MODIFIED DOUBLE STRANDED OLIGONUCLEOTIDE

FIELD OF THE INVENTION

The invention relates to dsRNA molecules having particular motifs that are advantageous for inhibition of target gene expression, as well dsRNA agent compositions, suitable for therapeutic use. Additionally, the invention provides methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity remained unknown.

There remains a need in the art for effective nucleotide or chemical motifs for dsRNA molecules, which are advantageous for inhibition of target gene expression. This invention is directed to that effort.

SUMMARY

This invention provides effective nucleotide or chemical motifs for dsRNA molecules, which are advantageous for inhibition of target gene expression, as well as RNAi compositions suitable for therapeutic use.

In one aspect the invention provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; and wherein the sense strand does not comprise a glycol nucleic acid (GNA).

It is understood that the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference. In other words, the dsRNA molecules of the invention are capable of inhibiting the expression of a target gene.

In some embodiments, the dsRNA comprises at least three 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at position 11 of the sense strand, counting from 5′-end of the sense strand.

In some embodiments, the dsRNA comprises at least five 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand.

In some embodiments, the dsRNA comprises at least seven 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand.

In some embodiments, the antisense strand comprises at least five 2′-deoxy modifications at positions 2, 5, 7, 12 and 14, counting from 5′-end of the antisense strand. In some further embodiments of this, the antisense strand has a length of 18-25 nucleotides, preferably, a length of 18-23 nucleotides.

In some embodiments, the dsRNA agent can comprise one or more non-natural nucleotides. For example, the dsRNA agent can comprise less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the dsRNA comprises no non-natural nucleotides. For example, the dsRNA agent comprises all natural nucleotides. Some exemplary non-natural nucleotides include, but are not limited to, acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

Accordingly, in some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid (GNA); and wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, at least one the sense strand and the antisence comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand or the antisense strand. Accordingly, in some embodiments, the invention provides a dsRNA agent comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; and wherein the sense strand and/or the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand and/or the antisense strand strand.

In some embodiment, the sense strand has length of 18 to 30 nucleotides and comprises at least two 2′-deoxy modifications in the central region of the sense strand. For example, the sense strand has length of 18 to 30 nucleotides and comprises at least two 2′-deoxy modifications within positions 7, 8, 9, 10, 11, 12, and 13, counting from 5′-end of the sense strand.

In some embodiments, the antisense strand has a length of 18 to 30 nucleotides and comprises at least two 2′-deoxy modifications in the central region of the antisense strand. For example, the antisense strand has length of 18 to 30 nucleotides and comprises at least two 2′-deoxy modifications within positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand.

In some embodiments, the invention provides a dsRNA agent comprising a sense strand and an antisense strand; wherein the sense strand has a length of 17-30 nucleotide and comprises at least one 2′-deoxy modification in the central region of the sense strand; wherein the antisense strand independently has a length of 17-30 nucleotides and comprises at least two 2′-deoxy modifications in the central region of the antisense strand.

In some embodiments, the invention provides a dsRNA agent comprising a sense strand and an antisense strand; wherein the sense strand has a length of 17-30 nucleotide and comprises at least two 2′-deoxy modifications in the central region of the sense strand; wherein the antisense strand independently has a length of 17-30 nucleotides and comprises at least one 2′-deoxy modification in the central region of the antisense strand.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand strand.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand strand.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the sense strand and/or the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand and/or the antisense strand strand.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy nucleotides on the sense and/or antisense strands; and wherein the dsRNA molecule has a double stranded (duplex) region of between 19 to 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand.

In some embodiments, when the dsRNA comprises less than 8 non-2′OMe nucleotides, the antisense stand comprises at least one DNA. For example, in any one of the embodiments of the invention when the dsRNA comprises less than 8 non-2′OMe nucleotides, the antisense stand comprises at least one DNA.

In some embodiments, when the antisense comprises two deoxy nucleotides and said nucleotides are at positions 2 and 14, counting from the 5′-end of the antisense strand, the dsRNA comprises 8 or less (e.g., 8, 7, 6, 5, 4, 3, 2, 1 or 0) non-2′OMe nucleotides. For example, in any one of the embodiments of the invention when the antisense comprises two deoxy nucleotides and said nucleotides are at positions 2 and 14, counting from the 5′-end of the antisense strand, the dsRNA comprises 0, 1, 2, 3, 4, 5, 6, 7 or 8 non 2′-OMe nucleotides.

