Double stranded rnai agents, compositions and methods of use

Abstract

Disclosed are, inter alia, therapeutic oligonucleotides for use in silencing a target gene in a cell or in a subject.

Description

PAT059650-WO-PCT DOUBLE STRANDED RNAi AGENTS, COMPOSITIONS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority and benefit to the U.S. Patent Application No. 63 / 670,337 filed July 12, 2024, and U.S. Patent Application No.63 / 724,713 filed November 25, 2024, the disclosure each of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Therapeutic oligonucleotides have been used to silencing a target gene in a cell or in an organism. Among others, an RNA interference (“RNAi”) agent having short length (e.g., about 20 to 25 nucleotides base pairs) double-stranded RNA (dsRNA) can inhibit gene expression by RNA-induced silencing complex (RISC) machinery to process (e.g., degeneration or degradation) messenger RNAs (mRNA) of the target gene. In this process, proficiency of activation of RISC process and following gene silencing may depend on how a passenger strand (sense strand) and a guide strand are designed for optimal metabolic stability and optimal loading to the RISC and cleavage (e.g., cleavage of the passenger strand or target mRNA) in the RISC.

[0003] Therefore, there are needs for a novel design for dsRNAs that can provide good gene-silencing properties for drug development based on RNA interference (RNAi). SUMMARY OF THE INVENTION

[0004] Provided herein are, inter alia, compounds that can inhibit expression of a target gene in a subject, e.g., human. The compounds include double stranded RNA that inhibits expression of a target gene in a subject, thereby suppressing a disease or disorder associated with the target gene.

[0005] In an aspect, provided is a double stranded RNAi (dsRNAi) agent for inhibiting expression of a target gene comprising: (i) a sense strand comprising monomers of N1 to N23 and having the Formula (III): 5′-N1-N2-N3-N4-N5-N6-N7-N8-N9-N10-N11-N12-N13-N14-N15-N16-N17-N18-N19-N20-N21- N22-N23-3′ (III), or a pharmaceutically acceptable salt thereof, wherein: N1 has a structure of Formula (A-1),PAT059650-WO-PCT a pharmaceutically acceptable salt thereof; a structure of Formula (B-1),a pharmaceutically acceptable salt thereof; of Formula (B-1),a pharmaceutically acceptable salt thereof; and, a pharmaceutically acceptable salt thereof; and(ii) an antisense strand comprising monomers of N1′to N25′and having the Formula (IV): 5′-N1′-N2′-N3′-N4′-N5′-N6′-N7′-N8′-N9′-N10′-N11′-N12′-N13′-N14′-N15′-N16′-N17′-N18′-N19′- N20′-N21′-N22′-N23′-N24′-N25′-3′ (IV), or a pharmaceutically acceptable salt thereof, wherein: N1′ has a structure of Formula (A-1),PAT059650-WO-PCT a pharmaceutically acceptable salt thereof; a structure of Formula (B-1),a pharmaceutically acceptable salt thereof; acid (GNA), or has a structure, Formula (B-2),N25′independently has a structure of Formula (C-1), a pharmaceutically acceptable salt thereof;2), and (C-1): in each occurrence, B is independently absent or a nucleobase; in each occurrence, each R21, R22, R23, and R24is independently hydrogen, halogen, - ORA, -SRA, -NRBRC, -C(O)RD, -OC(O)RD, -C(O)NRBRC, -NREC(O)RD, -PAT059650-WO-PCT NREC(O)ORD, -NREC(O)NRBRC, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl, wherein each RA, RB, RC, RD, and RFis independently hydrogen, or C1-C10 alkyl optionally substituted with halogen, -OH, -O-C1-4alkyl, or -O-C1-4haloalkyl; in each occurrence, R25is hydrogen, -OH, a detectable moiety, a first ligand, or a functional group capable of forming a covalent bond with a first ligand; in each occurrence, R26is hydrogen, a detectable moiety, -P(O)(OH)-OH, -P(O)(SH)- OH, a second ligand, or a functional group capable of forming a covalent bond with a second ligand; in each occurrence, L21is independently absent, -O-, -O-L22-, -P(O)(OH)-O-L22-, - P(O)(SH)-O-L22-, -P(O)(OH)-L23-, or -P(O)(SH)-L23-, wherein each L22is independently a substituted or unsubstituted C1-C4 alkylene and each L23is independently a substituted or unsubstituted C2-C4 alkenylene, and in each occurrence, is an attachment point to the adjacent monomers, provided that:(i) in at least one of N1 and N23, R22is -O-CH2CH2-OCH3, (ii) in N1′, R22is not -O-CH2CH2-OCH3,(iii) in N3 to N21, (a) R22is not -O-CH2CH2-OCH3, (b) at least one of R22is not hydrogen, and (c) in no more than six nucleotides, R22is -F, and (iv) no more than eight monomers from N3 to N21 are absent and no more than ten monomers from N3′to N23′are absent.

[0006] In some embodiments, N1has a structure of , or a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0007] In some embodiments, N23 has a , or apharmaceutically acceptable salt thereof.

[0008] In some embodiments, N1 has a apharmaceutically acceptable salt thereof, andapharmaceutically acceptable salt

[0009] In some embodiments, each N2 and N22 independently has a structure of a pharmaceutically acceptable salt thereof.has a structure ofPAT059650-WO-PCT a pharmaceutically acceptable, or a pharmaceutically acceptable salt thereof;-SH or -OH.

[011] In some

[0012] In some, or a pharmaceutically acceptable salt thereof.

[0013] In some embodiments, one to six monomers from N3to N21are absent.PAT059650-WO-PCT

[0014] In some embodiments, two monomers from N3 to N21 are absent.

[0015] In some embodiments, one to eight monomers from N3′to N23′are absent.

[0016] In some embodiments, two monomers from N3′ to N23′ are absent.

[0017] In some embodiments, N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4wherein n is an integer from 3 to 17, and in two, three or four monomers of Nn, Nn+2, Nn+3, and Nn+4 and n is an integer from 3 to 17, R22is -F.

[0018] In some embodiments, N3 to N21 include Nn, Nn+1, Nn+2, Nn+3, and Nn+4 wherein n is an integer from 5 to 10, and in two, three or four monomers of Nn, Nn+2, Nn+3, and Nn+4when n is an integer from 5 to 10, R22is -F.

[0019] In some embodiments, N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4wherein n is an integer from 3 to 17, wherein: in each monomer of Nn, Nn+2, and Nn+3, R22is -F and in Nn+4, R22is hydrogen.

[0020] In some embodiments, N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4wherein n is an integer from 5 to 10, wherein: in each monomer of Nn, Nn+2, and Nn+3, R22is -F and in Nn+4, R22is hydrogen.

[0021] In some embodiments, in only one monomer from N3′ to N23′, R22is -F, and in two to four monomer from N3′to N23′, R22is -H.

[0022] In some embodiments, in two to six monomers from N3′ to N23′, R22is -F.

[0023] In some embodiments, in more than six monomers from N3′to N23′, R22is -F.

[0024] In some embodiments, in N3 to N21, if present, R22is independently hydrogen, -F, - OH, -OCH3, -OCH2CH3,-CH3, -CH2CH3,-C(O)OH, -C(O)NH2, and -NH2.

[0025] In some embodiments, in N3 to N21, if present, R22is independently hydrogen, -F, or - OCH3.

[0026] In some embodiments, in N3 to N21, if present, L21is independently -P(O)(OH)-O- CH2-, or -P(O)(SH)-O-CH2-.

[0027] In some embodiments, N2 and N3 are present and in N2 and N3, L21is independently - P(O)(SH)-O-CH2-.

[0028] In some embodiments, N22 and N23 are present and in N22 and N23, L21is independently -P(O)2-O-CH2- or -P(O)(SH)-O-CH2-.PAT059650-WO-PCT

[0029] In some embodiments, in two to six of N3 to N21, L21are independently -P(O)(SH)- O-CH2-.

[0030] In some embodiments, in N2′, R22is -F or -OCH3.

[0031] In some embodiments, one or two of N3′to N23′are each a glycol nucleic acid (GNA).

[0032] In some embodiments, one or two of N3′to N23′each independently have a structure of Formula (B-1), , wherein R22is hydrogen, or a pharmaceutically acceptablesome one or two of N3′to N23′in the antisense strand of Formula (IV) each have a structure of Formula (B-2), a pharmaceutically acceptable salt thereof. N to N , if presen212′ 25′t, L is independently -P(O)(OH)-O- CH2-, or -P(O)(SH)-O-CH2-.

[0035] In some embodiments, N2′and N3′are present and in N2′and N3′,L21is independently -P(O)(SH)-O-CH2-.

[0036] In some embodiments, N24′and N25′are present and in N24′and N25′, L21is independently -P(O)(SH)-O-CH2-.

[0037] In some embodiments, in two to six of N3′to N23′, L21are independently -P(O)(SH)- O-CH2-.

[0038] In some embodiments, R21, R23, R24are hydrogen.

[0039] In an aspect, the disclosure also provides a double stranded RNAi (dsRNAi) agent for inhibiting expression of a target gene, comprising:PAT059650-WO-PCT (i) a sense strand comprising monomers of Y1 to Y23 and having a Formula (I′); 5′-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15-Y16-Y17-Y18-Y19-Y20-Y21- Y22-Y23-3′ (I′) or a pharmaceutically acceptable salt thereof, and (ii) an antisense strand comprising monomers of Y1′ to Y25′ and having a Formula (II′): 5′-Y1′-Y2′-Y3′-Y4′-Y5′-Y6′-Y7′-Y8′-Y9′-Y10′-Y11′-Y12′-Y13′-Y14′-Y15′-Y16′-Y17′-Y18′-Y19′- Y20′-Y21′-Y22′-Y23′-Y24′-Y25′-3′ (II′), or a pharmaceutically acceptable salt thereof, wherein: each Y1, Y2, Y22, and Y23is independently a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′- halo modified nucleotide, a 2′-amino modified nucleotide, a 2′-aminoalkyl modified nucleotide, a locked RNA (LNA), or a glycol motif nucleotide (GNA), wherein each alkyl, amino, or amino alkyl may be substituted or unsubstituted; Y3to Y21and Y1′to Y25′, are each, independently absent, a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, a 2′- aminoalkyl modified nucleotide, a locked RNA (LNA), or a glycol motif nucleotide (GNA), wherein each alkyl, amino, or amino alkyl may be substituted or unsubstituted; provided that: (i) at least one of Y1 and Y23 are 2′-O-methoxyethyl (MOE) modified nucleotides; (ii) Y1′is not 2′-MOE modified nucleotide; (iii) in Y3 to Y21, (a) none of Y3to Y21are 2′-MOE modified nucleotide, (b) at least one of Y3 to Y21 is not deoxynucleotide (DNA), and (c) no more than six monomers are 2′-F modified nucleotides; and (iv) no more than eight monomers from Y3 to Y21 are absent and no more than ten monomers from Y3′to Y23′are absent.

[0040] In some embodiments, Y1 and Y23 are 2′-MOE modified nucleotides.

[0041] In some embodiments, Y2and Y22are 2′-MOE modified nucleotides.PAT059650-WO-PCT

[0042] In some embodiments, each Y3 to Y21 and Y1′ to Y25′ is independently absent, a 2′-F modified nucleotide, a 2′-O-methyl (OMe) modified nucleotide, an abasic nucleotide, RNA, DNA, GNA, or LNA.

[0043] In some embodiments, Y1′comprises a 5′-(E)-vinyl phosphonate group.

[0044] In some embodiments, one to six monomers from Y3 to Y21 are absent.

[0045] In some embodiments, two monomers from Y3to Y21are absent.

[0046] In some embodiments, one to eight monomers from Y3′ to Y23′ are absent.

[0047] In some embodiments, two monomers from Y3′to Y23′are absent.

[0048] In some embodiments, Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4 wherein n is an integer from 3 to 17, and two, three or four monomers of Yn, Yn+2, Yn+3, and Yn+4are 2′- F modified nucleotides.

[0049] In some embodiments, Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4 wherein n is an integer from 5 to 10, and (i) Yn, Yn+2, Yn+3, and Yn+4 are 2′-F modified nucleotides, or (ii) Yn+2, Yn+3, and Yn+4are 2′-F modified nucleotides.

[0050] In some embodiments, Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4 wherein n is an integer from 3 to 17, and Yn, Yn+2, and Yn+3are 2′-F modified nucleotides, and Yn+4is a DNA.

[0051] In some embodiments, Y3to Y21comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4wherein n is an integer from 5 to 10, Yn, Yn+2, and Yn+3 are 2′-F modified nucleotides, and Yn+4 is a DNA.

[0052] In some embodiments, one to six monomers from Y3′ to Y23′ are 2′-F modified nucleotides.

[0053] In some embodiments, more than six monomers from Y3′ to Y23′ are 2′-F modified nucleotides.

[0054] In some embodiments, one or two of Y3′ to Y23′ are each a GNA.

[0055] In some embodiments, one to four of Y3′to Y23′each independently have a DNA.

[0056] In some embodiments, one or two of Y3′ to Y23′ each independently are each a TNA.

[0057] In some embodiments, Y1and Y2is connected via a phosphorothioate linkage.

[0058] In some embodiments, Y2 and Y3 is connected via a phosphorothioate linkage.

[0059] In some embodiments, Y21and Y22is connected via a phosphate or phosphorothioate linkage.PAT059650-WO-PCT

[0060] In some embodiments, Y22 and Y23 is connected via a phosphate or phosphorothioate linkage.

[0061] In some embodiments, at least one to six monomers between Y3 and Y21 are connected via a phosphorothioate linkage.

[0062] In some embodiments, Y1′ and Y2′ is connected via a phosphorothioate linkage.

[0063] In some embodiments, Y2′and Y3′is connected via a phosphorothioate linkage.

[0064] In some embodiments, Y23′ and Y24′ is connected via a phosphorothioate linkage.

[0065] In some embodiments, Y24′and Y25′is connected via a phosphorothioate linkage.

[0066] In some embodiments, at least one to six monomers between Y3′ and Y24′ are connected via a phosphorothioate linkage.

[0067] In some embodiments, the dsRNAi agent as described herein further comprises one or more ligands and each ligand comprises a GalNAc moiety.

[0068] In some embodiments, the first ligand and / or the second ligand comprise a GalNAc moiety.

[0069] In some embodiments, the GalNAc moiety has a structure of: ,each LAis an independently a linker which may be same or different in each occurrence; LBis a linker; m is an integer from 1 to 3; and is an attachment point to the sense strand or antisense strand.

[0070] In some embodiments, the GalNAc moiety comprises the following structure ofPAT059650-WO-PCT ,Each p1, p2, p3, q1, q2, r1, r2 and r3 is independently an integer from 0 to 12; Each m1, m2, and m3 is independently an integer from 1 to 3; and “*” is an attachment point to LB.

[0071] In some embodiments, the GalNAc moiety has a structure of: ,PAT059650-WO-PCT wherein: each LC, LD, LE, LF, and LGis an independently a linker; LBis a linker as described above; is an attachment point to the sense strand or the antisense strand, or to a conjugate linker conjugated to the sense strand or the antisense strand.

[0072] In some embodiments, the ligand has a structure of: , ,each p11 and q11 is independently an integer from 0 to 12; each z1, z2, and z3 is independently an integer of 0 to 12; and is an attachment point to the sense strand or the antisense strand, or to a conjugate linker conjugated to the sense strand or the antisense strand.

[0073] In some embodiments, the GalNAc moiety comprises the following structure:PAT059650-WO-PCT , ,

[0074] In some embodiments, the dsRNAi agent is in a pharmaceutically acceptable salt form.PAT059650-WO-PCT

[0075] In some embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0076] In some embodiments, the dsRNAi agent has improved half-life relative to a reference dsRNAi agent that does not contain chemical modification in nucleotides.

[0077] In certain aspects, the disclosure provides a pharmaceutical composition comprising the dsRNAi agent as described herein.

[0078] In certain aspects, the disclosure provides a method of inhibiting expression of a target gene in a subject comprising administering to the subject a therapeutically effective amount of the dsRNAi agent as described herein, or the pharmaceutical composition as described herein.

[0079] In certain aspects, the disclosure provides a method of treating or preventing a disorder or disease associated with a target gene in a subject, and the method comprises administering to the subject a therapeutically effective amount of the dsRNAi agent as described herein, or the pharmaceutical composition as described herein, wherein the dsRNAi agent inhibits the expression of the target gene.

[0080] In some embodiments, the dsRNAi agent or the pharmaceutical composition is administered subcutaneously or intravenously.

[0081] In some embodiments, the subject is a human.

