by Formula (I-A-III):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

11. The compound of claim 1, wherein the compound is represented by Formula (I-A-IV- A):Formula (I-A-IV-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

12. The compound of claim 1, wherein the compound is represented by Formula (I-A- IV):HO OHOHHO OHOHHN HN A1or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

13. The compound of claim 1, wherein the compound is represented by Formula (I-A-V- A):ormu a ( - -V- ) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

14. The compound of claim 1, wherein the compound is represented by Formula (I-A-V):o u a - - or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

15. The compound of claim 1, wherein n is 2.

16. The compound of claim 1, wherein each occurrence of a and b is 3.

17. The compound of claim 1, wherein each occurrence of d and e is 1.

18. The compound of claim 1, wherein each occurrence of X is independently selected from -OH and -SH, or a pharmaceutically acceptable salt thereof.

19. The compound of claim 1, wherein the compound is selected from a compound of Formula (I-A-VI-A) and a compound of Formula (I-A-VII-A):ndFormula (I-A-VI-A)ormua (- -V - ) or a pharmaceutically acceptable salt thereof.

20. The compound of claim 1, wherein the compound is selected from a compound of Formula (I-A-VI) and a compound of Formula (I-A-VII):Formula (I-A-VII) or a pharmaceutically acceptable salt thereof.

21. The compound of claim 1, wherein the compound is represented by Formula (I-B-A):Formula (I-B-A) or a pharmaceutically acceptable salt thereof.

22. The compound of claim 1, wherein the compound is represented by Formula (I-B):or a pharmaceutically acceptable salt thereof.

23. The compound of claim 1, wherein the compound is represented by Formula (I-B-I- A):or a pharmaceutically acceptable salt thereof.

24. The compound of claim 1, wherein the compound is represented by Formula (I-B-I):ormua - - or a pharmaceutically acceptable salt thereof.

25. The compound of claim 1, wherein the compound is represented by Formula (I-B-II- A):ormua - - - or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

26. The compound of claim 1, wherein the compound is represented by Formula (I-B-II):Formula (I-B-II) or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

27. The compound of claim 1, wherein the compound is represented by Formula (I-B-III- A):Formula (I-B-III-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

28. The compound of claim 1, wherein the compound is represented by Formula (I-B-III):or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.

29. The compound of claim 1, wherein the compound is represented by Formula (I-B-IV- A):or a pharmaceutically acceptable salt thereof.

30. The compound of claim 1, wherein the compound is represented by Formula (I-B-IV):o u a - - or a pharmaceutically acceptable salt thereof.

31. The compound of claim 1, wherein LAis absent.

32. The compound of claim 1, wherein LAis a cleavable linker.

33. The compound of any claim 1, wherein LAis a non- cleavable linker.

34. The compound of any claim 1, wherein A1is a double-stranded RNA (dsRNA) molecule, wherein LAis attached to only one strand of the dsRNA.

35. A compound represented by Formula (II):HO O HO O L H N A2or a pharmaceutically acceptable salt thereof, wherein: A2is an oligonucleotide; X is selected from hydroxy and thiol; each occurrence of L and L1is a linker; and LAis absent or a linker.

36. The compound of claim 35, wherein the compound is represented by Formula (II-A- I):.Formu a (II-A-I) or a pharmaceutically acceptable salt thereof.

37. The compound of claim 35, wherein each occurrence of L is independently selected from the group consisting of alkylene, heteroalkylene, and –(CH2)j-C(O)NH-(CH2)k-, wherein each of j and k is independently 1 to 10.

38. The compound of claim 35, wherein the compound is represented by Formula (II-A- II):Formula (II-A-II) or a pharmaceutically acceptable salt thereof, wherein each occurrence of n is an integer from 1-20.

39. The compound of claim 35, wherein the compound is represented by Formula (II-A- III):Formula (II-A-III) or a pharmaceutically acceptable salt thereof, wherein each occurrence of n is an integer from 1-20.

40. The compound of claim 35, wherein L1is alkylene.

41. The compound of claim 35, wherein the compound is represented by Formula (II-A- IV):or a pharmaceutically acceptable salt thereof.

42. A compound represented by Formula (III):Formula (III) or a pharmaceutically acceptable salt thereof, wherein: each occurrence of L1and L is a linker;n is an integer from 1 to 3; A1is an oligonucleotide; X is selected from hydroxy and thiol; and LAis absent or a linker.