In another aspect, the invention further provides a method for delivering the dsRNA molecule of the invention to a specific target in a subject by subcutaneous or intravenous administration. The invention further provides the dsRNA molecules of the invention for use in a method for delivering said agents to a specific target in a subject by subcutaneous or intravenous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1-4 show in vivo efficacy of some exemplary dsRNAs of the invention in mice.

FIG. 5-8 show in vivo efficacy of some exemplary dsRNA of the invention in non-human primates.

DETAILED DESCRIPTION

In one aspect, the invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting expression of a target gene. Without limitations, the dsRNA agents of the invention can be substituted for the dsRNA molecules and can be used in RNA interference based gene silencing techniques, including, but not limited to, in vitro or in vivo applications.

Generally, the dsRNA molecule comprises a sense strand (also referred to as passenger strand) and an antisense strand (also referred to as guide strand). Each strand of the dsRNA molecule can range from 15-35 nucleotides in length. For example, each strand can be between, 17-35 nucleotides in length, 17-30 nucleotides in length, 25-35 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. Without limitations, the sense and antisense strands can be equal length or unequal length. For example, the sense strand and the antisense strand independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

In some embodiments, the antisense strand is of length 15-35 nucleotides. In some embodiments, the antisense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some particular embodiments, the antisense strand is 23 nucleotides in length.

Similar to the antisense strand, the sense strand can be, in some embodiments, 15-35 nucleotides in length. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some particular embodiments, the sense strand is 21 nucleotides in length.

In some embodiments, the sense strand can be 15-35 nucleotides in length, and the antisense strand can be independent from the sense strand, 15-35 nucleotides in length. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length, and the antisense strand is independently 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some particular embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

The sense strand and antisense strand typically form a double-stranded or duplex region. Without limitations, the duplex region of a dsRNA agent described herein can be 12-35 nucleotide pairs in length. For example, the duplex region can be between 14-35 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotides in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length. In some preferred embodiments, the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.

As described herein, the dsRNA agent can comprise one or more non-natural nucleotides. For example, the dsRNA agent comprises no non-natural nucleotides or comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides. For clarification, by a “natural nucleotide” is meant a 2′-deoxy, 2′-OH, or 2′-OMe nucleotide with a nucleobase selected from adenine, guanine, cytosine, uracil, and thymine. In other words, a natural nucleotide has nucleobase selected from adenine, guanine, cytosine, uracil, and thymine, and a sugar selected from a 2′-deoxy, 2′-OH, or 2′-OMe ribose. By a “non-natural nucleotide” is meant a nucleotide having a nucleobase other than adenine, guanine, cytosine, uracil, or thymine, and/or a sugar other than a 2′-deoxy, 2′-OH, or 2′-OMe ribose. For clarity, when a non-natural nucleotide has a 2′-deoxy, 2′-OH, or 2′-OMe ribose sugar, then the nucleobase is not adenine, guanine, cytosine, uracil, or thymine.

Exemplary nucleobases for the non-natural nucleotide include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyl adenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

In some embodiments, nucleobase for the non-natural nucleotide is selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N⁶-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N⁶-(isopentyl)adenine, N⁶-(methyl)adenine, N⁶, N⁶-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N⁴-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N³-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)i socarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N²-substituted purines, N⁶-substituted purines, O⁶-substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

Some exemplary non-natural nucleotides include, but are not limited to, acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

Thus, in some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides. In some embodiments, the sense and antisense strand the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably the sense strand and the antisense strand are independently 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, more preferably, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

Central Region

As described herein, the dsRNA can comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand and/or the antisense strand. As used herein, a “central region” of a strand refers to positions 5-17, e.g., positions 6-16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5′-end of the strand. For example, the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand. A preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5′-end of the sense strand. A more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand. A preferred central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 15 16 and 17, counting from 5′-end of the antisense strand. A more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand.