[0082] In certain aspects, the disclosure provides a kit comprising the dsRNAi agent as described herein, or the pharmaceutical composition as described herein.

[0083] Other aspects of the invention are disclosed infra. BRIEF DESCRIPTION OF DRAWINGS

[0084] Figure 1: Compounds 1-4 as outlined in Table 1 of the disclosure are depicted and each modification (e.g., 2′ modified nucleosides and linkages) are depicted. “PO” means a chemical group to form a phosphate linkage and “PS” means linking group to form a phosphorothioate linkage, and “L96” refers to a ligand that is connected to the 3′-end of the sense strand via phosphate (phosphodiester) linkage. Figure discloses SEQ ID NOS 139-146, respectively, in order of appearance.

[0085] Figures 2A to 2B: Liver HMGCR mRNA concentrations for male C57BL / 6 mice (n=4 or 5 per group) administered a single subcutaneous dose of PBS (white), Compound 5 (white / black), Compound 6 (black) or Compound 7 (gray) at 3 mg / kg are shown in Figure 3A. The siRNAs share a same nucleotide sequence but have different chemical modifications. Statistical significance was determined by ordinary one-way ANOVA and Sidak′s multiple comparisons test. At day 35 post-dose, statistically significant reductions in hepatic HMGCRPAT059650-WO-PCT mRNA abundance versus vehicle (PBS) were observed in mice dosed with Compound 6 (- 55%, P<0.05 vs. PBS) and Compound 7 (-70%, P<0.01 vs. PBS). In mice dosed with Compound 7, liver HMGCR mRNA levels were significantly reduced vs. vehicle through day 56 post-dose (-51%, P<0.05). These results show durable HMGCR knockdown in mice, with Compound 7 performing better than Compound 6 in this experiment. Incorporation of guide strand into liver RISC over time is illustrated in Figure 2B. Male C57BL / 6 mice (n= 4 or 5 per group) were administered a single subcutaneous dose of PBS (white) or Compound 5 (white / black), Compound 6 (black) or Compound 7 (gray) at 3 mg / kg. Statistical significance was determined by ordinary one-way ANOVA and Sidak′s multiple comparisons test. Consistent with the liver HMGCR mRNA results, Compound 7 showed the highest RISC- loading at both timepoints evaluated. The difference observed between Compound 5 and Compound 7 at day 35 was robust and statistically significant (P<0.0001). These results show durable RISC-incorporation (through day 56 post-dose) for Compound 7 in mice.

[0086] Figure 3: HMGCR protein levels in a human liver cell line (Huh7) treated with Compound 1 at a concentration of 12.5 or 25 nM are provided in Figure 4. Briefly, Huh7 cells were transfected with test or control siRNA using lipofectamine and incubated at 37°C for 6 hours. After changing the culture media, cells were incubated for another 24 hours prior to processing for the measurement of HMGCR protein levels by Western blotting. Relative to the control siRNA, Compound 1 reduced HMGCR protein content by 51% and 73% at concentrations of 12.5 and 25 nM, respectively. These results demonstrate that Compound 1 significantly, and dose-dependently, reduces HMGCR protein content in a human liver cell line.

[0087] Figure 4: A target gene was suppressed in A431 cells (ATCC#CRL-1555) using different siRNA compounds in Table 3. Graphs show normalized.

[0088] Figures 5A-5B: Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D1, D2, D3, D4, D5, or D6 to humanized PCSK9 mice on (Figure 5A) plasma PCSK9 levels over time and (Figure 5B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 56 post-dose.

[0089] Figures 6A-6B: Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D7, D8, D9, or D10 to humanized PCSK9 mice on (Figure 6A) plasma PCSK9 levels over time and (Figure 6B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 77post-dose.PAT059650-WO-PCT

[0090] Figures 7A-7B: Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D1, D4, D11, or D12 to humanized PCSK9 mice on (Figure 7A) plasma PCSK9 levels over time and (Figure 7B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 63 post-dose.

[0091] Figures 8A-8B: Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D4, D14, D15, D16, or D17 to humanized PCSK9 mice on (Figure 8A) plasma PCSK9 levels over time and (Figure 8B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 77 post-dose.

[0092] Figures 9A-9B: Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D4, D13, D24, D25, D26, D27, or D28 to humanized PCSK9 mice on (Figure 9A) plasma PCSK9 levels over time and (Figure 9B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 63 post- dose.

[0093] Figures 10A-10B: Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D25, D29, D30, D31, D32, D33, or D34 to humanized PCSK9 mice on (Figure 10A) plasma PCSK9 levels over time and (Figure 10B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 64 post-dose.

[0094] Figures 11A-11B. Effects of a single 3 mg / kg subcutaneous administration of PCSK9 siRNAs D4, D15, D35, D36, D37, or D38 to humanized PCSK9 mice on (Figure 11A) plasma PCSK9 levels over time and (Figure 11B) liver human PCSK9 mRNA level, liver siRNA level, and liver RISC loading (i.e., the level of siRNA incorporated into RISC) on day 77 post-dose.

[0095] Figure 12 shows exemplary PCSK9 dsRNAi agents.

[0096] Figure 13: Effects of TNA at position 3 on the antisense strand in human HMGCR siRNAs (H1) by measuring HMGCR mRNA fold in human hepatocyte compared to siRNA not including TNA in the antisense strand..

[0097] Figures 14A-14B: Effects of MOE clamps in the sense strand of the AGT siRNAs (A2, A4) in comparison to the effects from the AGT siRNA without MOE clamps (A1, A3) and reference agent.

[0098] Figure 15: Effects of siRNAs targeting gene X at position A (“siRNA A”) and containing different modifications (FA-1 to FA-3) in suppressing target protein X (plasma protein) observed in transgenic mice expressing the human gene X.PAT059650-WO-PCT

[0099] Figures 16A-16C: Effects of siRNAs targeting gene X at position B (“siRNA B”) and containing different modifications (FB-1 to FB-9) in suppressing target protein X (plasma protein) observed in transgenic mice expressing the human gene X.

[0100] Figure 17: Effects of siRNAs targeting gene X at position C (“siRNA C”) and containing different modifications (FC-1 to FC-2) in suppressing target protein X (plasma protein) observed in transgenic mice expressing the human gene X. DETAILED DESCRIPTION DEFINITIONS

[0101] Unless defined otherwise, all technical terms, scientific terms, abbreviations, chemical structures, and chemical formulae used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise.

[0102] All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed.

[0103] Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. As used herein, the term "a,” "an,” "the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0104] Unless otherwise indicated, all numbers, values, and / or expressions referring to nucleotide lengths, inhibition, activities, dosages, contents, and formulations used herein are toPAT059650-WO-PCT be understood as modified in all instances by the term “about” as such numbers are inherently approximations that are reflective of, among other things, the various uncertainties of measurement encountered in obtaining such values. Further, unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the “mean. “About” may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0105] The term “nucleic acid” means a compound containing at least two nucleotide monomers covalently linked together. Nucleic acids include polynucleotides and oligonucleotides, including double-stranded oligonucleotides and single-stranded oligonucleotides, and modified versions thereof.

[0106] The term “nucleotide” as used herein means a compound or chemical group including (i) a nucleoside and (ii) a phosphate group, or a variant of phosphate group, e.g., phosphorothioate (PS) group or vinyl phosphonate (VP) group, which are covalently attached at 5′ position or 3′ position of the pentofuranosyl sugar (e.g., ribose or deoxyribose). In certain aspects, the nucleotide is a ribonucleotide (RNA) having the ribose as the pentofuranosyl sugar. In certain aspects, a nucleotide is a deoxyribonucleotide (DNA) having the deoxyribose (2′- deoxyribose) as the pentofuranosyl sugar. Unless otherwise specifically indicated, when referring a “nucleotide” in a chain of nucleotides (e.g., oligonucleotides), e.g., Y1 to Y23 and Y1′to Y25′, a nucleotide is meant by a nucleoside and a phosphate group that is covalently attached at 3′ position of the pentofuranosyl sugar (e.g., ribose or deoxyribose).

[0107] The term “nucleoside” means a monomer consisting of a nucleobase and a pentofuranosyl sugar (e.g., ribose or deoxyribose). A nucleoside including a ribose sugar ringhas to a a pharmaceutically acceptable salt thereof and anucleotide including a deoxyribose sugar ring has a , wherein in each structure, “Base” is a nucleobase.

[0108] The term “nucleobase” or “base,” as used herein, means the heterocyclic base moiety of a nucleoside or nucleotide. Non-limiting examples of nucleobases includes cytosinePAT059650-WO-PCT or a derivative thereof (e.g., cytosine analogue), guanine or a derivative thereof (e.g., guanine analogue), adenine or a derivative thereof (e.g., adenine analogue), thymine or a derivative thereof (e.g., thymine analogue), uracil or a derivative thereof (e.g., uracil analogue), hypoxanthine or a derivative thereof (e.g., hypoxanthine analogue), xanthine or a derivative thereof (e.g., xanthine analogue), 7-methylguanine or a derivative thereof (e.g., 7- methylguanine analogue), deaza-adenine or a derivative thereof (e.g., deaza-adenine analogue), deaza-guanine or a derivative thereof (e.g., deaza-guanine), deaza-hypoxanthine or a derivative thereof, 5,6-dihydrouracil or a derivative thereof (e.g., 5,6-dihydrouracil analogue), 5- methylcytosine or a derivative thereof (e.g., 5-methylcytosine analogue), or 5- hydroxymethylcytosine or a derivative thereof (e.g., 5-hydroxymethylcytosine analogue) moieties. In some embodiments, the nucleobase is adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, or isoguanine, which may be optionally substituted or modified. In some embodiments, the nucleobase is,which may be optionally substituted or modified, wherein “ ” denotes the point ofattachment to a pentofuranosyl sugar ring (e.g., 1′ position).

[0109] The term “phosphate,” or “phosphate group” as used herein a chemical species made of one phosphorus atom and four oxygen or esters, salts, or acids thereof. In certain aspects, when the phosphatebetween adjacent nucleosides in RNA or DNA strand and form a “backbone” of the oligonucleotides, these termsPAT059650-WO-PCT “phosphate,” or “phosphate group” may be interchangeable used as “phosphate group,” “phosphate linkage,” “phosphodiester linkage,” or “linkage.” For example, the phosphate orphosphodiester linkage in the backbone of RNA or DNA may have theor esters, salts (e.g., pharmaceutically acceptable salts), or ) thereof,wherein “ ” denotes the point of attachment to 5′ and 3′adjacent nucleosides. In certain aspects, a variant of a phosphate or phosphodiester linkage, e.g., phosphorothioate (PS) linkage, can replace a phosphate group (or phosphodiester linkage) in the backbone and connect two adjacent nucleosides. In certain aspects, a variant of a phosphate or phosphodiester linkage, e.g., phosphorothioate (PS) linkage or vinyl phosphonate (VP) group, may be additionally attached at 3′ end or 5′ end of the oligonucleotides (e.g., RNA or DNA), e.g., 3′-OH or 5′-OH position of the terminal pentofuranosyl sugar (e.g., ribose or deoxyribose), so as to act as chemically or biologically functional group. In certain aspects, a variant of phosphate or phosphodiester linkage may also be referred as a phosphorus-derived internucleoside linkage that includes at least one phosphorus atom in the backbone.

[0110] Unless otherwise indicated herein, an unmodified RNA (or “ribonucleotide”) in a chain of nucleotides (e.g., mRNA, rRNA, or sense strand or antisense strand of siRNA) as disclosed refers to a structure of a pharmaceutically acceptable salt thereof. Likewise, an unmodifiedin a chain of nucleotides (e.g., genomic DNA or cDNA) as disclosed herein specifically refers to a structure ofPAT059650-WO-PCT a pharmaceutically acceptable salt thereof. In each structure “Base” is attachment point to the adjacent nucleotides.indicated herein, when an unmodified RNA is the first nucleotide from the 5′ end of an RNA chain (e.g., mRNA, or sense strand or antisense strand of siRNA), that nucleotide has a a pharmaceutically acceptable salt thereof. Likewise, when an from the 5′ end of a DNAchain (e.g., genomic DNA or cDNA), that nucleotide has a or a pharmaceutically acceptable salt thereof. In each structureis an attachment point (5′ oxygen) to the adjacent nucleotides. Alternatively but equivalently, for example, the first nucleotide from the 5′ end of an RNA chain (e.g., mRNA, or sense strand or antisense strand of siRNA), that nucleotide has a a pharmaceutically acceptable salt thereof and the firstPAT059650-WO-PCT chain (e.g., genomic DNA or cDNA), that nucleotide has a or a pharmaceutically acceptable salt thereof, when is an theadjacent nucleotides.

[0112] Unless otherwise indicated herein, when an unmodified RNA is the first nucleotide from the 3′ end of an RNA chain (e.g., mRNA, or sense strand or antisense strand of siRNA), that nucleotide has a a pharmaceutically acceptable salt. Likewise, when an from the 3′ end of a DNA chain(e.g., genomic DNA or cDNA), that nucleotide has a a pharmaceutically acceptable salt thereof. In certainis the first nucleotide from the 3′ end of an RNA chain (e.g., mRNA, or sense strand or antisense strand of siRNA) that nucleotide does not include 3′ end phosphate group or phosphodiester linkage, for example, which has been removed during hydrolysis or synthesis, has a structure a pharmaceutically acceptable salt. Likewise, when an unmodifiedfrom the 3′ end of a DNA chain (e.g., genomic DNA or cDNA), that nucleotide does not include 3′ end phosphate group, for example, which has been removed during hydrolysis or synthesis, has a a pharmaceuticallyPAT059650-WO-PCT acceptable salt thereof. In each structure “Base” is a nucleobase and is an attachment point (e.g., phosphorus of the phosphate linkage) to the adjacent nucleotides.

[0113] A code “A”, “G”, “C”, “U”, or “T” presented in a sequence list as disclosed herein stand for a RNA nucleotide that contains adenine, guanine, cytosine, uracil and thymine as a base, respectively. A code “dA”, “dG”, “dC” or “dT” presented in a sequence list as disclosed herein stand for a DNA nucleotide that contains adenine, guanine, cytosine, and thymine as a base, respectively.

[0114] The term “oligonucleotide” means a shorter length nucleic acid, e.g. of less than 100 nucleotides in length. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, an oligonucleotide may include naturally occurring ribonucleotides, naturally occurring deoxyribonucleotides, and / or nucleotides having one or more modifications to a naturally occurring terminus, sugar, nucleobase, and / or internucleoside linkage. Non- limiting examples of oligonucleotides include double-stranded oligonucleotides (e.g., dsRNA), single-stranded oligonucleotides (e.g., single stranded RNA or ssRNA), antisense oligonucleotides (“ASO”), small interfering RNA (siRNA), microRNA mimics, short hairpin RNAs (shRNA), single-strand small interfering RNA (ssRNAi), RNaseH oligonucleotides, anti-microRNA oligonucleotides, steric blocking oligonucleotides, exon-skipping oligonucleotides, CRISPR guide RNAs, and aptamers. In certain aspects, the oligonucleotide is a dsRNA and each strand has a length less than 100 nucleotides (“nt”), less than 90 nt, less than 80 nt, less than 70 nt, less than 60 nt, less than 50 nt, less than 40 nt, less than 35 nt, less than 30 nt, less than 28 nt, less than 26 nt, less than 25 nt, less than 24 nt, less than 23 nt, less than 22 nt, less than 21 nt, less than 20 nt, less than 19 nt, less than 18 nt, less than 17 nt, less than 16 nt, or 15 nt.

[0115] The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript (mRNA) via an RNA-induced silencing complex (RISC) pathway. An RNAi agent directs the sequence-specific degradation of mRNA through a process and thereafter inhibits expression of the gene encoded by the mRNA in a cell in vivo, e.g., in a subject (e.g., any vertebrate, mammal, or human).

[0116] The term “small interfering RNA” or “siRNA” means a double-stranded oligonucleotide (dsRNA) formed with two anti-parallel, and partially, substantially or fully complementary nucleic acid strands (e.g., a first strand and a second strand; or a “sense” strand and an “antisense” strand), which interferes with the expression of genes in a sequence-specificPAT059650-WO-PCT manner by facilitating mRNA degradation before translation through the RNA interference pathway. In some embodiments, depending on the context, the first strand can be a “guide” or antisense strand, and the second strand can be a “passenger” or sense strand. In some embodiments, depending on the context, the “first” strand can be a passenger or sense strand, and the “second” strand can be a guide or antisense. In certain aspects, an “RNAi agent” or “siRNA agent,” as used herein, refers a double-stranded RNA (dsRNA) with or without a ligand or other conjugate, and may be interchangeably used with a term “double stranded RNAi agent (dsRNAi agent),” or “dsRNA agent.” In certain aspect of the disclosure, the term “siRNA” can be used to describe a dsRNA with specific nucleotide sequences (unmodified or modified nucleotide sequences), without a ligand or other conjugate.