43. The compound of claim 42, wherein the compound is represented by Formula (III-A):ormu a ( - ) or a pharmaceutically acceptable salt thereof.

44. The compound of claim 42, wherein L1is -CH2C(H)m-, wherein m is an integer from 0 to 2, provided that the sum of m and n is 3.

45. The compound of claim 42, wherein each occurrence of L is:wherein p is an integer from 1 to 4.

46. The compound of claim 45, wherein p is 2.

47. A compound represented by Formula (IV):ormu a or a pharmaceutically acceptable salt thereof, wherein: A1is an oligonucleotide; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl substituted with –(CH2CH2O)q-, wherein q is an integer from 1 to 20; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

48. The compound of claim 47, wherein each occurrence of T1and T2is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C1–6alkylene.

49. The compound of claim 47, wherein the compound is represented by Formula (IV-A- A):or a pharmaceutically acceptable salt thereof.

50. The compound of claim 47, wherein the compound is represented by Formula (IV-A):Formula (IV-A) or a pharmaceutically acceptable salt thereof.

51. The compound of claim 47, wherein the compound is represented by Formula (IV-A- I-A):HOOHHOOHOH OH Hormua (V- -- ) or a pharmaceutically acceptable salt thereof.

52. The compound of claim 47, wherein the compound is represented by Formula (IV-A- I): OHHOHHOOH O OHA1or a pharmaceutically acceptable salt thereof.

53. The compound of claim 47, wherein the compound is represented by Formula (IV-A- II-A):Formula (IV-A-II-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

54. The compound of claim 47, wherein the compound is represented by Formula (IV-A- II):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

55. The compound of claim 47, wherein the compound is represented by Formula (IV-A- III-A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

56. The compound of claim 47, wherein the compound is represented by Formula (IV-A- III):ormu a ( - - ) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

57. The compound of claim 47, wherein the compound is represented by Formula (IV-A- IV-A):ormua (V- -V- ) or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

58. The compound of claim 47, wherein the compound is represented by Formula (IV-A- IV):or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

59. The compound of claim 47, wherein the compound is represented by Formula (IV-A- V-A):ormua ( - - - ) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

60. The compound of claim 47, wherein the compound is represented by Formula (IV-A- V):or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein each occurrence of d and e is an integer from 1-20.

61. The compound of claim 47, wherein n is 2.

62. The compound of claim 47, wherein each occurrence of a and b is 3.

63. The compound of claim 47, wherein each occurrence of d and e is 1.

64. The compound of claim 47, wherein each occurrence of X is independently selected from -OH and -SH, or a pharmaceutically acceptable salt thereof.

65. The compound of claim 47, wherein q is an integer from 2 to 6.

66. The compound of claim 47, wherein q is 2.

67. The compound of claim 47, wherein the compound is represented by A1-LA-D, wherein D is selected from:or a pharmaceutically acceptable salt thereof.

68. The compound of claim 1, 45, 42, or 47, wherein LAis absent.

69. The compound of claim 1, 45, 42, or 47, wherein LAis a cleavable linker.

70. The compound of claim 1, 45, 42, or 47, wherein LAis a non- cleavable linker.

71. The compound of claim 1, 35, 42, or 47, wherein A2is a double-stranded RNA (dsRNA) molecule, wherein LAis attached to only one strand of the dsRNA.

72. The compound of claim 1, 35, 42, or 47, wherein LAis attached to A2at the 5’ end, the 3’ end, or both ends of A2.

73. The compound of claim 1, 35, 42, or 47, wherein LAis attached to A2at the 5’ end, the 3’ end, or both ends of A2.

74. A compound selected from the group consisting of BCR-0000739, BCR-0000740, BCR-0000741, BCR-0000742, BCR-0000743, BCR-0000744, BCR-0000745, BCR- 0000746, BCR-0000747, BCR-0000748, BCR-0000749, BCR-0000750, BCR-0000751, BCR-0000752, BCR-0000753, BCR-0000754, BCR-0001075, BCR-0001076, BCR- 0001077, and BCR-0001078, or a pharmaceutically acceptable salt thereof.

75. A pharmaceutical composition comprising the compound of claim 1, 45, 42, or 47, a or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

76. A method of treating a disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the compound of claim 1, 45, 42, or 47, or a pharmaceutically acceptable salt thereof.

77. The method of claim 76, wherein the disorder is a liver disorder.

78. The method of claim 76, wherein the disorder is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), obesity, hypertension, and hypercholesteremia.