Accordingly, at least one of the sense stand and the antisense can comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modification in positions 5-17, e.g., positions 6-16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5′-end of the sense strand or the antisense strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14 (preferably positions 7, 8, 9, 10, 11, 12 and 13) of the sense strand, counting from the 5′-end of the sense strand, and/or at positions 9, 10, 11, 12, 13, 14, 15 16 and 17 (preferably positions 10, 11, 12, 13, 14, 15 and 16) of the antisense strand counting from 5′-end of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the sense comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand. In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 7, 8, 9, 10, 11, 12 and 13 of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15 and 16 of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

The antisense strand comprises one at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand, and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand

As used herein, a “non-central region” means a region of a strand that is not a central region. For example, the non-central region can be a terminal region, e.g., 1, 2, 3, 4, 5 or 6 nucleotides from either end of the strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least one 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15, and 16 of the antisense strand, and at least one 2′-deoxy in positions 1, 2, 3, 4, 5 or 6 from either one of the 5′-end or the 3′-end. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the antisense strand comprises at least five 2′-deoxy modifications. For example, the antisense strand comprises at least five 2′-deoxy modifications and wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14, counting from 5′-end of the antisense strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense strand comprises at least five, at least six, at least seven or more, 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5-′end of the antisense strand. In some embodiments, the antisense strand is 18-23 nucleotides in length and comprises at least five 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5′-end of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises at least three 2′-deoxy modifications, wherein at least two of the 2′-deoxy modifications are in the antisense strand and at least one of the 2′-deoxy modification is in the sense strand. For example, the antisense strand comprises at least two 2′-deoxy modifications and the sense strand comprises at least one 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at position 11 of the sense strand, counting from 5′-end of the sense strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein at least two of the 2′-deoxy modifications are in the antisense strand, and at least one of the 2′-deoxy modification is in the sense strand. In some embodiments, the antisense strand comprises at least two 2′-deoxy modifications and the sense strand comprises at least one 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at position 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises at least five 2′-deoxy modifications, wherein at least three of the 2′-deoxy modifications are in the antisense strand and at least two of the 2′-deoxy modifications are in the sense strand. For example, the antisense strand comprises at least three 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein at least three of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the antisense strand comprises at least three 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises at least seven 2′-deoxy modifications, wherein at least five of the 2′-deoxy modifications are in the antisense strand and at least two of the 2′-deoxy modification are in the sense strand. For example, the antisense strand comprises at least five 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein at least five of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the antisense strand comprises at least five 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

A wide variety of entities can be coupled to the dsRNA agents described herein. Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether. Generally, a ligand alters the distribution, targeting or lifetime of the molecule, e.g., a dsRNA described herein, into which it is incorporated. In some embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Ligands providing enhanced affinity for a selected target are also termed targeting ligands herein.

Some ligands can have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In some embodiments, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972, which is incorporated by reference in its entirety), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586, which is incorporated by reference in its entirety), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68, which is incorporated by reference in its entirety). In some embodiments, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

Ligands can improve transport, hybridization, and specificity properties and can also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer. Table 2 shows some examples of targeting ligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelating agent (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, j aplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake of the dsRNA into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or gamma interferon.

In some embodiments, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In some embodiments, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL) and high-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 1). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 2)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 3)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 4) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-94, 1991, which is incorporated by reference in its entirety). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002, which is incorporated by reference in its entirety). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001, which is incorporated by reference in its entirety). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing αvβ₃(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, which is incorporated by reference in its entirety). Peptides that target markers enriched in proliferating cells can be used. For example, RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type ligands that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003, which is incorporated by reference in its entirety).

In some embodiments, a targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number of helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, (3, or y peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, or an aptamer. A cluster is a combination of two or more sugar units. The targeting ligands also include integrin receptor ligands, Chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligands can also be based on nucleic acid, e.g., an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulator include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g. oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).

In addition, aptamers that bind serum components (e.g. serum proteins) are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S. patent applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which are incorporated by reference in their entireties for all purposes.

When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In a preferred embodiment, all the ligands have different properties.