[0117] The term “antisense strand,” as used herein, refers an oligonucleotide (e.g., RNA) of an siRNA or a dsRNAi that is complementary (e.g., partially, substantially, or fully complementary) to the target mRNA and is incorporated into the RNA-induced silencing complex (RISC) to direct gene silencing in a sequence-specific manner through the RNA interference pathway. An antisense strand may also be referred to as the “guide strand.” In some embodiments, the antisense strand may have a length from 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19 nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30 nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, 21-22 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, or 36 nt.

[0118] The term “sense strand,” as used herein, refers an oligonucleotide that is complementary (e.g., partially, substantially, or fully complementary) to the antisense strand. The sense strand is typically degraded following incorporation of the antisense strand into RISC. The sense strand may also be referred to as the “passenger strand.” In some embodiments, the sense strand may have a length from 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19 nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30 nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, 21-22 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, or 36 nt.PAT059650-WO-PCT

[0119] The term “complementary” means that a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides are capable of base pairing non-covalently via hydrogen bonding with another nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine or uridine and the complementary (matching) nucleotide of guanosine is cytidine. The complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. For example, two sequences that are complementary to each other, may have a specified percentage of nucleotides that participate in nucleobase-pairing (i.e., about 50% complementarity, preferably 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater complementarity over a specified region). In some embodiments, two sequences are partially complementary when the percentage of nucleotides that participate in nucleobase-pairing is about 50%, about 55%, about 65%, about 70%, about 75%, or about 80%, or ranges from about 50% to about 80%. In some embodiments, two sequences are substantially complementary when the percentage of nucleotides that participate in nucleobase- pairing is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 92%, about 93%, about 94%, or about 95%, or ranges from about 80% to about 95%.

[0120] Examples of complementary (e.g., partially, substantially, or fully complementary) sequences are sense and antisense sequences, wherein the sense sequence contains complementary (e.g., partially, substantially, or fully complementary) nucleotides to the antisense sequence and thus forms the complement of the antisense sequence. In certain aspects, a sense strand and an antisense strand of a double-stranded oligonucleotide (e.g., double stranded RNA) are substantially or fully complementary over their entire lengths. In some embodiments, a sense strand and an antisense strand of dsRNA are substantially or fully complementary over the entire length of the double-stranded region of the siRNA, and one or both termini of either strand comprises single-stranded nucleotides.

[0121] Another examples of complementary (e.g., partially, substantially, or fully complementary) sequences are an antisense strand and its target mRNA sequence. In certain aspects, an antisense strand is substantially or fully complementary to its target mRNA. For example, the complementary (e.g., partially, substantially, or fully complementary) sequences may be between an antisense strand and a coding region of the target mRNA, or a non-coding sequence of the target mRNA. In certain aspects, an antisense strand is substantially, or fullyPAT059650-WO-PCT complementary to its target mRNA to reduce or eliminate off-target profile for and to improve down-regulation of the target gene (e.g., gene of the target mRNA sequence).

[0122] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 60% identity, or at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or within a range defined by any of two of the preceding values, identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). This definition also refers to, or may be applied to, the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps, insertions and the like. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0123] As used herein, “target sequence” or “target gene” refer to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi- directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides (“nt”) in length, e.g., 15-30 nt in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence may have a length from 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19- 20 nt, 19 nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30 nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, 21-22 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt,PAT059650-WO-PCT 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, or 36 nt.

[0124] The term “ligand,” as used herein, refers to a compound or moiety that can impose characteristics to provide additional properties, e.g., affinity or cell delivery efficiency, to an RNAi (e.g., dsRNAi) as described herein. The ligand may be coupled or conjugated directly to the RNAi (e.g., sense strand or antisense strand of dsRNA), or indirectly to the RNAi agent (e.g., sense strand or antisense strand of dsRNA) via an intervening linker (“linker”). When a ligand is conjugated or coupled indirectly to the RNAi (e.g., dsRNA) via a linker, the ligand may be formed of a core moiety (e.g., targeting moiety) that has specific function to provide affinity or efficacy and the linker that provides merely an optimal distance, e.g., between the core moiety and the RNAi agent (dsRNA). In certain aspects, the term “ligand” embraces the ligand in combination with the linker. Examples of ligands or targeting moieties thereof may include, but not be limited to, one or more selected from a synthetic or natural compound, a peptide, an antibody, a carbohydrate (e.g., sugar moiety), or an additional nucleic acid.

[0125] The term “modified nucleotide” means a nucleotide having one or more modifications relative to a naturally occurring nucleotide, e.g., RNA. The modified nucleotide may be selected over an unmodified form because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, increased stability in the presence of nucleases, and / or reduced immune stimulation. In certain aspects, the modification may be present in at least one of (i) an internucleoside linkage (“linkage”), (ii) a nucleobase, and (iii) a sugar moiety of the nucleotide. In certain aspects, the modification is present in the internucleoside linkage, e.g., by chemically modifying a phosphate group or replacing a phosphate group with other linking groups. In certain aspects, the modification is present in a sugar moiety, i.e., ribose ring, by substituting hydroxyl group on 2′ position of the ribose ring with other chemical group or by replacing a ring structure withother heterocycloalkyl or cycloalkyl, glycol group having a , bicyclic orbridged ring on the ribose such as locked nucleic acida moiety of , or the like.PAT059650-WO-PCT

[0126] In certain aspects, the modification is present in a nucleobase (e.g., A, G, C, T, or U) by chemical modification in a nucleobase by replacing the nucleobase with other moiety, for example, by replacing one naturally occurring nucleobase with another naturally occurring nucleobase. In certain aspects, a modified nucleotide may contain a modification in a sugar moiety and an unmodified phosphate group. In certain aspects, a modified nucleotide may have a modification in a sugar moiety but with an unmodified nucleobase. In certain aspects, a modified nucleotide may have a modification in a sugar moiety and a nucleobase. In certain aspects, a modified nucleotide may have a modification in a sugar moiety and a phosphate group. In certain aspects, a modified nucleotide may have a modification in a sugar moiety, a phosphate group and a nucleobase. In certain aspects, a modified nucleotide may have an unmodified sugar moiety and an unmodified phosphate group. In certain aspects, a modified nucleotide may have an unmodified sugar moiety and an unmodified nucleobase. In certain aspects, a modified nucleotide may have an unmodified sugar moiety and a modified nucleobase. In certain aspects, a modified nucleotide may have an unmodified sugar moiety and a modified phosphate group. In certain aspects, a modified nucleotide may have a modified sugar moiety, a modified phosphate group and a modified nucleobase.

[0127] The term “modified phosphate group,” or “modified phosphodiester linkage” as used herein refers to a chemical group in place of a phosphate group (or phosphodiester linkage) in a nucleotide as being attached to the 3′ end (3′ carbon) of the pentofuranosyl group.

[0128] The term “monomer” as used herein refers to a monomeric unit in an oligonucleotide or a nucleic acid. In certain aspects, the monomer inclusively refers to a nucleotide (e.g., RNA or DNA), a modified nucleotide (e.g., 2′-F modified nucleotide, abasic nucleotide, 3′- phosphorothioate (PS) modified nucleotide, etc), or other variants thereof (e.g., LNA, GNA, or TNA). In certain aspects, a plurality of monomers can be connected (e.g., covalently connected) to form a linear strand (e.g., sense strand or antisense strand).

[0129] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 60% identity, or at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or within a range defined by any of two of the preceding values, identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) asPAT059650-WO-PCT measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). This definition also refers to, or may be applied to, the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps, insertions and the like. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0130] Throughout the disclosure, nucleotide positions or coordinates are relative to the beginning (5′ end) of the reference transcript.

[0131] The term “overhang” or “nucleotide overhang” herein refers to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of the duplex structure of an RNAi agent. In some embodiments, when a 3′-end of one strand extends beyond the 5′-end of the other strand, or vice versa, this forms a nucleotide overhang, e.g., the unpaired nucleotide(s) form the overhang.

[0132] “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAi agent is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

[0133] A “mismatch” is defined herein as a difference between the base sequence (e.g., A instead of G) or length when two sequences are maximally aligned and compared.

[0134] The term “non-end” herein refers to a position between the 3′ end and the 5′ end of the sense or antisense strand.

[0135] The term “Compound” may refer to a double stranded RNA that is conjugated with a ligand or a delivery moiety, while a term “compound” denotation may refer to a substance or a molecule that can be chemically defined and / or identifiable.

[0136] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like mean negatively affecting (e.g. decreasing) activity, expression or function relative to the activity, expression or function in the absence of an inhibitor. In certain aspects, inhibition can mean negatively affecting (e.g. decreasing) the concentration or levels of a biomolecule, such as aPAT059650-WO-PCT protein or mRNA, relative to the concentration or level of the biomolecule in the absence of an inhibitor. In certain aspects, inhibition includes, partially or totally, blocking stimulation, decreasing, preventing, or delaying activation; inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity; or decreasing the amount of a biomolecule target (e.g., protein target or mRNA target). In certain aspects, inhibition refers to a reduction in the expression of a particular biomolecule target, such as a protein target or an mRNA target (e.g., target mRNA). In certain aspects, inhibition refers to a reduction of amount of a target biomolecule (e.g., target protein or mRNA) resulting from a down-regulating protein expression (e.g. directly inhibiting translation or transcription). In certain aspects, inhibition refers to a reduction of activity of a target biomolecule (e.g., target protein or mRNA) from an indirect interaction (e.g., inhibiting or regulating other transcriptional or translational factors).

[0137] The term “inhibitor” also refers to a compound, composition, or substance capable of detectably negatively affecting (e.g. decreasing) activity, expression or function of a given protein or gene. For example, an inhibitor may decrease activity, expression or function by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater in comparison to a control in the absence of the inhibitor. Inhibitors include, for example, synthetic or biological molecules, such as oligonucleotides. In some embodiments, the inhibitors include RNAi agent, e.g., siRNA agent, dsRNAi agent, or dsRNA agent.

[0138] As used herein, the “level or degree of inhibiting or decreasing expression” of a given gene refers to the at least partial suppression of the expression of a target gene, as manifested by a reduction of the amount of the target gene mRNA or protein encoded by the target gene, which may be isolated from or detected in a group of cells (“a first cell”) in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is inhibited, as compared to group of cells substantially identical to the first cell but without treated (“control cells” or “a second cell”).

[0139] In some embodiments, the level or expression of the target gene can be measured by evaluation of mRNA (e.g., via Northern blots or PCR). The effect of an RNAi agent on the target gene expression can be determined by measuring the gene transcription rates (e.g., via Northern blots; or reverse transcriptase polymerase chain reaction or real-time polymerase chain reaction). In some embodiments, the degree of inhibition can be calculated as the following equation: (mRNAin control cells) - (mRNA in treated cells)•100 % (mRNAin control cells)PAT059650-WO-PCT

[0140] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, alteration in expression of the protein whose expression is dependent on the target gene, alteration in an activity of the protein encoded by the target gene. In some embodiments, the level or expression of the protein from the target gene can be evaluated by measuring the expressed protein amount (e.g., Western blots). In some embodiments, the level or expression of the protein from the target gene can be measured by the enzymatic assay (e.g., kinetic assay) of the protein.

[0141] As used herein, the term “down-regulate” or “down-regulating” refers to any statistically significant decrease in a biological activity and / or expression of the target protein, including full blocking of the activity (i.e., complete inhibition) and / or expression. For example, “down-regulation” can refer to a decrease of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in the target gene expression, activity of the target protein.

[0142] As used herein, the terms “salt” or “salts” refers to an acid addition or base addition salt of a compound of the present invention. “Salts” include in particular “pharmaceutical acceptable salts”. The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto. When both a basic group and an acid group are present in the same molecule, the compounds of the present invention may also form internal salts, e.g., zwitterionic molecules. In certain aspects, pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Examples of the inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Examples of the organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. In certain aspects, the pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Examples of the inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table, such as sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium,PAT059650-WO-PCT potassium, sodium, calcium and magnesium salts. Examples of the organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0143] In certain aspects, the term “pharmaceutically acceptable salt” as used herein may include the salts forms in acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, caprate, chloride / hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate trifenatate, trifluoroacetate or xinafoate.

[0144] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance useful in the preparation or use of a pharmaceutical composition and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp.1049-1070).

[0145] As used herein, the term “treat,” “treating,” or “treatment” of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient. In some embodiments, treating does not include preventing.

[0146] As used herein, the term “prevent”, “preventing" or “prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.PAT059650-WO-PCT

[0147] The term “therapy,” as used herein refers to an application of one or more specific procedures used for the amelioration of at least one indicator or a disease or condition. In certain aspects, the specific procedure is the administration of one or more pharmaceutical or therapeutic agents.

[0148] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g. a protein associated disease) means that the disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function (e.g., protein activity or function). Thus, as used herein, what is described as “being associated” with a disease, if a causative agent, could be a target for treatment of the disease.

[0149] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal) compatible with the preparation. Parenteral administration includes, e.g, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0150] Herein, the terms "subject" and “patient” are used interchangeably. The term subject includes a human or non-human animal, preferably a vertebrate, and more preferably a mammal. In certain aspects, the subject is a human. In certain aspects, the subject is a human patient.

[0151] As used herein, a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0152] The term "a therapeutically effective amount" of a compound (e.g., siRNA) as disclosed herein refers to an amount of the compound that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In certain aspects, the term “a therapeutically effective amount” refers to the amount of the compound (e.g., siRNA) of the disclosure that, when administered to a subject, is effective to (1) at least partially alleviate, prevent and / or ameliorate a condition, or a disorder or a disease (i) mediated by the target gene (e.g., protein encoded by the target gene), or (ii)PAT059650-WO-PCT associated with its activity, or (iii) characterized by activity (normal or abnormal) of the protein encoded by the target gene; or (2) reduce or inhibit the activity of the protein encoded by the target gene; or (3) reduce or inhibit the expression of the target gene. In certain aspects, the term “a therapeutically effective amount” refers to the amount of the compound that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of the protein encoded by the target gene; or at least partially reducing or inhibiting the expression of the protein encoded by the target gene. The meaning of the term “a therapeutically effective amount” as illustrated in the above embodiment for the target gene expression also applies by the same means to any other relevant proteins / peptides / enzymes.

[0153] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art. Therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. An example of an “therapeutically effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease. For example, for the given parameter (e.g., biomarker), a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0154] The term “control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. Typically, a control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is the measurement of the expression of a protein or mRNA in the absence of RNAi agents as described herein.

[0155] Unless defined otherwise, the chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.PAT059650-WO-PCT

[0156] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which is fully saturated (i.e., molecule by only single bonds) and include mono-, di- and multivalent radicals. As used herein, the alkyl is an uncyclized chain. The alkyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons). Examples of alkyl include, but are not limited to, groups such as C1-30alkyl, C1-25alkyl, C1-20alkyl, C1-15alkyl, C1-12 alkyl, C1-10 alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl. For example, C1-6 alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n- butyl, n-pentyl and 1,1-dimethylethyl (t-butyl), and their isomers.

[0157] A term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, - CH2CH2CH2CH2-.

[0158] As used herein, the term "alkenyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which is mono- or polyunsaturated (i.e., molecule including at least one double bond) and include mono-, di- and multivalent radicals. As used herein, the alkenyl is an uncyclized chain. Like the alkyl, the alkenyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons). Examples of alkenyl include, but are not limited to, groups such as C1-30alkenyl, C1-25alkenyl, C1-20alkenyl, C1-15alkenyl, C1-12alkenyl, C1-10alkenyl, C1-8 alkenyl, C1-6 alkenyl, C1-4 alkenyl, or C1-3 alkenyl. For example, C2-6 alkenyl include, but are not limited to, ethenyl (vinyl), prop-1-enyl, but-1-enyl, pent-1-enyl, pent-4- enyl and penta-1,4-dienyl, and their isomers. A term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkenyl, as exemplified, but not limited by, -CH=CHCH2CH2-.