79. The method of claim 78, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).

80. A method of treating a disease mediated by expression of ANGPTL3 or AGT in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the compound of any one of claim 1, 45, 42, or 47, or a pharmaceutically acceptable salt thereof .

81. The method of claim 76, comprising administering to the patient one or more additional therapeutic agents.

82. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of Angiotensinogen (AGT), wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein:a. the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 613; b. the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 614; or c. the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 618, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO:

615.

83. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of AGT, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: d. the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 622, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 619; e. the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 623, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 620; or f. the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 624, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO:

621.

84. The dsRNA of claim 82 or claim 83, wherein the AGT is human AGT.

85. The dsRNA of claim 82 or claim 83, wherein the AGT is human AGT comprising the sequence shown in SEQ ID NO: 441 (NM_001384479.1).

86. The dsRNA of claim 82 or claim 83, wherein the sense strand is 70%, 80%, 90%, 95% or more identical to the sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO:

621.

87. The dsRNA of claim 82 or claim 83, wherein the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence comprising thesequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO:

621.

88. The dsRNA of claim 82, wherein the sense strand comprises: g. 21 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621; h. 22 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621; and / or i. 23 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 615, SEQ ID NO: 619, or SEQ ID NO:

621.

89. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO:

624.

90. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises: j. 21 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO: 624; k. 22 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO: 624; and / or l. 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO:

624.

91. The dsRNA of claim 82 or claim 83, wherein the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO: 621, and the antisense strand is selected from an antisense strand sequence comprising the sequence ofSEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO:

624.

92. The dsRNA of claim 82 or claim 83, wherein the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 620, or SEQ ID NO:

621.

93. The dsRNA of claim 82 or claim 83, wherein the antisense strand is selected from an antisense strand sequence comprising the sequence of SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO:

624.

94. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises the sequence of SEQ ID NO: 616 and the sense strand comprises the sequence of SEQ ID NO:

613.

95. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises the sequence of SEQ ID NO: 617 and the sense strand comprises the sequence of SEQ ID NO:

614.

96. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises the sequence of SEQ ID NO: 618 and the sense strand comprises the sequence of SEQ ID NO:

615.

97. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises the sequence of SEQ ID NO: 622 and the sense strand comprises the sequence of SEQ ID NO:

619.

98. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises the sequence of SEQ ID NO: 623 and the sense strand comprises the sequence of SEQ ID NO:

620.

99. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises the sequence of SEQ ID NO: 624 and the sense strand comprises the sequence of SEQ ID NO:

621.

100. The dsRNA of claim 82 or claim 83, wherein at least one nucleotide of the dsRNA is a modified nucleotide selected from the group consisting of: a 5’-vinyl phosphonatenucleotide, a 2'-O-methyl modified nucleotide, an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’-5’ linked nucleotide), a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide, and a nucleotide comprising a modified nucleotide component represented by Formula (I):wherein: B1is a nucleobase; and R1is selected from the group consisting of hydrogen and C1–6alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide.

101. The dsRNA of claim 82 or claim 83, wherein the antisense strand has a 3’ end nucleotide overhang compared to the sense strand.

102. The dsRNA of claim 101, wherein the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand.

103. The dsRNA of claim 82 or claim 83, wherein the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary.

104. The dsRNA of claim 82 or claim 83, wherein the antisense strand and the sense strand are at least 80% complementary.

105. The dsRNA of claim 82 or claim 83, wherein the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.

106. The dsRNA of claim 82 or claim 83, wherein the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of AGT mRNA.

107. The dsRNA of claim 82 or claim 83, wherein the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the AGT mRNA.

108. The dsRNA of claim 82 or claim 83, wherein the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the AGT mRNA.

109. The dsRNA of claim 82 or claim 83, wherein at least one nucleotide of the dsRNA is a modified nucleotide.

110. The dsRNA of claim 109, wherein the modified nucleotide is at least one of a modified nucleotide selected from the group consisting of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’-5’ linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-Isomer and a nucleotide comprising a modified nucleotide component represented by Formula (I):Formula (I) wherein: B1is a nucleobase; and R1is selected from the group consisting of hydrogen and C1–6alkyl.

111. The dsRNA of claim 110, wherein B1is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof.

112. The dsRNA of claims 110 or 111, wherein B1is independently selected from uracil, cytosine, and modified analogs thereof.