Ligands can be coupled to the dsRNA at various places, for example, 3′-end, 5′-end, and/or at an internal position of the sense and/or antisense strand. In preferred embodiments, the ligand is attached to the sense and/or antisense strand of the dsRNA via a linker or tether. The ligand or tethered ligand can be present on a monomer when said monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated via coupling to a “precursor” monomer after said “precursor” monomer has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_nNH₂may be incorporated into a growing oligonucleotide strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having complementary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.

The ligands can be attached to one or both strands. In some embodiments, a dsRNA described herein comprises a ligand conjugated to the sense strand. In some embodiments, a dsRNA described herein comprises a ligand conjugated to the antisense strand.

In some embodiments, ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic linkages of nucleic acid molecules. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2′, 3′, and 5′ carbon atoms. The 1′ position can also be attached to a conjugate moiety, such as in an abasic residue. Internucleosidic linkages can also bear conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

In some embodiments, the ligand is conjugated to the sense strand. As described herein, the ligand can be conjugated at the 3′-end, 5′-end or at an internal position of the sense strand. In some embodiments, the ligand is conjugated to the 3′-end of the sense strand. Further, the ligand can be conjugated to a nucleobase, sugar moiety or internucleotide linkage of the sense strand.

Any suitable ligand in the field of RNA interference may be used, although the ligand is typically a carbohydrate e.g. monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more carbohydrates, e.g., GalNAc (N-acetylgalactosamine) derivatives attached through a monovalent, bivalent or trivalent branched linker.

In some embodiments, the dsRNA of the invention is conjugated to a bivalent and trivalent branched linkers include the structures shown in any of Formula (IV)-(VII):

embedded image

wherein:

q^2A, q^2B, q^3A, q^3B, q^4A, q^4B, q^5A, q^5Band q^5Crepresent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^2A, P^2B, P^3A, P^3B, P^4A, P^4B, P^5A, P^5B, P^5C, T^2A, T^2B, T^3A, T^3B, T^4A, T^4B, T^5A, T^5B, T^5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^2A, Q^2B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^5B, Q^5Care independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^N), C(R′)═C(R″), C≡C or C(O);

R^2A, R^2B, R^3A, R^3B, R^4A, R^4B, R^5A, R^5B, R^5Care each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), —C(O)—CH(R^a)—NH—, CO, CH═N—O,

embedded image

or heterocyclyl;

L^2A, L^2B, L^3A, L^3B, L^4A, L^4B, L^5A, L^5B, L^5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and

R^ais H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for use with dsRNA agents described herein for inhibiting the expression of a target gene, such as those of Formula (VII):

embedded image

wherein L^5A, L^5Band L^5Crepresent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the following compounds:

embedded image

In some embodiments, a dsRNA described herein comprises Ligand 1, i.e., a ligand having the following structure:

embedded image

In some embodiments, a dsRNA described herein comprises a ligand described in U.S. Pat. No. 5,994,517 or 6,906,182, content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the ligand can be a tri-antennary ligand described in FIG. 3 of U.S. Pat. No. 6,906,182. For example, a dsRNA described herein can comprise a ligand selected from the following tri-antennary ligands:

embedded image

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

In one embodiment the dsRNA molecule of the invention is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end or both ends. For instance, the ligand can be conjugated to the sense strand, in particular, the 3′-end of the sense strand.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); and wherein the sense strand does not comprise a glycol nucleic acid. In some embodiments, the sense and antisense strand the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably the sense strand and the antisense strand are independently 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, more preferably, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); and wherein the sense strand does not comprise a glycol nucleic acid (GNA). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense and antisense strand the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably the sense strand and the antisense strand are independently 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, more preferably, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense and antisense strand the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably the sense strand and the antisense strand are independently 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, more preferably, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14 (preferably positions 7, 8, 9, 10, 11, 12 and 13) of the sense strand, counting from the 5′-end of the sense strand, and/or at positions 9, 10, 11, 12, 13, 14, 15 16 and 17 (preferably positions 10, 11, 12, 13, 14, 15 and 16) of the antisense strand counting from 5′-end of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14 (preferably positions 7, 8, 9, 10, 11, 12 and 13) of the sense strand, counting from the 5′-end of the sense strand, and/or at positions 9, 10, 11, 12, 13, 14, 15 16 and 17 (preferably positions 10, 11, 12, 13, 14, 15 and 16) of the antisense strand counting from 5′-end of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14 (preferably positions 7, 8, 9, 10, 11, 12 and 13) of the sense strand, counting from the 5′-end of the sense strand, and/or at positions 9, 10, 11, 12, 13, 14, 15 16 and 17 (preferably positions 10, 11, 12, 13, 14, 15 and 16) of the antisense strand counting from 5′-end of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the sense strand comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 7, 8, 9, 10, 11, 12 and 13 of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 7, 8, 9, 10, 11, 12 and 13 of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the sense comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 7, 8, 9, 10, 11, 12 and 13 of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the antisense strand comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15, and 16 of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15, and 16 of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least two 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15 and 16 of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand, and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least one 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15, and 16 of the antisense strand, and at least one 2′-deoxy in positions 1, 2, 3, 4, 5 or 6 from either one of the 5′-end or the 3′-end. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least one 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15, and 16 of the antisense strand, and at least one 2′-deoxy in positions 1, 2, 3, 4, 5 or 6 from either one of the 5′-end or the 3′-end. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in a central region of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least one 2′-deoxy modifications in a central region, e.g., positions 10, 11, 12, 13, 14, 15, and 16 of the antisense strand, and at least one 2′-deoxy in positions 1, 2, 3, 4, 5 or 6 from either one of the 5′-end or the 3′-end. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the antisense strand comprises at least five, at least six, at least seven or more, 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5-′end of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-23 nucleotides in length and comprises at least five 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5′-end of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the antisense strand comprises at least five, at least six, at least seven or more, 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5-′end of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-23 nucleotides in length and comprises at least five 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5′-end of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the antisense strand comprises at least five, at least six, at least seven or more, 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5-′end of the antisense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand is 18-23 nucleotides in length and comprises at least five 2′-deoxy modifications, e.g., at positions 2, 5, 7, 12 and 14, counting from 5′-end of the antisense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein at least two of the 2′-deoxy modifications are in the antisense strand, and at least one of the 2′-deoxy modification is in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least two 2′-deoxy modifications and the sense strand comprises at least one 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at position 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein at least two of the 2′-deoxy modifications are in the antisense strand, and at least one of the 2′-deoxy modification is in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least two 2′-deoxy modifications and the sense strand comprises at least one 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at position 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein at least two of the 2′-deoxy modifications are in the antisense strand, and at least one of the 2′-deoxy modification is in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least two 2′-deoxy modifications and the sense strand comprises at least one 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at position 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein at least three of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the antisense strand comprises at least three 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein at least three of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least three 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein at least three of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least three 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least seven 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of between 18 to 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein at least five of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modification is in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least five 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least seven 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII); wherein the sense strand does not comprise a glycol nucleic acid; and wherein at least five of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein. In some embodiments, the antisense strand comprises at least five 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modification, wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In some embodiments, the dsRNA comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5′ end of the antisense strand; at least three, four, five or six 2′-deoxy modifications on the sense and/or antisense strands; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA molecule comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein at least five of the 2′-deoxy modifications are in the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand. In some embodiments, the antisense strand comprises at least five 2′-deoxy modifications and the sense strand comprises at least two 2′-deoxy modifications, wherein the 2′-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5′-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5′-end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the non-natural nucleotides are selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the antisense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); and wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand); wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the sense strand does not comprise a glycol nucleic acid.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; and wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; and wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; and wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′ end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the sense strand does not comprise a glycol nucleic acid; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; wherein the dsRNA comprises at least two, e.g., at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, preferably positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5′-end of the sense strand), and at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2′-deoxy modifications in the central region of the sense strand (e.g., at positions 9, 10, 11, 12, 13, 14, 15 16 and 17, preferably positions 10, 11, 12, 13, 14, 15 and 16, counting from 5′-end of the antisense strand), and at least one 2′-deoxy modification in a non-central region, e.g., within 1, 2, 3, 4, 5 or 6 nucleotides from either 5′-end and/or 3′-end of the antisense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; antisense strand comprises at least two 2′-deoxy modifications at positions 2 and 14 of the antisense strand, counting from wherein the sense strand comprises at least one 2′-deoxy modification at position 11, counting from 5′-end of the sense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; antisense strand comprises at least three 2′-deoxy modifications at positions 2, 12 and 14 of the antisense strand, counting from wherein the sense strand comprises at least two 2′-deoxy modifications at positions 9 and 11, counting from 5′-end of the sense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; antisense strand comprises at least three 2′-deoxy modifications at positions 2, 12 and 14 of the antisense strand, counting from wherein the sense strand comprises at least two 2′-deoxy modifications at positions 9 and 11, counting from 5′-end of the sense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; antisense strand comprises at least five 2′-deoxy modifications at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from wherein the sense strand comprises at least two 2′-deoxy modifications at positions 9 and 11, counting from 5′-end of the sense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