[0159] As used herein, the term " alkynyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which is mono- or polyunsaturated (i.e., molecule including at least one triple bond) and include mono-, di- and multivalent radicals. As used herein, the alkynyl is an uncyclized chain. Like the alkyl, the alkynyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons). Examples of alkynyl include, but are not limited to, groups such as C1-30alkynyl, C1-25alkynyl, C1-20alkynyl, C1-15alkynyl, C1-12alkynyl, C1-10 alkynyl, C1-8 alkynyl, C1-6 alkynyl, C1-4 alkynyl, or C1-3 alkynyl. For example, C2-6 alkynyl include, but are not limited to, alkynyl, and their isomers. A term “alkynyl,” by itself or as partPAT059650-WO-PCT of another substituent, means, unless otherwise stated, a divalent radical derived from an alkenyl, as exemplified, but not limited by, -CCH2CH2-.

[0160] As used herein, the term “alkoxy” refers to a radical of the formula -ORawhere Rais an alkyl (e.g., C1-30alkyl, C1-25alkyl, C1-20alkyl, C1-15alkyl, C1-12alkyl, C1-10alkyl, C1-8alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl) radical as generally defined above. For example, C1-6 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, and hexoxy.

[0161] As used herein, the term " alkoxyalkyl" refers to a radical of the formula -Ra-O-Rbwhere each Raand Rbis independently an alkyl (e.g., C1-30 alkyl, C1-25 alkyl, C1-20 alkyl, C1-15 alkyl, C1-12alkyl, C1-10alkyl, C1-8alkyl, C1-6alkyl, C1-4alkyl, or C1-3alkyl) radical as defined above and oxygen atom may be bonded to any carbon atom in either alkyl radical. For example, C1-6alkoxy C1-6alkyl include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy- ethyl, 1-ethoxy-propyl and 2-methoxy-butyl.

[0162] As used herein, the term “alkylcarbonyl” refers to a radical of the formula -C(=O)- Rawhere Rais an alkyl (e.g., C1-30alkyl, C1-25alkyl, C1-20alkyl, C1-15alkyl, C1-12alkyl, C1-10alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl) radical as defined above.

[0163] As used herein, the term "alkyl-carbonyl alkyl” refers to a radical of the formula - Ra-C(=O)-Rbwhere each Raand Rbis independently an alkyl (e.g., C1-30 alkyl, C1-25 alkyl, C1-20alkyl, C1-15alkyl, C1-12alkyl, C1-10alkyl, C1-8alkyl, C1-6alkyl, C1-4alkyl, or C1-3alkyl) radical as defined above. The carbon atom of the carbonyl group may be bonded to any carbon atom in either alkyl radical.

[0164] As used herein, the term "alkylaminocarbonyl" refers to a radical of the formula - C(=O)-NH-Rawhere Rais an alkyl (e.g., C1-30alkyl, C1-25alkyl, C1-20alkyl, C1-15alkyl, C1-12alkyl, C1-10 alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl) as defined above.

[0165] As used herein, the term "alkoxycarbonyl” refers to a radical of the formula - C(=O)-O-Rawhere Rais an alkyl (e.g., C1-30 alkyl, C1-25 alkyl, C1-20 alkyl, C1-15 alkyl, C1-12 alkyl, C1-10alkyl, C1-8alkyl, C1-6alkyl, C1-4alkyl, or C1-3alkyl) radical as defined above.

[0166] As used herein, the term “alkoxycarbonyl alkyl” refers to a radical of the formula -Ra-C(=O)-O-Rbwhere each Raand Rbis independently an alkyl (e.g., C1-30alkyl, C1-25alkyl, C1-20 alkyl, C1-15 alkyl, C1-12 alkyl, C1-10 alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl) radical as defined above.

[0167] As used herein, the term "haloalkyl" refers to an alkyl (e.g., C1-30 alkyl, C1-25 alkyl, C1-20 alkyl, C1-15 alkyl, C1-12 alkyl, C1-10 alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl)PAT059650-WO-PCT radical, as defined above, substituted by one or more halo radicals, as defined above. Examples of halogenC1-6alkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,3-dibromopropan-2-yl, 3-bromo-2- fluoropropyl and 1,4,4-trifluorobutan-2-yl.

[0168] As used herein, the term "hydroxyalkyl” refers to an alkyl (e.g., C1-30 alkyl, C1-25 alkyl, C1-20alkyl, C1-15alkyl, C1-12alkyl, C1-10alkyl, C1-8alkyl, C1-6alkyl, C1-4alkyl, or C1-3alkyl) radical as defined above, wherein one of the hydrogen atoms of the alkyl radical is replaced by OH. Examples of hydroxyC1-6alkyl include, but are not limited to, hydroxy- methyl, 2-hydroxy-ethyl, 2-hydroxy-propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl.

[0169] As used herein, the term “aminoalkyl” refers to an alkyl (e.g., C1-30alkyl, C1-25alkyl, C1-20 alkyl, C1-15 alkyl, C1-12 alkyl, C1-10 alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl) radical as defined above, wherein one of the hydrogen atoms of the C1-6alkyl group is replaced by a primary amino group. Examples of amino C1-6 alkyl include, but are not limited to, amino-methyl, 2-amino-ethyl, 2-amino-propyl, 3-amino-propyl, 3-amino-pentyl and 5- amino-pentyl.

[0170] As used herein, the term “alkylamino” refers to a radical of the formula -NH-Rawhere Rais an alkyl (e.g., C1-30alkyl, C1-25alkyl, C1-20alkyl, C1-15alkyl, C1-12alkyl, C1-10alkyl, C1-8 alkyl, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl) radical as defined above.

[0171] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combination thereof, which is fully saturated (i.e., molecule by only single bonds) and include mono-, di- and multivalent radicals, including at least one carbon atom and at least one heteroatom (e.g., O, N, S, Si, or P), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. The heteroalkyl may include a designated number of carbons and heteroatoms (e.g., “2 to 10 membered heteroalkyl” means two to 10 atoms including carbons and heteroatoms).

[0172] Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).PAT059650-WO-PCT

[0173] As used herein, the term "heteroalkenyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which is mono- or polyunsaturated (i.e., molecule including at least one double bond between carbon and carbon) and include mono-, di- and multivalent radicals. As used herein, the alkenyl is an uncyclized chain. Like the alkenyl, the heteroalkenyl may include a designated number of carbons and heteroatoms (e.g., “2 to 10 membered heteroalkenyl” means two to 10 atoms including carbons and heteroatoms).

[0174] As used herein, the term " heteroalkynyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which is mono- or polyunsaturated (i.e., molecule including at least one triple bond between carbon and carbon) and include mono-, di- and multivalent radicals. As used herein, the alkynyl is an uncyclized chain. The heteroalkynyl may include a designated number of carbons and heteroatoms (e.g., “2 to 10 membered heteroalkynyl” means two to 10 atoms including carbons and heteroatoms).

[0175] For alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R′- represents both -C(O)2R′- and -R′C(O)2-.

[0176] A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien- 3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0177] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3PAT059650-WO-PCT rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzooxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4- biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5- indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6- quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0178] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.PAT059650-WO-PCT

[0179] The symbol “ ” denotes the point of attachment of a chemical moiety to theremainder of a molecule or chemical formula.

[0180] The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.

[0181] Each of the above terms (e.g., “alkyl,” “alkenyl,” “alkynyl,” “heteroalkyl,” “cycloalkyl,” “heterocycloalkyl,” “aryl,” and “heteroaryl”, and divalent or trivalent radicals thereof) includes both substituted and unsubstituted forms of the indicated radical. Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, — C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, — NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —NR′NR″R′″, —ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, — NO2, —NR′SO2R″, —NR′C(O)R″, —NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R, R′, R″, R′″, and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ group when more than one of these groups is present. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, —NR′R″ includes, but is not limited to, 1-pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).

[0182] Certain compounds provided herein possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids, and individual isomers arePAT059650-WO-PCT encompassed within the scope of the present disclosure. The compounds of provided herein do not include those that are known in art to be too unstable to synthesize and / or isolate. Compounds provided herein include those in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds (vinyl group) and unless specified otherwise, it is intended that the compounds include both (E) and (Z) geometric isomers.

[0183] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[0184] As used herein, the term “ detectable moiety” refers to a molecule or substance that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence (i.e. qualitative analysis) and / or concentration (i.e. quantitative analysis) of the label in a sample. In some embodiments, the detectable moiety includes a label that generate an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signal which may be detected by a suitable device. In some embodiments, the detectable moiety includes an isotope (e.g., radioactive isotope) or a molecule including an isotope (e.g., radioactive isotope) that may be detected by measuring radiation or emission. DOUBLE STRANDED (ds) RNAi AGENTS

[0185] In an aspect, the disclosure provides an RNAi agent including a double stranded RNA (dsRNA). In an aspect, also provided is a dsRNA interference (dsRNAi) agent that includes a dsRNA consisting of (i) a sense strand and (ii) an antisense strand, and a ligand attached to at least one of the sense strand and the antisense strand.

[0186] A dsRNA is a complex of ribonucleic acid (RNA) molecules formed in a duplex structure. In certain aspects, the dsRNA may be a small interfering RNA (siRNA) that has 10 to 30, or particularly 15-25 nucleotides in each RNA molecule, respectively, “passenger strand” and “guide strand”, and can be incorporated into an RNA-induced silencing complex (RISC). The siRNA is dissociated or unwounded in the RISC, and the passenger strand is degraded while the guide strand remains in the RISC pathway. The guide strand can subsequently bind to an mRNA molecule that includes a complementary sequence to the guide strand and induce or initiate cleavage or degradation of the mRNA molecule. In certain aspects, the mRNA encodes a target gene (e.g., mRNA transcript of a target gene) such that expression of the target gene is suppressed or inhibited through a post-transcriptional gene-silencing (“RNAPAT059650-WO-PCT silencing”). A guide RNA molecule has a complementary sequence to a target mRNA sequence and has anti-parallel orientation to the target gene, so it is interchangeably referred to as an antisense strand. A passenger RNA molecule forming a duplex with the guide RNA and having a complementary sequence to the guide strand (antisense strand) has the same orientation with the target mRNA sequence, so it is interchangeably referred to as a sense strand.

[0187] In an aspect, the disclosure provides a dsRNA interference (dsRNAi) agent that is capable of interacting or recruiting a target mRNA sequence in the RISC thereby cleaving the target mRNA. The dsRNAi agent can silence the target gene, e.g., by inhibiting, downregulating, or suppressing the expression of the target gene (protein). Gene-silencing (e.g., inhibiting, downregulating, or suppressing of the gene) may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with the target gene expression compared with a control level. The control level may be any type obtained from, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or untreated or treated subject with inactive agents (e.g., PBS buffer). In some embodiments, the level of silencing the target gen may be demonstrated by a reduction of the amount of a total target gene mRNA in a cell. In some embodiments, the level of silencing the target gene may be demonstrated by a reduction of the amount of a total target protein in a cell.

[0188] In some embodiments, expression of the target gene is inhibited by any dsRNAi agent disclosed here for at least about 10%, about 15%, about 20%, about 25%, about 30 %, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% based on the expression level of the same gene in untreated cell or subject. In some embodiments, expression of the target gene is inhibited by at least about 20% based on the expression level of the same gene in untreated cell or subject. In some embodiments, expression of the target gene is inhibited by at least about 30% based on the expression level of the same gene in untreated cell or subject. In some embodiments, expression of the target gene is inhibited by at least about 40% based on the expression level of the same gene in untreated cell or subject. In some embodiments, expression of the target gene is inhibited by at least about 50% based on the expression level of the same gene in untreated cell or subject. In some embodiments, expression of the target gene is inhibited by at least about 60% based on the expression level of the same gene in untreated cell or subject. In some embodiments, expression of the target gene is inhibited by at least about 70% based on the expression level of the same gene in untreated cell or subject.PAT059650-WO-PCT

[0189] In some embodiments, inhibition of the expression of the target gene may be manifested by a reduction of the amount of mRNA expressed in a first cell or a first group of cells obtained from a subject that has been treated, e.g., by contacting the cell or by administering the dsRNAi agent as described herein, as compared to a second cell or a second group of cells obtained from a subject that has not been treated but is identical to the first cell or the first group of cells. For example, the level of gene-silencing (e.g., inhibiting, downregulating, or suppressing of the gene) of the target gene may be presented as a percentage of remaining mRNA in the treated cells (first cell or group of cells) compared to the mRNA amount in the control (untreated) cells, as shown in the following equation: (mRNAin control cells) - (mRNA in treated cells)•100 % (mRNAin control cells) .

[0190] In some embodiments, the level of gene-silencing (e.g., inhibiting, downregulating, or suppressing of the gene) of the target gene may be assessed by measuring a parameter or biomarker, e.g., the expressed protein level, in a biological sample (e.g., e.g., a blood, serum or liver tissue obtained from a subject), which may be treated or untreated. Conventional analytical methods as known in the art such as electrophoresis (e.g., SDS or capillary electrophoresis), chromatography (e.g., high performance liquid chromatography (HPLC)), spectroscopy, western blotting, enzyme- linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like can be used without limitation, but examples are not limited thereto. In some embodiments, reduced level of gene- silencing (e.g., inhibiting, downregulating, or suppressing of the gene) of the target protein may be observed or assessed by in a tissue biopsy of the target organ in the treated subject.

[0191] In certain aspects, the dsRNAi agent is a free acid. In certain aspects, the RNAi agent is in salt form (e.g., pharmaceutically acceptable salt form). It will be understood that references to dsRNAi agent are meant to also include the pharmaceutically acceptable salts of the dsRNAi agent. If the dsRNAi agent has, for example, at least one basic center, they can form acid addition salts. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center. Active substances having an acid group, e.g., COOH, can form salts with bases. The dsRNAi agent or pharmaceutically acceptable salts thereof may also be used in form of a hydrate or include other solvents used for crystallization. In some embodiments, the RNAi agent is a sodium salt. In some embodiments, the dsRNAi agent is in a salt form (e.g., a pharmaceutically acceptable salt form), where the salt is sodium (Na+), ammonium (NH+), calcium (Ca2+), iron (Fe2+or Fe3+), magnesium (Mg2+), potassiumPAT059650-WO-PCT (K+), pyridinium (C5H5NH+), quaternary ammonium (NR4+, R being an alkyl group or an aryl group as described herein), or copper (Cu2+). dsRNA MODIFICATIONS

[0192] In an aspect, the disclosure provides a dsRNA having sequences (e.g., antisense strand sequence) that is designed to recognize a specific region of a target gene mRNA and lead cleavage of that mRNA and silencing of that gene. The dsRNA includes a sense strand and an antisense strand and each strand may range from 12 to 30 nucleotides in length. In some embodiments, each strand may have 15 to 30 nucleotides in length. In some embodiments, each strand may have 15 to 25 nucleotides in length. In some embodiments, the antisense strand may have 18 to 25 nucleotides in length. In some embodiments, the sense strand may have 18 to 25 nucleotides in length.

[0193] In some embodiments, the sense strand may have 19 to 23 nucleotides in length. In some embodiments, the sense strand may have 19 to 21 nucleotides in length. In some embodiments, the sense strand may have 19 nucleotides in length. In some embodiments, the sense strand may have 20 nucleotides in length. In some embodiments, the sense strand may have 21 nucleotides in length. In some embodiments, the sense strand may have 22 nucleotides in length. In some embodiments, the sense strand may have 23 nucleotides in length.

[0194] In some embodiments, the antisense strand may have 19 to 25 nucleotides in length. In some embodiments, the antisense strand may have 19 to 23 nucleotides in length. In some embodiments, the antisense strand may have 19 nucleotides in length. In some embodiments, the antisense strand may have 20 nucleotides in length. In some embodiments, the antisense strand may have 21 nucleotides in length. In some embodiments, the antisense strand may have 22 nucleotides in length. In some embodiments, the antisense strand may have 23 nucleotides in length. In some embodiments, the antisense strand may have 24 nucleotides in length. In some embodiments, the antisense strand may have 25 nucleotides in length.

[0195] In an aspect, a dsRNA as described herein forms a double-stranded (or “duplex”) region made between a sense strand and an antisense strand and having 10 to 25 nucleotide pairs in length. The double stranded or duplex region are loaded into the RISC and subsequent specific degradation of the sense strand occurs during the RISC pathway. In some embodiments, the double stranded region has 10 nucleotide base pairs in length. In some embodiments, the double stranded region has 11 nucleotide base pairs in length. In some embodiments, the double stranded region has 12 nucleotide base pairs in length. In some embodiments, the double stranded region has 13 nucleotide base pairs in length. In some embodiments, thePAT059650-WO-PCT double stranded region has 14 nucleotide base pairs in length. In some embodiments, the double stranded region has 15 nucleotide base pairs in length. In some embodiments, the double stranded region has 16 nucleotide base pairs in length. In some embodiments, the double stranded region has 17 nucleotide base pairs in length. In some embodiments, the double stranded region has 18 nucleotide base pairs in length. In some embodiments, the double stranded region has 19 nucleotide base pairs in length. In some embodiments, the double stranded region has 20 nucleotide base pairs in length. In some embodiments, the double stranded region has 21 nucleotide base pairs in length. In some embodiments, the double stranded region has 22 nucleotide base pairs in length. In some embodiments, the double stranded region has 23 nucleotide base pairs in length.