113. The dsRNA of claim 110, wherein R1is C1–6alkyl.

114. The dsRNA of claim 110, wherein R1is -CH3.

115. The dsRNA of claim 110, wherein B1is uracil.

116. The dsRNA of claim 110, wherein R1is -CH3and B1is uracil.

117. The dsRNA of claim 110, wherein the sense strand comprises an inverted deoxyribonucleotide at the 5’ end; optionally wherein the inverted deoxyribonucleotide is a 5'-5' linked deoxythymidine.

118. The dsRNA of claim 110, wherein the sense strand comprises an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide is a 3'-3' linked deoxythymidine.

119. The dsRNA of claim 110, wherein the sense strand comprises an inverted deoxyribonucleotide at the 5’ end and an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide at the 5’ end is a 5'-5' linked deoxythymidine and the inverted deoxyribonucleotide at the 3’ end is a 3'-3' linked deoxythymidine.

120. The dsRNA of claim 110, wherein the sense strand comprises a nucleotide comprising the modified nucleotide component represented by Formula (I) at the 3’ end; optionally wherein R1is -CH3and B1is uracil.

121. The dsRNA of claim 109, wherein the modified nucleotide is at least one of: 5’-vinyl phosphonate nucleotide, a 5’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and / or a 2’-arabino fluoro (2’-araF) nucleotide.

122. The dsRNA of claim 121, wherein the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O- phosphonate.

123. The dsRNA of claim 109, wherein the modified nucleotide is at least one of: a 2'- deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, a 2’-amino-modified nucleotide, a 2’-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and / or a non-natural base comprising nucleotide.

124. The dsRNA of claim 109, wherein the antisense strand and / or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage.

125. The dsRNA of claim 124, wherein the antisense strand and / or the sense strand comprises at least one nucleotide modified linkage.

126. The dsRNA of claim 124, wherein all the nucleotide linkages in the antisense strand are modified linkages.

127. The dsRNA of claim 124, wherein the antisense strand and / or the sense strand comprises at least one a phosphorothioate (PS) bond.

128. The dsRNA of claim 82 or 83, further comprising a ligand or targeting moiety.

129. The dsRNA of claim 128, wherein the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA.

130. The dsRNA of claim 128, wherein the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA.

131. The dsRNA of claim 128, wherein the ligand or targeting moiety is conjugated to the 5’ end of the sense strand of the dsRNA.

132. The dsRNA of claim 128, wherein the ligand or targeting moiety is at least one N- Acetyl-Galactosamine (GalNAc).

133. The dsRNA of claim 128, wherein the ligand or targeting moiety is represented by represented by Formula (I):or a pharmaceutically acceptable salt thereof, wherein: A1is the point of attachment to the dsRNA; each occurrence of T1and T2is independently selected from 5-membered heterocyclyl and alkylene; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; LAis absent or a linker; and n is an integer from 1 to 6.

134. The dsRNA of claim 128, wherein each occurrence of T1and T2is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C1–6alkylene.

135. The dsRNA of claim 128, wherein the compound is represented by Formula (I-A-A):Formula (I-A-A) or a pharmaceutically acceptable salt thereof.

136. The dsRNA of claim 128, wherein the compound is represented by Formula (I-A-I-A):or a pharmaceutically acceptable salt thereof.

137. The dsRNA of claim 128, wherein the compound is represented by Formula (I-A-II- A):ormu a ( - - - ) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

138. The dsRNA of claim 128, wherein the compound is represented by Formula (I-A-III- A):or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.

139. The dsRNA of claim 128, wherein the ligand or targeting moiety is tri-GalNAc6.

140. A cell comprising the dsRNA of claim 82 or 83.

141. A vector encoding at least one unmodified strand of the dsRNA of any one of claim 82 or 83, optionally both strands.

142. A cell comprising the vector of claim 141.

143. A pharmaceutical composition for inhibiting expression of AGT comprising the dsRNA of claim 82 or 83, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

144. A method of inhibiting AGT expression in a cell, the method comprising: (a) contacting the cell with the dsRNA of claim 82 or 83; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an AGT gene, thereby inhibiting expression of the AGT gene in the cell, optionally wherein the method is in vivo.

145. The method of claim 144, wherein the AGT expression is inhibited by at least 30% relative to a control.

146. A method of treating a disorder mediated by or associated with AGT comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim 82 or 83.

147. The method of claim 146, wherein the disorder is a cardiovascular disorder.

148. The method of claim 146, wherein the disorder is cardiovascular disease.

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