In some embodiments, the invention provides a dsRNA comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, e.g., independently 17-30 nucleotides in length, independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, preferably independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; at least two phsophorothioate internucleotide linkages between the first five nucleotides, counting from the 5′end of the antisense strand; antisense strand comprises at least five 2′-deoxy modifications at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from wherein the sense strand comprises at least two 2′-deoxy modifications at positions 9 and 11, counting from 5′-end of the sense strand; wherein the dsRNA molecule has a double stranded (duplex) region of 18, 19, 21, 22, 23, 24 or 25 base pairs; wherein the sense strand does not comprise a glycol nucleic acid; the dsRNA comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or the dsRNA agent comprises all natural nucleotides; and wherein the dsRNA molecule comprises a ligand, e.g., a ligand of any one of Formula (IV)-(VII). In some embodiments, the ligand binds with or targets a liver cell or receptor, e.g., the ligand binds with or target the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of Formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from the Ligands 1-8 disclosed herein.

Overhangs and Blunt Ends

In some embodiments, the dsRNA molecule of the invention comprises one or more overhang regions and/or capping groups of dsRNA molecule at the 3′-end, or 5′-end or both ends of a strand. The overhang can be 1-10 nucleotides in length. For example, the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In some embodiments, the overhang is 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target sequence or it can be complementary to the gene sequences being targeted or it can be the other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In some embodiments, the nucleotides in the overhang region of the dsRNA molecule of the invention can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2′-Fluoro 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine, 2′-O-methoxyethyladenosine, 2′-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h′ GNA, and any combinations thereof. For example, dTdT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule of the invention may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3′-end of the sense strand, antisense strand or both strands. In some embodiments, this 3′-overhang is present in the antisense strand. In some embodiments, this 3′-overhang is present in the sense strand.

The dsRNA molecule of the invention may comprise only a single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability. For example, the single-stranded overhang is located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The dsRNA can also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa.

Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process. For example, the single overhang is at least one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in length. In some embodiments, the dsRNA has a 2 nucleotide overhang on the 3′-end of the antisense strand and a blunt end at the 5′-end of the antisense strand.

Modified Nucleotides

The dsRNA of the invention can comprise one or more modified nucleotides. For example, every nucleotide in the sense strand and antisense strand of the dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar; replacement of the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a central region, may only occur at a non-terminal t region, or may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide.

In some embodiments, the dsRNA molecule of the invention comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNA molecule of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

The dsRNA molecule of the invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5 or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3 or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the dsRNA molecule of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s).

In some embodiments, the dsRNA molecule of the invention further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In some embodiments, compound of the invention comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

In some embodiments, compound of the invention comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

In some embodiments, compound of the invention comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.

In some embodiments, compound of the invention comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

Various publications describe multimeric siRNA which can all be used with the dsRNA of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 which are hereby incorporated by their entirely.

5′-Modifications

In some embodiments dsRNA molecules of the invention are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)₂(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). In one example, the modification can in placed in the antisense strand of a dsRNA molecule.

Thermally Destabilizing Modifications.

The dsRNA agents of the invention can comprise thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. Without wishing to be bound by a theory, dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, thermally destabilizing modification of the duplex is located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at position 5, 6, 7 or 8 from the 5′-end of the antisense strand.

In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, 6, 7, 8 or 9 from the 5′-end of the antisense strand.

The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA). For example, the thermally destabilizing modifications can include, but are not limited to, mUNA and GNA building blocks as follows:

embedded image

In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5′-mUNA, 4′-mUNA, 3′-mUNA, and 2′-mUNA.