[0196] In certain aspects, the dsRNA described herein comprise one or more mismatches, for example, in the duplex region or in other regions. A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide, which comprises a phosphate- internucleoside linkage but no base). In a certain aspect, a nucleotide modification in the sugar or phosphate is also not considered a mismatch. For example, if one sequence comprises a G, and the complementary sequence comprises a modified C (e.g., 2′-modification) at the same position, no mismatch would be counted.

[0197] In an aspect, a dsRNA as described herein may include at least one single-stranded nucleotide overhang, for example, for increasing in vivo effectiveness of the dsRNA and having substantially improved inhibition of the target genes. In certain aspects, the dsRNA may contain one or more extra nucleotides constituting overhang regions that locate other than the double stranded region at the 3′-end, 5′-end, or both ends of either stand or both strands (sense and antisense strands). In some embodiments, the overhang region may exist at the 3′-end, 5′- end, or both ends of the sense strand. In some embodiments, the overhang region may exist at the 3′-end, 5′-end, or both ends of the antisense strand. In some embodiments, the antisense strand may have a greater length than a length in the sense strand. In some embodiments, the antisense strand may have a shorter length than a length in the sense strand.

[0198] In some embodiments, the dsRNA may contain one or more extra nucleotides constituting overhang regions at the 3′-end, 5′-end, or both ends of the antisense strand. In some embodiments, the overhang region in the antisense strand may consist of 1 to 6 nucleotides in length, for example, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides in length. In some embodiments, the dsRNA may contain one orPAT059650-WO-PCT more extra nucleotides constituting overhang regions at the 3′-end, 5′-end, or both ends of the sense strand. In some embodiments, the overhang region may consist of 1 to 6 nucleotides in length, for example, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides in length.

[0199] In some embodiments, the antisense strand may include one-nucleotide overhang at the 5′ end. In some embodiments, the antisense strand may include one-nucleotide overhang at the 3′ end. In some embodiments, the antisense strand may include two- nucleotides overhang. In some embodiments, the antisense contains two-nucleotides overhang at the 5′ end. In some embodiments, the antisense contains two-nucleotides overhang at the 3′ end. In some embodiments, the antisense contains one-nucleotide overhang at the 5′ end and one-nucleotide overhang at the 3′ end. In some embodiments, the antisense strand may include three-nucleotide overhang. In some embodiments, the antisense contains three-nucleotides overhang at the 5′ end. In some embodiments, the antisense contains three-nucleotides overhang at the3′ end. In some embodiments, the antisense contains two-nucleotides overhang at the 5′ end and one-nucleotide overhang at the 3′ end. In some embodiments, the antisense contains two nucleotides overhang at the 3′ end and one- nucleotide overhang at the 5′ end.

[0200] In certain aspects, a dsRNA as described herein may include at least one blunt end, e.g., for increasing in vivo stability with resistance to degradation in physiological surroundings. In some embodiments, the dsRNA may have a blunt end at the 3′-end, 5′-end, or both ends of the duplex. In some embodiments, the dsRNA includes one overhang (e.g., at 3′ end of antisense strand) and one blunt end (e.g., at 5′ end of antisense strand). In some embodiments, the dsRNA includes a blunt end at the 5′-end of the sense strand (and at 3′ end of the antisense strand) and contain overhang nucleotide(s) at the other end. In some embodiments, the dsRNA may have a blunt end at the 3′-end of the sense strand (and at 5′ end of the antisense strand) and contain overhang nucleotide(s) at the other end.

[0201] The sequences of the single strands (i.e., sense strand and antisense strand) of the dsRNA can be selected by selecting a target region (or target sequence) and a length in the target gene. In certain aspects, the antisense strand includes a region that is substantially or fully complementary to the target sequence and the target sequence is typically referred to an mRNA transcribed from the target gene.

[0202] The dsRNA disclosed herein may include a complementary sequence that can bind or hybridize with one or more of the regions in the open reading frame (ORF), 5′ untranslatedPAT059650-WO-PCT region (5′ UTR), 3′ UTR, exons, and introns in the mRNA of the target gene. In certain aspects, the target region where the antisense strand can bind or hybridize may include sequences, which can be identified as “hot spot” regions for strong downregulation of the target gene expression and / or activity. In certain aspects, the target sequence includes at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence of the hot region. In certain aspects, the target sequence may include all the nucleotides in the hot spot region.

[0203] In certain aspects, the antisense strand includes a complementary (e.g., partially, substantially, or fully complementary) sequence to the target sequence (e.g., the hot spot region or the target region in the mRNA of the target gene). In certain aspects, the antisense strand of the dsRNA includes a nucleotide sequence complementary (e.g., partially, substantially, or fully complementary) to about 10 or more (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of the target sequence (e.g., the hot spot region in the mRNA of the target gene).

[0204] In certain aspects, the sense strand of the dsRNA includes a nucleotide sequence that is identical or substantially identical to about 15 or more (e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of an mRNA sequence comprising at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a target sequence (e.g., mRNA sequence of the target gene).

[0205] In an aspect, the disclosure provides a set of modification patterns determined or arranged by modified nucleotides in dsRNAs described herein. Aside from or in addition to the nucleobase sequences, various arrangements of modified nucleotides and the modification patterns thereof can be introduced, for example, to increase stability in a biological or physiological surrounding, to facilitate or promote cleavage by the RNA-induced silencing complex, and / or to mitigate or reduce off-targeting risk (e.g., to off-targeting risk).

[0206] In an aspect, the disclosure provides a dsRNA that is partially (e.g., greater than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of the total nucleotides), substantially (e.g., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total nucleotides), or entirely made of modified nucleotides, which can provide improved resistance to chemical and / or nuclease digestion and increased in vivo stability thereby imposing a longer in vivo half-life. Further, increasing the in vivo half-life of the dsRNA results in enhanced bioavailability and enhanced effectiveness in inhibiting expression or activity of a target gene. For example, the stability of dsRNA in blood or serum may bePAT059650-WO-PCT determined, e.g., by its susceptibility to degradation by the cellular enzymes, which may be dependent on the characteristics (e.g., sequences, modification, modification pattern, or other chemical moieties) of each strand (i.e., sense strand or antisense strand) of the dsRNA. Thus, In certain aspects, the efficiency of dsRNA as a therapeutic agent may be improved by increasing the in vivo stability (e.g., in blood or serum) of the dsRNA while maintaining the ability of the dsRNA to mediate RNA interference in vivo.

[0207] The modified nucleotides as used herein contain one or more modifications, for example, the modified nucleotides contain at least one chemical modification or replacement in an internucleoside linkage (“linkage”), a nucleobase, and / or a sugar moiety of the nucleotide. Non-limiting examples include a 2′-modification on a ribose sugar ring (e.g., 2′-deoxy, 2′-O- alkyl, 2′-halo, 2′-O-alkoxylalkyl, 2′-O-amino alkyl, etc.), 3′-modification (e.g., substitution) in backbone phosphate group, or 4′-modification on a ribose sugar ring (e.g., 4′-thio RNA). Also, other non-limiting examples of the modified nucleotides may include a deoxy-nucleotide (e.g., deoxy at 2′, 3′, and / or 4′ position of a ribose ring), a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy- modified nucleotide, a 2′-5′-linked ribonucleotide (3′-RNA), a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl- modified nucleotide, 2′- hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a modified nucleotide with a non-natural nucleobase, a tetrahydropyran modified nucleotide, a 1,5- anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′- phosphate mimic, a thermally destabilizing nucleotide, a glycol nucleic acid (GNA), a nucleotide comprising a 2′ phosphate, and a 2-O-(N-methylacetamide) modified nucleotide.

[0208] In some embodiments, each of the modified nucleotides is independently selected from LNA, GNA, 2′-O-alkoxyalkyl modified nucleotide, 2′-O-alkyl modified nucleotide, 2′- O-allyl modified nucleotide, 2′-C-allyl modified nucleotide, 2′-halo modified nucleotide, and 2′-deoxy modified nucleotide (DNA). The term alkyl, alkoxyl, allyl, amino, and halo can be interpreted as described above. In some embodiments, the modified nucleotides include at least one LNAs. In some embodiments, the modified nucleotides include at least one GNAs. In some embodiments, the modified nucleotides include at least one 2′-O-alkoxyalkyl modifiedPAT059650-WO-PCT nucleotides. In some embodiments, the modified nucleotides include at least one 2′-O-alkyl modified nucleotides. In some embodiments, the modified nucleotides include at least one 2′- O-allyl modified nucleotides. In some embodiments, the modified nucleotides include at least one 2′-C-allyl modified nucleotides. In some embodiments, the modified nucleotides include at least one 2′-halo (e.g., -F) modified nucleotides. In some embodiments, the modified nucleotides include at least one 2′-deoxy modified nucleotides (DNA).

[0209] In some embodiments, each of the modified nucleotides contain independently selected from LNA modification, GNA modification, 2′-O-alkoxyalkyl modification, 2′-O- alkyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-halo modification, and 2′-deoxy modification (DNA). The term alkyl, alkoxyl, allyl, amino, and halo can be interpreted as described above. In some embodiments, the modified nucleotides include at least one LNAs. In some embodiments, the modified nucleotides include at least one GNAs. In some embodiments, the modified nucleotides include at least one 2′-O-alkoxyalkyl modifications. In some embodiments, the modified nucleotides include at least one 2′-O-alkyl modifications. In some embodiments, the modified nucleotides include at least one 2′-O-allyl modifications. In some embodiments, the modified nucleotides include at least one 2′-C-allyl modifications. In some embodiments, the modified nucleotides include at least one 2′-halo (e.g., -F) modifications. In some embodiments, the modified nucleotides include at least one 2′-deoxy modifications (DNA).

[0210] In some embodiments, the modified nucleotide may be a bicyclic (or bridged) nucleic acid (“BNA”) having a covalent linkage between the 2′ and 4′ carbons on a ribose sugar. In some embodiments, the modified nucleotide is a locked RNA (“LNA”) having covalent linkage of a bicyclic sugar modification is a 4′-CH2-O-2′ linkage (methylene oxy),also known as “LNA having a a pharmaceuticallyacceptable salt thereof.

[0211] In some embodiments, a ribose ring may be replaced with a glycol moiety linkedto phosphate and the GNA includes a a pharmaceutically acceptablePAT059650-WO-PCTsalt thereof. In some embodiments, the GNA may have a structureor apharmaceutically acceptable salt thereof. In some embodiments, the phosphodiester linkage in the GNA may be modified, e.g., with phosphorothioate group and modified GNA may have the structure a pharmaceutically acceptable salt thereof. The GNA may further replacing hydrogen(s) and such modified GNA maybe of GNA herein.

[0212] In some embodiments, a ribose pentofuranosyl ring may be replaced with a threofuranosyl ring linked to the phosphate and the threofuranosyl nucleotide (TNA) may include a a pharmaceutically acceptable salt thereof. In someembodiments, the TNA may have a a pharmaceutically acceptable salt thereof. In some embodiments,in the TNA may be modified, e.g., with phosphorothioate group and modified TNA may include a structure of a pharmaceutically acceptable salt thereof. The TNA may further includeat 1′, 3′ and / or 4′ positions and such modified TNA may be encompassed by the definition of TNA herein.PAT059650-WO-PCT

[0213] In some embodiments, a ribose ring may not include a base and an abasicnucleotide has a a pharmaceutically acceptable salt thereof.

[0214] In certain may include a heterocyclic group (e.g., 5 to 6 memberedof a ribose ring. In some embodiments, the ribose ring may be replaced with a morpholinyl ring, e.g., to form an morpholino oligonucleotide. In some embodiments, the ribose ring may be replaced with an arabinose ring.

[0215] In certain aspects, the modified nucleotides contain one or more modification groups at 2′ position on the ribose ring by replacing 2′-OH. In some embodiments, the modification group may be hydrogen (i.e. deoxy), halogen (e.g., -F), substituted or unsubstituted alkyl (e.g., C1-C12 alkyl), or substituted or unsubstituted heteroalkyl (e.g., -O- (C1-C12alkyl), -N-(C1-C12alkyl), -C(O)NH-(C1-C12alkyl), -NHC(O)-(C1-C12alkyl), or -C(O)- (C1-C12 alkyl)). In some embodiments, the modification group may be hydrogen, -F, -O-alkyl (e.g., C1-C4alkyl), or -O-alkoxyalkyl (e.g., -O-(C1-C4alkylene)-(C1-C4alkoxyl)). Any of the alkyl, heteroalkyl, alkylene in the disclosure are optionally substituted with one or more of hydroxyl (-OH), C1-C3alkyl (e.g., methyl, or ethyl), amine (e.g., monoamine or diamine), alkoxyl (e.g., -O-CH3 (OMe) or -O-CH2CH3 (OEt)), halogen (e.g., -F) or the like.

[0216] In certain aspects, the modified nucleotides may include one or more of 2′-deoxy modification, 2′-O-alkyl modification, 2′-O-subsituted alkyl modification, 2′-O-alkoxyalkyl modification, and 2′-O-aminoalkyl modification. In some embodiments, the modified nucleotides may include one or more of 2′-deoxy modification, 2′-O-alkyl modification, 2′-O- subsituted alkyl modification, 2′-O-alkoxyalkyl modification, and 2′-O-aminoalkyl modification. In some embodiments, the modified nucleotides include at least one GNAs. In some embodiments, the modified nucleotides include at least one 2′-O-alkoxyalkyl modifications. In some embodiments, the modified nucleotides include at least one 2′-O-alkyl modifications. In some embodiments, the modified nucleotides include at least one 2′-O-allyl modifications. In some embodiments, the modified nucleotides include at least one 2′-C-allyl modifications. In some embodiments, the modified nucleotides include at least one 2′-halo (e.g., -F) modifications. In some embodiments, the modified nucleotides include at least onePAT059650-WO-PCT 2′-deoxy modifications (DNA). In some embodiments, the modified nucleotides do not include 2′-deoxy modifications (DNA).

[0217] In certain aspects, the modified nucleotides may include one or more of 2′-deoxy nucleotide (DNA), 2′-O-methyl (2′-OMe) modification, 2′-flouro (2′-F) modification, 2′-O- methoxyethyl (2′-O-MOE or “2′-MOE”) modification, 2′-O-aminopropyl (2′-O-AP) modification, 2′-O-dimethylaminoethyl (2′-O-DMAOE) modification, 2′-O- dimethylaminopropyl (2′-O-DMAP) modification, 2′-O-dimethylaminoethyloxyethyl (2′-O- DMAEOE) modification, and 2′-O-N-methylacetamido (2′-O-NMA) modification. In some embodiments, the modified nucleotides may include at least one 2′-deoxy modification (DNA). In some embodiments, the modified nucleotides may include at least one 2′-O-methyl (2′-OMe) modification. In some embodiments, the modified nucleotides may include at least one 2′- flouro (2′-F) modification. In some embodiments, the modified nucleotides may include at least one 2′-O-methoxyethyl (2′-O-MOE or “2′-MOE”) modification. In some embodiments, the modified nucleotides may include at least one 2′-O-aminopropyl (2′-O-AP) modification. In some embodiments, the modified nucleotides may include at least one 2′-O- dimethylaminoethyl (2′-O-DMAOE) modification. In some embodiments, the modified nucleotides may include at least one 2′-O-dimethylaminopropyl (2′-O-DMAP) modification. In some embodiments, the modified nucleotides may include at least one 2′-O- dimethylaminoethyloxyethyl (2′-O-DMAEOE) modification. In some embodiments, the modified nucleotides may include at least one 2′-O-N-methylacetamido (2′-O-NMA) modification.

[0218] In some embodiments, each modified nucleotide containing a modification on a 2′ sugar ring may optionally contain a phosphorothioate group at 5′ or 3′ linkage. In some embodiments, each modified nucleotide containing a modification on a 2′ sugar ring may optionally contain a modification such as an abasic modification (absence of a nucleobase) or methylated nucleobase modification at nucleobase (e.g., thymine (T) or 5-methyl cytosine (5mC)).

[0219] In certain aspects, the dsRNA is partially (e.g., greater than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of the total nucleotides), substantially (e.g., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total nucleotides), or entirely made of modified nucleotides containing the modification on 2′ sugar ring. In some embodiments, the dsRNA is partially (e.g., greater than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of the total nucleotides) made of modified nucleotides containing thePAT059650-WO-PCT modification on 2′ sugar ring. In some embodiments, the dsRNA is substantially (e.g., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total nucleotides) made of modified nucleotides containing the modification on 2′ sugar ring. In some embodiments, the dsRNA includes greater than about 80% of modified nucleotides containing the modification on 2′ sugar ring based on the total nucleotides. In some embodiments, the dsRNA includes greater than about 85% of modified nucleotides containing the modification on 2′ sugar ring based on the total nucleotides. In some embodiments, the dsRNA includes greater than about 90% of modified nucleotides containing the modification on 2′ sugar ring based on the total nucleotides. In some embodiments, the dsRNA includes greater than about 95% of modified nucleotides containing the modification on 2′ sugar ring based on the total nucleotides. In some embodiments, the dsRNA is entirely made of modified nucleotides containing the modification on 2′ sugar ring.

[0220] In certain aspects, the modified nucleotide may include a modification in a phosphate group or, in other words, an internucleoside linkage modification (e.g., phosphorothioate, phosphorodithioate, methylphosphonate, methylene phosphonate, or vinyl phosphonate (VP) linkage). In some embodiments, the linkage modification may include phosphorothioate (PS) having a structure or its pharmaceutically acceptable salt form, which may be an Rp isomer orIn some embodiments, the linkage modification may include phosphorothioate (PS) having a structure or its pharmaceutically acceptable salt form, which may be a stereopureIn some embodiments, the linkage modification may include phosphorothioate (PS) having a structure O of or its pharmaceutically acceptable salt form, which may be a stereopure SpPAT059650-WO-PCT

[0221] For example, the modified nucleotide including 3′-PS modification can be a pharmaceutically acceptable salt thereof, wherein R represents -F, -CH3, -OMe, or MOE) and is an attachment pointto the some the 3′-PS group may be a stereopure Sp isomer. In some embodiments, the 3′-PS group may be a stereopure Rp isomer .

[0222] In certain aspects, the first nucleotide from the 5′ end of each strand (e.g., sense strand and antisense strand) may include an additional phosphate group or a variant thereof (e.g., phosphorothioate, phosphorodithioate, methylphosphonate, methylene phosphonate, or vinyl phosphonate (VP)) attached or linked to the 5′ terminal group of the first nucleotide.

[0223] In some embodiments, the first nucleotide from the 5′ end of each strand (e.g., sense strand and antisense strand) includes a 5′-vinyl phosphonate (5′-VP) group that is a chemical moiety having the structure of , or a pharmaceutically acceptable salt thereof, wherein represents the point of attachment to the 5′ carbon of the pentofuranosyl sugar of a nucleotide. In some embodiments, the first nucleotide from the 5′ end of each strand (e.g., sense strand and antisense strand) may include (E)-vinyl phosphonate (VP) having a structure , or a pharmaceutically acceptable salt thereof, wherein represents the pointto the 4′ carbon of the pentofuranosyl sugar. In some embodiments, the first nucleotide from the 5′ end of each strand (e.g., sense strand and antisense strand) may include (Z)-vinyl phosphonate having a structure of , or a pharmaceutically acceptable salt thereof, wherein represents the point of attachment to the 4′ carbon of the pentofuranosyl sugar.

[0224] In certain aspects, one or more of the modified nucleotides contain a 2′ modification (e.g., 2′-OMe, 2′-F, 2′-MOE, 2′-deoxy, etc.) and an internucleoside linkagePAT059650-WO-PCT modification (e.g., phosphorothioate or (E)-vinyl phosphonate). In some embodiments, one or more of the modified nucleotides contain 2′-OMe modification and phosphorothioate group. In some embodiments, one or more of the modified nucleotides contain 2′-OMe modification and (E)-vinyl phosphonate group. In some embodiments, one or more of the modified nucleotides contain 2′-F modification and phosphorothioate group. In some embodiments, one or more of the modified nucleotides contain 2′-F and (E)-vinyl phosphonate group. In some embodiments, one or more of the modified nucleotides contain 2′-MOE modification and phosphorothioate group. In some embodiments, one or more of the modified nucleotides contain 2′-MOE modification and (E)-vinyl phosphonate group. In some embodiments, one or more of the modified nucleotides contain 2′-deoxy modification and phosphorothioate group. In some embodiments, one or more of the modified nucleotides contain 2′-OMe modification and (E)-vinyl phosphonate group. In some embodiments, one or more of the modified nucleotides are GNA containing (E)-vinyl phosphonate group. In some embodiments, one or more of the modified nucleotides are GNA containing a phosphorothioate group.

[0225] In certain aspects, the modified nucleotides contain one or more modifications on a modified nucleobase. In some embodiments, one or more of the modified nucleotides may include thymine (“T”) nucleobase (“ribothymidine” or “5-methyluridine”) in the ribonucleotide (e.g., including 2′-OH). In some embodiments, one or more of the modified nucleotides may include methylcytosine nucleobase (e.g., 5-methylcytidine (5mC) or N4- methylcytidine (4mC)). In certain aspects, one or more of the modified nucleotides may contain no nucleobase (base). Sense Strand (SS)

[0226] In certain aspects, a sense strand of the dsRNA as described herein are substantially (e.g., greater than about 80%, 85%, 90%, or 95% of the total nucleotides) made of modified nucleotides. In another certain aspect, the sense strand is entirely made of modified nucleotides.

[0227] In certain aspects, a sense strand of the dsRNA as described herein includes two or more 2′MOE modifications. In some embodiments, the sense strand includes two, four, six or eight 2′-MOE modifications. In some embodiments, the sense strand includes two 2′- MOE modifications. In some embodiments, the sense strand includes four 2′-MOE modifications. In some embodiments, the sense strand includes six 2′-MOE modifications. In some embodiments, the sense strand includes eight 2′-MOE modifications.PAT059650-WO-PCT

[0228] In some embodiments, the 2′-MOE modified nucleotides in the sense strand as described herein include a , or a pharmaceutically acceptable salt thereof, whereinto a linkage (e.g., phosphodiester or phosphorothioate linkage) adjacent nucleotides and “Base” is a nucleobase.

[0229] In some the 2′-MOE modified nucleotide at the first position from the 3′-end of the sense strand as described herein includes a structure ofadjacent nucleotides and “Base” is a nucleobase. In some embodiments, the 2′-MOE modified nucleotide at the first position from the 3′-end of the sense strand as described ,PAT059650-WO-PCT

[0230] In some embodiments, the 2′-MOE modified nucleotides include a structure ofnucleotides. In embodiments, the 2′-MOE modified nucleotides include a structure ofa pharmaceutically acceptable salt thereof. the 2′-MOE modified nucleotides include a structure ofa pharmaceutically acceptable saltinclude a structure of a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0232] In some embodiments, the 2′-MOE modified nucleotides include a nucleotide aa terminal group (e.g., H or salt) or the adjacent some embodiments, the 2′-MOEmodified nucleotides include a nucleotide having a or a pharmaceutically acceptable salt thereof.

[0233] In some embodiments, the 2′-MOE modified nucleotides include a nucleotide amodifiedPAT059650-WO-PCT nucleotides include a nucleotide having a a pharmaceutically acceptable salt thereof.

[0234] In some embodiments, the 2′-MOEas described herein include a nucleotide having a ,a pharmaceutically acceptable salt thereof, wherein is(e.g., H or salt) or the adjacent nucleotides. In some embodiments, the 2′-MOE modified nucleotides in the dsRNA as described herein include a nucleotide having a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0235] In some embodiments, the 2′-MOE modified nucleotides include a nucleotide aterminal group (e.g., H or salt) or the adjacent some embodiments, the 2′-MOEmodified nucleotides include a nucleotide having a , or a pharmaceutically acceptable salt thereof.

[0236] In certain aspects, at least one of the 2′-MOE modified nucleotides in the sense strand as described herein has a apharmaceutically acceptable salt group (e.g., H or salt) or the adjacentcertain aspects, at least one of the 2′-PAT059650-WO-PCT MOE modified nucleotides in the sense strand as described herein has a structure of a pharmaceutically acceptable salt thereof. the first nucleotide from the 5′ end of the sense strandincludes a a pharmaceutically acceptable saltadjacent nucleotide. In some embodiments, the firstfrom the 5′ end of the sense strand includes a structure of a pharmaceutically acceptable salt thereof.the first nucleotide from the 3′ end of the sense strand a pharmaceutically acceptable salt thereof.the 3′ end of the sense strand includes aPAT059650-WO-PCT a pharmaceutically acceptable salt thereof, wherein some embodiments, the first nucleotide from endof the sense strand includes a acceptable salt thereof.

[0239] In certain aspects, at least one sense strand as described herein has a ,a pharmaceutically acceptable salt thereof, is angroup (e.g., H, OH or salt) or the adjacentIn some embodiments, at least one of the 2′-MOE modified nucleotides in the sense strand asPAT059650-WO-PCT described herein has a acceptable salt thereof. In of thesense strand has a a pharmaceutically acceptable salt thereof. In some the 3′ end of the sense stranda pharmaceutically acceptable salt thereof,In some embodiments, the first nucleotide from the 3′ end of the sense strand has a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0240] In some embodiments, the 2′-MOE modified nucleotides in the sense strand as agroup (e.g., H, OH, or salt) or the adjacent and “Base” is a nucleobase. In someembodiments, the 2′-MOE modified nucleotides in the sense strand as described herein include a a pharmaceutically acceptable salt thereof.

[0241] In modified nucleotides include a structure ofa pharmaceutically acceptable saltgroup (e.g., H, OH, or salt) or the adjacentIn some embodiments, the 2′-MOE modified nucleotides include a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0242] In some embodiments, the 2′-MOE modified nucleotides include a structure of a pharmaceutically acceptable salt include a structure ofinclude a nucleotidehaving a , or a pharmaceuticallyto a terminal group (e.g., H, OH, or salt) or the adjacentIn some embodiments, the 2′-MOE modified nucleotides include a nucleotide having a or a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0244] In some embodiments, the 2′-MOE modified nucleotides include a nucleotide a modifiednucleotides include a nucleotide having a , or a pharmaceutically acceptable salt thereof.

[0245] In some embodiments, the 2′-MOE modified nucleotides in the sense strand as described herein include a nucleotide having a ,a pharmaceutically acceptable salt thereof, is(e.g., H, OH, or salt) or the adjacentIn some embodiments, the 2′-MOE modified nucleotides in the sense strand as described hereinPAT059650-WO-PCT include a nucleotide having a , or a pharmaceutically acceptable salt

[0246] In some embodiments, a nucleotide aterminal group (e.g., H, OH, or salt) or the adjacentIn some embodiments, the 2′-MOE modified nucleotides include a nucleotide having a or a pharmaceutically acceptable salt thereof.

[0247] In certain aspects, at least one of the 2′-MOE modified nucleotides in the sense strand as described herein has a ,PAT059650-WO-PCT a pharmaceutically acceptable salt thereof, wherein is an group (e.g., H, OH, or salt) or the adjacent Inone of the 2′-MOE modified nucleotides in the sense strand as described herein has a a pharmaceutically acceptable salt thereof.

[0248] In some embodiments, the first nucleotide from the 5′ end of the sense strand includes a a pharmaceutically acceptable saltfrom the 5′ end of the sense strand includes a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0249] In some embodiments, the first nucleotide from the 3′ end of the sense strand includes a a pharmaceutically acceptable salt the 3′ end of theasome embodiments, the first nucleotide fromend of the sense strand includes a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0250] In certain aspects, at least one of the 2′-MOE modified nucleotides in the sense strand as described herein has a ,a pharmaceutically acceptable salt thereof, wherein is an group (e.g., H, OH, or salt) or the adjacentInone of the 2′-MOE modified nucleotides in the sense strand as described herein has a a pharmaceutically acceptable salt thereof.

[0251] In some embodiments, the first nucleotide from the 5′ end of the sense strand includes a a pharmaceutically acceptable saltthe 5′ end of thePAT059650-WO-PCT sense strand includes a a pharmaceutically acceptable salt thereof.

[0252] In some3′ end of the sense strand, anPAT059650-WO-PCT attachment point to a ligand. In some embodiments, the first nucleotide from the 3′ end of the sense strand includes a acceptable salt thereof.

[0253] In certain aspects, atsense strand as described herein has aadjacentIn some embodiments, at least one of the 2′-MOE modified nucleotides in the sense strand as described herein has a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT

[0254] In some embodiments, the first nucleotide from the 5′ end of the sense strand includes a a pharmaceutically from the5′ end of the sense strand includes a a pharmaceutically acceptable salt thereof.

[0255] In some embodiments, the first sense strand includes a a pharmaceuticallyfrom the3′ end of the sense strand includes a ,PAT059650-WO-PCTnucleotide3′ end of the sense strand includes a structure of a pharmaceutically acceptable salt thereof.at least one of the 2′-MOE modified nucleotides in the sensePAT059650-WO-PCT acceptable salt thereof, wherein is an attachment point to a terminal group (e.g., H, OH, or salt) or the adjacent In some embodiments, at least one of the 2′-MOE modifiednucleotides in the sense strand as described herein has a structure of a pharmaceutically acceptable salt thereof. first nucleotide from the 5′ end of the sense stranda the5′ end of the sense strand includes a a pharmaceutically acceptable salt thereof.

[0258] In some embodiments, the first nucleotide from the 3′ end of the sense strand orPAT059650-WO-PCT the 3′ end of the sense strand includes a ,end of the sense strand includes a a pharmaceutically acceptable salt

[0259] In certain aspects, the 2′-MOE modified nucleotides locate at both 5′ and 3′ ends of a sense strand so as to form a structural confinement (“2′-MOE clamp”) at the sense strand termini. In some embodiments, the 2′-MOE clamps may be symmetric and having the samePAT059650-WO-PCT number of 2′-MOE modified nucleotides at both 5′ and 3′ ends of the sense strand. For example, the sense strand includes one 2′-MOE modified nucleotide at 5′ end and one 2′- MOE modified nucleotide at 3′ end; two 2′-MOE modified nucleotides at 5′ end and two 2′- MOE modified nucleotides at 3′ end; or three 2′-MOE modified nucleotides at 5′ end and three 2′-MOE modified nucleotides at 3′ end. In some embodiments, the 2′-MOE clamps may be asymmetric and having different numbers of 2′-MOE nucleotides at 5′ and 3′ ends of the sense strand. For example, the sense strand includes one 2′-MOE modified nucleotide at 5′ end only; one 2′-MOE modified nucleotide at 3′ end only; two 2′-MOE modified nucleotides at 5′ end only; two 2′-MOE modified nucleotides at 3′ end only; one 2′-MOE modified nucleotide at 5′ end and two 2′-MOE modified nucleotides at 3′ end; or two 2′-MOE modified nucleotides at 5′ end and one 2′-MOE modified nucleotide at 3′ end.

[0260] In certain aspects, the sense strand includes one 2′-MOE modified nucleotide at 5′ end and one 2′-MOE modified nucleotide at 3′end. In some embodiments, the sense strand includes only one 2′-MOE modified nucleotide at 5′ end and only one 2′-MOE modified nucleotide at 3′end. In some embodiments, the sense strand includes only one 2′-MOE modified nucleotide at 5′ end. In some embodiments, the sense strand includes only one 2′- MOE modified nucleotide at 3′end.

[0261] In certain aspects, the sense strand includes at least two contiguous 2′-MOE modified nucleotides at 5′ end and at least two 2′-MOE modified nucleotides at 3′end. In some embodiments, the sense strand includes only two 2′-MOE modified nucleotides at 5′ end and only two 2′-MOE modified nucleotides at 3′end. In some embodiments, the sense strand includes only two 2′-MOE modified nucleotides at 5′ end. In some embodiments, the sense strand includes only two 2′-MOE modified nucleotides at 3′end.

[0262] In certain aspects, the sense strand includes two or four of the 2′-MOE modified nucleotides. In some embodiments, the sense strand includes only two of the 2′-MOE modified nucleotides. In some embodiments, the sense the sense strand includes only four of the 2′-MOE modified nucleotides.

[0263] In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand includes one, two, three, or four 2′-MOE modified nucleotides positioned at the 1st, 2nd, 20th, and 21st nucleotides from the 5′ end of the sense strand. In some embodiments, the sense strand includes two 2′-MOE modified nucleotides positioned at the 1st, 2nd, 20th, and 21st nucleotides from the 5′ end of the sense strand. In some embodiments, the sense strand includes three 2′-MOE modified nucleotides positioned at thePAT059650-WO-PCT 1st, 2nd, 20th, and 21st nucleotides from the 5′ end of the sense strand. In some embodiments, the sense strand includes 2′-MOE modified nucleotides positioned at the 1st, 2nd, 20th, and 21st nucleotides from the 5′ end of the sense strand. In some embodiments, the sense strand does not include a 2′-MOE modified nucleotide at the 3rd to 19th nucleotides from 5′ end of the sense strands.

[0264] In certain aspects, a sense strand of the dsRNA as described herein includes two or more 2′-F modifications. In some embodiments, the sense strand of the dsRNA includes two, three, four, five, six, seven, or eight 2′-F modified nucleotides. In some embodiments, the sense strand includes two 2′-F modified nucleotides. In some embodiments, the sense strand includes three 2′-F modified nucleotides. In some embodiments, the sense strand includes four 2′-F modified nucleotides. In some embodiments, the sense strand includes five 2′-F modified nucleotides. In some embodiments, the sense strand includes six 2′-F modified nucleotides. In some embodiments, the sense strand includes seven 2′-F modified nucleotides. In some embodiments, the sense strand includes eight 2′-F modified nucleotides. In some embodiments, two contiguous 2′-F modified nucleotides locate in the sense strand. In some embodiments, three contiguous 2′-F modified nucleotides locate in the sense strand. In some embodiments, four contiguous 2′-F modified nucleotides locate in the sense strand.

[0265] In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, 2′-F modified nucleotides locate at 5th, 7th, 8th, and / or 9th positions from the 5′ end of the sense strand. In some embodiments, 2′-F modified nucleotides locate at 6th, 8th, 9th, and / or 10th positions from the 5′ end of the sense strand. In some embodiments, 2′-F modified nucleotides locate at 7th, 9th, 10th, and / or 11th positions from the 5′ end of the sense strand. In some embodiments, 2′-F modified nucleotides locate at 8th, 10th, 11th, and / or 12th positions from the 5′ end of the sense strand. In some embodiments, 2′-F modified nucleotides locate at 9th, 11th, 12th, and / or 13th positions from the 5′ end of the sense strand.

[0266] In certain aspect, a sense strand of the dsRNA as described herein includes one, two, three, or four 2′-deoxy modifications (DNA). In some embodiments, the sense strand of the dsRNA includes one 2′-deoxy modified nucleotide. In some embodiments, the sense strand includes two 2′-deoxy modified nucleotides. In some embodiments, the sense strand includes three 2′-deoxy modified nucleotides. In some embodiments, the sense strand includes four 2′-deoxy modified nucleotides.PAT059650-WO-PCT

[0267] In certain aspect, a sense strand of the dsRNA as described herein includes one, two, three, or four deoxythymidines (dT). In some embodiments, the sense strand of the dsRNA includes one deoxythymidine (dT). In some embodiments, the sense strand includes two deoxythymidines. In some embodiments, the sense strand includes three deoxythymidines (dT). In some embodiments, the sense strand includes four deoxythymidines.

[0268] In some embodiments, the sense strand is 21 nucleotides in length. In some embodiments, one or more 2′-deoxy modified nucleotides locate at 5th, 7th, 8th, and / or 9th positions from the 5′ end of the sense strand. In some embodiments, one or more 2′-deoxy modified nucleotides locate at 6th, 8th, 9th, and / or 10th positions from the 5′ end of the sense strand. In some embodiments, one or more 2′-deoxy modified nucleotides locate at 7th, 9th, 10th, and / or 11th positions from the 5′ end of the sense strand. In some embodiments, one or more 2′-deoxy modified nucleotides locate at 8th, 10th, 11th, and / or 12th positions from the 5′ end of the sense strand. In some embodiments, one or more 2′-deoxy modified nucleotides locate at 9th, 11th, 12th, and / or 13th positions from the 5′ end of the sense strand.

[0269] In some embodiments, the sense strand is 21 nucleotides in length and one 2′- deoxy modified nucleotide locates at 7th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 8th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 9th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 10th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 11th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 12th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 13th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one 2′-deoxy modified nucleotide locates at 14th position from the 5′ end of the sense strand.

[0270] In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 7th position from the 5′ end of the sense strand. In somePAT059650-WO-PCT embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 8th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 9th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 10th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 11th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 12th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 13th position from the 5′ end of the sense strand. In some embodiments, the sense strand is 21 nucleotides in length and one deoxythymidine (dT) locates at 14th position from the 5′ end of the sense strand.

[0271] In some embodiments, when the sense strand is 21 nucleotides in length, 2′-F modified nucleotides locate at 5th, 7th, and 8th from the 5′ end of the sense strand and one deoxythymidine (dT) locates at 9th position from the 5′ end of the sense strand. In some embodiments, when the sense strand is 21 nucleotides in length, 2′-F modified nucleotides locate at 6th, 8th, and 9th from the 5′ end of the sense strand and one deoxythymidine (dT) locates at 10th position from the 5′ end of the sense strand. In some embodiments, when the sense strand is 21 nucleotides in length, 2′-F modified nucleotides locate at 7th, 9th, and 10th from the 5′ end of the sense strand and one deoxythymidine (dT) locates at 11th position from the 5′ end of the sense strand. In some embodiments, when the sense strand is 21 nucleotides in length, 2′-F modified nucleotides locate at 8th, 10th, and 11th from the 5′ end of the sense strand and one deoxythymidine (dT) locates at 12th position from the 5′ end of the sense strand. In some embodiments, when the sense strand is 21 nucleotides in length, 2′-F modified nucleotides locate at 9th, 11th, and 12th from the 5′ end of the sense strand and one deoxythymidine (dT) locates at 13th position from the 5′ end of the sense strand.

[0272] In some embodiments, the sense strand includes 2′-OMe modified nucleotides in the remaining positions in the sense strand.

[0273] In certain aspects, the sense strand includes one to six phosphorothioate (PS) linkages between nucleosides. In some embodiments, the sense strand includes one, two, three, or four phosphorothioate (PS) linkages between nucleosides.PAT059650-WO-PCT

[0274] In some embodiments, the sense strand includes a 3′-PS linkage in the 1st nucleotide from 5′-end of the sense strand. In some embodiments, the sense strand includes two 3′-PS linkages in the 1st and 2nd, nucleotides from 5′-end of the sense strand. In some embodiments, the sense strand includes three 3′-PS linkages in the 1st, 2nd, and 3rd nucleotides from 5′-end of the sense strand. In some embodiments, the sense strand includes four 3′-PS linkages in the 1st, 2nd, 3rd, and 4th nucleotides from 5′-end of the sense strand.

[0275] In some embodiments, the sense strand includes a 3′-PS linkage in the 1st nucleotide from 3′-end of the sense strand. In some embodiments, the sense strand includes two 3′-PS linkages in the 1st and 2nd, nucleotides from 3′-end of the sense strand. In some embodiments, the sense strand includes three 3′-PS linkages in the 1st, 2nd, and 3rd nucleotides from 3′-end of the sense strand. In some embodiments, the sense strand includes four 3′-PS linkages in the 1st, 2nd, 3rd, and 4th nucleotides from 3′-end of the sense strand.

[0276] In some embodiments, the sense strand includes 3′-PS modified nucleotides at the 1st and 2nd positions from 5′-end of the sense strand. In some embodiments, the sense strand includes only two 3′-PS modified nucleotides positioned at the 1st and 2nd nucleotides from 5′-end of the sense strand. In some embodiments, the sense strand includes only two 3′-PS modified nucleotides at the 1st and 2nd positions from 3′-end of the sense strand. In some embodiments, the sense strand includes two 3′-PS modified nucleotides at the 1st and 2nd positions from 5′-end of the sense strand and two 3′-PS modified nucleotides at the 1st and 2nd positions from 3′-end of the sense strand.

[0277] In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand includes two 3′-PS modified nucleotides at the 1st, 2nd, 19th and / or 20th positions from 5′ end of the sense strand. In some embodiments, the sense strand includes three 3′-PS modified nucleotides at the 1st, 2nd, 19th and / or 20th positions from 5′ end of the sense strand. In some embodiments, the sense strand includes 3′-PS modified nucleotides at the 1st, 2nd, 19th and 20th positions from 5′ end of the sense strand.

[0278] In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand includes 3′-PS modified nucleotides at the 1st and 2nd positions from 5′ end of the sense strand. In some embodiments, the sense strand includes a 3′-PS modified nucleotide at the 19th and 20th position from 5′ end of the sense strand. In some embodiments, the sense strand includes 3′-PS modified nucleotides at the 1st and 20th positions from 5′ end of the sense strand. In some embodiments, the sense strand includes 3′-PAT059650-WO-PCT PS modified nucleotides at the 1st, 2nd, 19th and 20th positions from 5′ end of the sense strand.

[0279] In certain aspects, the sense strand includes two to eight phosphorothioate (PS) groups or linkages between nucleosides. In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand includes two 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 17th, 18th, 19th and / or 20th nucleotides from 5′ end of the sense strand. In some embodiments, the sense strand includes four 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 17th, 18th, 19th and / or 20th nucleotides from 5′ end of the sense strand. In some embodiments, the sense strand includes six 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 17th, 18th, 19th and / or 20th nucleotides from 5′ end of the sense strand. In some embodiments, the sense strand includes 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 17th, 18th, 19th and 20th nucleotides from 5′ end of the sense strand.

[0280] In some embodiments, at least one of the 3′-PS groups of the sense strand described above is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 1st nucleotide from 5′-end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 5′-end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 3rd nucleotide from 5′-end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 4th nucleotide from 5′-end of the sense strand is a stereopure Rp isomer.

[0281] In some embodiments, the 3′-PS group at the 1st nucleotide from 3′-end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 3′-end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 3rd nucleotide from 3′-end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 4th nucleotide from 3′-end of the sense strand is a stereopure Rp isomer.

[0282] In some embodiments, at least one of the 3′-PS groups of the sense strand described above is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 1st nucleotide from 5′-end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 5′-end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 3rd nucleotide from 5′-end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 4th nucleotide from 5′-end of the sense strand is a stereopure Sp isomer.PAT059650-WO-PCT

[0283] In some embodiments, the 3′-PS group at the 1st nucleotide from 3′-end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 3′-end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS groups at the 3rd nucleotide from 3′-end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 4th nucleotide from 3′-end of the sense strand is a stereopure Sp isomer.

[0284] In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 3rd, 4th, 17th, 18th, 19th and / or 20th nucleotides from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 19th and / or 20th nucleotides from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st and / or 20th nucleotides from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 1st nucleotide from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 19th nucleotide from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 20th nucleotide from 5′ end of the sense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS groups at the 1st and 20th nucleotides from 5′ end of the sense strand are stereopure Rp isomers. In some embodiments, the 3′-PS groups at the 1st, 2nd, 19thand 20th nucleotides from 5′ end of the sense strand are stereopure Rp isomers.

[0285] In certain aspects, the sense strand is 21 nucleotides in length. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 3rd, 4th, 17th, 18th, 19th and / or 20th nucleotides from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 19th and / or 20th nucleotides from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st and / or 20th nucleotides from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 1st nucleotide from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 19th nucleotide from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 20nd nucleotide from 5′ end of the sense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS groups atPAT059650-WO-PCT the 1st and 20th nucleotides from 5′ end of the sense strand are stereopure Sp isomers. In some embodiments, the 3′-PS groups at the 1st, 2nd, 19th, and 20th nucleotides from 5′ end of the sense strand are stereopure Sp isomers.

[0286] In certain aspects, a sense strand of the dsRNA as described herein includes one or more of MOE modified nucleotides, one or more of 2′-F modified nucleotides, one or more of 2′-deoxy modified nucleotides, and one or more of 2′-OMe modified nucleotides. In certain aspects, a sense strand of the dsRNA as described herein consists of one or more of MOE modified nucleotides, one or more of 2′-F modified nucleotides, one or more of 2′- deoxy modified nucleotides, and one or more of 2′-OMe modified nucleotides.

[0287] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and / or 21st nucleotides from the 5′ end of the sense strand; and (ii) 3′-PS modifications at the 1st, 2nd, 19th, and / or 20th nucleotides from 5′ end of the sense strand.

[0288] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and / or 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′ end of the sense strand; 2′-deoxy modification at 11th nucleotide; and 2′-OMe modifications in the remaining nucleotides.

[0289] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and / or 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′ end of the sense strand; and 2′-deoxy modification at 11th nucleotide; and (ii) 3′-PS modifications at the 1st and 2nd nucleotides from 5′ end of the sense strand.

[0290] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and / or 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′ end of the sense strand; and 2′-deoxy modification at 11th nucleotide; and (ii) 3′-PS modifications at the 1st, 2nd, 19th, and 20th nucleotides from 5′ end of the sense strand.

[0291] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and / or 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′PAT059650-WO-PCT end of the sense strand; 2′-deoxy modification at 11th nucleotide; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st and 2nd nucleotides from 5′ end of the sense strand.

[0292] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and / or 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′ end of the sense strand; 2′-deoxy modification at 11th nucleotide; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st, 2nd, 19th, and 20th nucleotides from 5′ end of the sense strand.

[0293] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′ end of the sense strand; 2′-deoxy modification at 11th nucleotide; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st and 2nd nucleotides from 5′ end of the sense strand.

[0294] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 7th, 9th, and 10th nucleotides from the 5′ end of the sense strand; 2′-deoxy modification at 11th nucleotide; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st, 2nd, 19th, and 20th nucleotides from 5′ end of the sense strand.

[0295] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 9th, 10th and 11th nucleotides from the 5′ end of the sense strand; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st and 2nd nucleotides from 5′ end of the sense strand.

[0296] In some embodiments, the sense strand having 21 nucleotides in length includes: (i) 2′-MOE modifications at the 1st, 2nd, 20th and 21st nucleotides from the 5′ end of the sense strand; 2′-F modifications at 9th, 10th and 11th nucleotides from the 5′PAT059650-WO-PCT end of the sense strand; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st, 2nd, 19th, and 20th nucleotides from 5′ end of the sense strand.

[0297] Exemplary modification patterns of sense strands are shown in Table A. Table A 21-mer SS 2′-MOE 2′-F modified 2′-deoxy 2′-OMe 3′-PS linkage modification modified nucleotide modified modified pattern No. nucleotide position nucleotide nucleotide position position position SS21-1 7, 9, 10, 11 1, 2, 3, 4, 5, 1, 2 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 SS21-2 1 7, 9, 10, 11 2, 3, 4, 5, 6, 1, 2 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 SS21-3 1 7, 9, 10 11 2, 3, 4, 5, 6, 1, 2 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 SS21-4 21 7, 9, 10, 11 1, 2, 3, 4, 5, 1, 2 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20 SS21-5 21 7, 9, 10 11 1, 2, 3, 4, 5, 1, 2 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20 SS21-6 1, 21 7, 9, 10, 11 2, 3, 4, 5, 6, 1, 2, 19, 20 8, 12, 13, 14, 15, 16, 17, 18, 19, 20 SS21-7 1, 21 7, 9, 10 11 2, 3, 4, 5, 6, 1, 2, 19, 20 8, 12, 13, 14, 15, 16, 17, 18, 19, 20 SS21-8 1, 2, 20, 21 7, 9, 10, 11 3, 4, 5, 6, 8, 1, 2 12, 13, 14, 15, 16, 17, 18, 19 SS21-9 1, 2, 20, 21 7, 9, 10 11 3, 4, 5, 6, 8, 1, 2 12, 13, 14, 15, 16, 17, 18, 19 SS21-10 1, 2, 20, 21 7, 9, 10, 11 3, 4, 5, 6, 8, 1, 2, 19, 20 12, 13, 14,PAT059650-WO-PCT 15, 16, 17, 18, 19 SS21-11 1, 2, 20, 21 7, 9, 10 11 3, 4, 5, 6, 8, 1, 2, 19, 20 12, 13, 14, 15, 16, 17, 18, 19 SS21-12 9, 10, 11 1, 2, 3, 4, 5, 1, 2 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 SS21-13 1, 2, 20, 21 9, 10, 11 3, 4, 5, 6, 7, 1, 2 8, 12, 13, 14, 15, 16, 17, 18, 19 SS21-14 1, 2, 20, 21 9, 10, 11 3, 4, 5, 6, 7, 1, 2, 19, 20 8, 12, 13, 14, 15, 16, 17, 18, 19 SS21-15 9, 10, 11 1, 2, 3, 4, 5, 1, 2, 19, 20 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21

[0298] In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-1. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-2. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-3. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-4. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-5. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-6. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-7. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-8. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-9. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-10. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-11. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-12. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-13. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-14. In some embodiments, the sense strand having 21 nucleotides in length has the modification pattern of SS21-15.PAT059650-WO-PCT Antisense Strand (AS)

[0299] In certain aspects, an antisense strand of the dsRNA as described herein are substantially (e.g., greater than about 80%, 85%, 90%, or 95% of the total nucleotides) made of modified nucleotides. In another certain aspect, the antisense strand is entirely made of modified nucleotides.

[0300] In certain aspects, the first nucleotide from the 5′ end of the antisense strand may contain an additional phosphate group or a variant thereof (e.g., phosphorothioate, phosphorodithioate, methylphosphonate, methylene phosphonate, or vinyl phosphonate (VP)) attached or linked to the 5′ terminal group of the first nucleotide) attached or linked to the 5′ terminal group of the first nucleotide.

[0301] In certain aspects, the antisense strand includes 5′-vinyl phosphonate (5′-VP) group at the first nucleotide from the 5′ end in the antisense strand. The “5′-VP” is a chemical moiety having the structure of , or a pharmaceutically acceptable salt thereof, where the wavy line represent the point of attachment to the 5′ carbon of the pentofuranosyl sugar of a nucleotide.

[0302] In some embodiments, the first nucleotide from the 5′ end in the antisense strand includes (E)-vinyl phosphonate (VP) having a structure , or a pharmaceutically acceptable salt thereof, wherein thethe point of attachment to the 4′ carbon of the pentofuranosyl sugar of a nucleotide. In some embodiments, the first nucleotide from the 5′ end in the antisense strand includes (Z)-vinyl phosphonate having a structure of , or a pharmaceutically acceptable salt thereof, wherein the wavy line presents the point of attachment to the 4′ carbon of the pentofuranosyl sugar of a nucleotide.PAT059650-WO-PCT

[0303] In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure of a pharmaceutically acceptable saltnucleotide (5′ carbon) and “Base” is a In some embodiments, the first nucleotide from the 5′end of the antisense strand has a acceptable salt thereof, wherein is ancarbon) and “Base” is aIn some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure of ,a pharmaceutically acceptable salt thereof, is annucleotide(5′ carbon) and “Base” is aIn some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure ofPAT059650-WO-PCT a pharmaceutically acceptable salt thereof, wherein is an nucleotide(5′ carbon) and “Base” is athe first nucleotide from the 5′ end of the strand O OH OH NH has a athe adjacent nucleotide (5′ carbon). In some embodiments,nucleotide from the 5′ end of the O OH NH antisense strand has a a pharmaceutically acceptable salt thereof. Infrom the 5′ end of the antisense strand has a ,PAT059650-WO-PCT or a pharmaceutically acceptable salt thereof, wherein is an attachment point to the adjacent nucleotide (5′ carbon). In some the first nucleotide from the 5′ end of the antisense strand has a acceptable salt thereof, whereincarbon).

[0305] In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a aadjacent nucleotide (5′ carbon). In some embodiments,nucleotide from the 5′ end of the antisense strand has a a pharmaceutically acceptable salt thereof. Infrom the 5′ end of thePAT059650-WO-PCT antisense strand has a , or a pharmaceuticallyadjacent nucleotide (5′ carbon). In some first nucleotide from the 5′ end ofthe antisense strand has a acceptable salt thereof, whereincarbon).

[0306] In some embodiments, the first nucleotide from the 5′ end of the antisense strand aadjacent nucleotide (5′ carbon). In some embodiments,nucleotide from the 5′ end of thePAT059650-WO-PCT a pharmaceutically from the 5′ end of theantisense strand has a ,a pharmaceutically acceptable salt thereof, wherein isnucleotide (5′ carbon). In some embodiments,first nucleotide from the 5′ end of the antisense strand has a structure of a pharmaceutically acceptable salt thereof, is an(5′ carbon).PAT059650-WO-PCT

[0307] In some embodiments, the first nucleotide from the 5′ end of the antisense strand ornucleotide (5′ carbon). In some embodiments, nucleotide from the 5′ end of theantisense strand has a a pharmaceutically acceptable salt thereof. Inthe 5′ end of the antisense strand has a ,a pharmaceutically acceptable salt thereof,PAT059650-WO-PCT is an attachment point to the adjacent nucleotide (5′ carbon). In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure of a pharmaceutically acceptable salt thereof, wherein nucleotide (5′ carbon).some first nucleotide from the 5′ end of the antisense strand has a structure of acceptable saltembodiments, the firstfrom the 5′ end of the antisense strand has a structure of a pharmaceutically acceptable salt thereof, wherein is annucleotide. In some embodiments, the firstfrom the 5′ end of the antisense strand has a structure of ,PAT059650-WO-PCT a pharmaceutically acceptable salt thereof, wherein is an nucleotide. In some embodiments, the first fromathe adjacent nucleotide.

[0309] In some embodiments, the first nucleotide from the 5′ end of the antisense strand athe adjacent nucleotide. In some embodiments, the firstfrom the 5′ end of the antisense strand has a a pharmaceutically acceptable salt thereof,wherein is an attachment point to the adjacent nucleotide. In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure ofPAT059650-WO-PCT a pharmaceutically acceptablenucleotide. In some embodiments, thenucleotide from the 5′ end of the antisense strand has a structure of a pharmaceutically acceptable salt thereof, wherein is an nucleotide.

[0310] In some embodiments, the first nucleotide from the 5′ end of the antisense strand aadjacent nucleotide. In some embodiments, the firstfrom the 5′ end of the antisense strand a pharmaceutically acceptable salt thereof,PAT059650-WO-PCT wherein is an attachment point to the adjacent nucleotide. In some embodiments, the first from the 5′ end of the antisense strand has a structure of a pharmaceutically acceptablenucleotide. In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure of a pharmaceutically acceptable salt thereof, wherein is annucleotide.

[0311] In some embodiments, the first nucleotide from the 5′ end of the antisense strand ,adjacent nucleotide. In some embodiments, the firstfrom the 5′ end of thePAT059650-WO-PCT antisense strand has a a pharmaceutically acceptable salt thereof,nucleotide. In some embodiments, the first from the 5′ end of the antisense strand has a structure ofathe adjacent nucleotide. In some embodiments, the firstfrom the 5′ end of the antisense strand has a a pharmaceutically acceptable saltthereof, nucleotide.PAT059650-WO-PCT

[0312] In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a anucleotide. In some embodiments, the first from the 5′ end of the antisense stranda pharmaceutically acceptable saltnucleotide. In some embodiments, the firstfrom the 5′ end of the antisense strand has a structure of a pharmaceuticallynucleotide. In some embodiments, the first nucleotide from the 5′ end of the antisense strand has a structure ofPAT059650-WO-PCT a pharmaceutically acceptable salt thereof, wherein is nucleotide.strand of the dsRNA as described herein two or more 2′-F modifications. In some embodiments, the antisense strand of the dsRNA includes two, three, four, five, six, seven, or eight 2′-F modified nucleotides. In some embodiments, the antisense strand includes two 2′-F modified nucleotides. In some embodiments, the antisense strand includes three 2′-F modified nucleotides. In some embodiments, the antisense strand includes four 2′-F modified nucleotides. In some embodiments, the antisense strand includes five 2′-F modified nucleotides. In some embodiments, the antisense strand includes six 2′-F modified nucleotides. In some embodiments, the antisense strand includes seven 2′-F modified nucleotides. In some embodiments, the antisense strand includes eight 2′-F modified nucleotides. In some embodiments, two contiguous 2′-F modified nucleotides locate in the antisense strand. In some embodiments, three contiguous 2′-F modified nucleotides locate in the antisense strand. In some embodiments, four contiguous 2′-F modified nucleotides locate in the antisense strand.

[0314] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense includes comprises two, three, or four 2′-F modifications positioned at the 2nd, 6th, 14th, and / or 16th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense includes two 2′-F modifications positioned at the 2nd, 6th, 14th, and / or 16th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense includes three 2′-F modifications positioned at the 2nd, 6th, 14th, and / or 16th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense includes 2′-F modifications positioned at the 2nd, 6th, 14th, and 16th nucleotide from 5′ end of the antisense strand.

[0315] In certain aspects, an antisense strand of the dsRNA as described herein does not include a 2′-MOE modification. Alternatively, in certain aspects, the antisense strand includes one to four 2′-MOE modified nucleotides. In some embodiments, the antisensePAT059650-WO-PCT strand includes one 2′-MOE modified nucleotide. In some embodiments, the antisense strand includes two 2′-MOE modified nucleotides. In some embodiments, the antisense strand includes three 2′-MOE modified nucleotides. In some embodiments, the antisense strand includes four 2′-MOE modified nucleotides.

[0316] In certain aspects, the antisense strand includes at least one GNA. In some embodiments, the antisense strand includes only one GNA.

[0317] In certain aspects, the antisense strand includes at least one GNA. In some embodiments, the antisense strand includes only one GNA. In some embodiments, the antisense strand includes only one GNA at the 3rd nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 4th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 5th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 6th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 7th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 8th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 9th nucleotide from 5′-end of the antisense strand.

[0318] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand includes only one GNA at the 3rd nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 4th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 5th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 6th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one GNA at the 7th nucleotide from 5′ end of the antisense strand.

[0319] In certain aspects, the antisense strand includes at least one TNA. In some embodiments, the antisense strand includes only one TNA.

[0320] In certain aspects, the antisense strand includes at least one TNA. In some embodiments, the antisense strand includes only one TNA. In some embodiments, the antisense strand includes only one TNA at the 3rd nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 4th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strandPAT059650-WO-PCT includes only one TNA at the 5th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 6th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 7th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 8th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 9th nucleotide from 5′- end of the antisense strand.

[0321] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand includes only one TNA at the 3rd nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 4th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 5th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 6th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one TNA at the 7th nucleotide from 5′ end of the antisense strand.

[0322] In certain aspects, the antisense strand includes at least one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC). In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC).

[0323] In certain aspects, the antisense strand includes at least one 2′-deoxy modified nucleotides (e.g., dT, dA, dG, or dC). In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC). In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 3rd nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 4th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 5th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 6th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 7th nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 8thPAT059650-WO-PCT nucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 9th nucleotide from 5′-end of the antisense strand.

[0324] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 3rd nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 4th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 5th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 6th nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes only one 2′-deoxy modified nucleotide (e.g., dT, dA, dG, or dC) at the 7th nucleotide from 5′ end of the antisense strand.

[0325] In certain aspects, the antisense strand includes one to eight phosphorothioate (PS) linkages between nucleosides. In some embodiments, the antisense strand includes one, two, three, or four phosphorothioate (PS) linkages between nucleosides.

[0326] In some embodiments, the antisense strand includes a 3′-PS linkage in the 1stnucleotide from 5′-end of the antisense strand. In some embodiments, the antisense strand includes two 3′-PS linkages in the 1stand 2nd, nucleotides from 5′-end of the antisense strand. In some embodiments, the antisense strand includes three 3′-PS linkages in the 1st, 2nd, and 3rdnucleotides from 5′-end of the antisense strand. In some embodiments, the antisense strand includes four 3′-PS linkages in the 1st, 2nd, 3rd, and 4thnucleotides from 5′-end of the antisense strand.

[0327] In some embodiments, the antisense strand includes a 3′-PS linkage in the 1stnucleotide from 3′-end of the antisense strand. In some embodiments, the antisense strand includes two 3′-PS linkages in the 1stand 2nd, nucleotides from 3′-end of the antisense strand. In some embodiments, the antisense strand includes three 3′-PS linkages in the 1st, 2nd, and 3rdnucleotides from 3′-end of the antisense strand. In some embodiments, the antisense strand includes four 3′-PS linkages in the 1st, 2nd, 3rd, and 4thnucleotides from 3′-end of the antisense strand.

[0328] In some embodiments, the antisense strand includes 3′-PS modified nucleotides at the 1stand 2ndpositions from 5′-end of the antisense strand. In some embodiments, thePAT059650-WO-PCT antisense strand includes only two 3′-PS modified nucleotides positioned at the 1stand 2ndnucleotides from 5′-end of the antisense strand. In some embodiments, the antisense strand includes only two 3′-PS modified nucleotides at the 1stand 2ndpositions from 3′-end. In some embodiments, the antisense strand includes two 3′-PS modified nucleotides at the 1stand 2ndpositions from 5′-end of the antisense strand and two 3′-PS modified nucleotides at the 1stand 2ndpositions from 3′-end of the antisense strand.

[0329] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand includes two 3′-PS modified nucleotides at the 1st, 2nd, 21st and / or 22nd positions from 5′ end of the antisense strand. In some embodiments, the antisense strand includes three 3′-PS modified nucleotides at the 1st, 2nd, 21st and / or 22nd positions from 5′ end of the antisense strand. In some embodiments, the antisense strand includes 3′-PS modified nucleotides at the 1st, 2nd, 21st and / or 22nd positions from 5′ end of the antisense strand.

[0330] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand includes 3′-PS modified nucleotides at the 1st and 2nd positions from 5′ end of the antisense strand. In some embodiments, the antisense strand includes a 3′-PS modified nucleotide at the 21st and 22nd position from 5′ end of the antisense strand. In some embodiments, the antisense strand includes 3′-PS modified nucleotides at the 1st and 20th positions from 5′ end of the antisense strand. In some embodiments, the antisense strand includes 3′-PS modified nucleotides at the 1st, 2nd, 21st and 22nd positions from 5′ end of the antisense strand.

[0331] In certain aspects, the antisense strand includes two to eight phosphorothioate (PS) groups or linkages between nucleosides. In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand includes two 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 19th, 20th, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand. In some embodiments, the antisense strand includes four 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 19th, 20th, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand. In some embodiments, the antisense strand includes six 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 19th, 20th, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand. In some embodiments, the antisense strand includes 3′-PS modified nucleotides positioned at the 1st, 2nd, 3rd, 4th, 19th, 20th, 21st, and 22nd nucleotides from 5′ end of the antisense strand.PAT059650-WO-PCT

[0332] In some embodiments, at least one of the 3′-PS groups of the antisense strand described above is a stereopure Rp isomer. In some embodiments, the 3′-PS groups at the 1stnucleotide from 5′-end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 2ndnucleotide from 5′-end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 3rdnucleotide from 5′- end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 4thnucleotide from 5′-end of the antisense strand is a stereopure Rp isomer.

[0333] In some embodiments, the 3′-PS group at the 1stnucleotide from 3′-end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 2ndnucleotide from 3′-end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 3rdnucleotide from 3′-end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 4thnucleotide from 3′- end of the antisense strand is a stereopure Rp isomer.

[0334] In some embodiments, at least one of the 3′-PS groups of the antisense strand described above is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 1stnucleotide from 5′-end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 2ndnucleotide from 5′-end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 3rdnucleotide from 5′- end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 4thnucleotide from 5′-end of the antisense strand is a stereopure Sp isomer.

[0335] In some embodiments, the 3′-PS group at the 1stnucleotide from 3′-end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 2ndnucleotide from 3′-end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS groups at the 3rdnucleotide from 3′-end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS groups at the 4thnucleotide from 3′- end of the antisense strand is a stereopure Sp isomer.

[0336] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 3rd, 4th, 18th, 19th, 20th, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 21st and / or 22nd nucleotides from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st and / or 22nd nucleotides from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group atPAT059650-WO-PCT the 1st nucleotide from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 21st nucleotide from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS group at the 22nd nucleotide from 5′ end of the antisense strand is a stereopure Rp isomer. In some embodiments, the 3′-PS groups at the 1st and 22nd nucleotides from 5′ end of the antisense strand are stereopure Rp isomers. In some embodiments, the 3′-PS groups at the 1st, 2nd, 21st and 22nd nucleotides from 5′ end of the antisense strand are stereopure Rp isomers.

[0337] In certain aspects, the antisense strand is 23 nucleotides in length. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 3rd, 4th, 18th, 19th, 20th, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st, 2nd, 21st and / or 22nd nucleotides from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, at least one of the 3′-PS groups at the 1st and / or 22nd nucleotides from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 1st nucleotide from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 2nd nucleotide from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 21st nucleotide from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS group at the 22nd nucleotide from 5′ end of the antisense strand is a stereopure Sp isomer. In some embodiments, the 3′-PS groups at the 1st and 22nd nucleotides from 5′ end of the antisense strand are stereopure Sp isomers. In some embodiments, the 3′-PS groups at the 1st, 2nd, 21st and 22nd nucleotides from 5′ end of the antisense strand are stereopure Sp isomers.

[0338] In certain aspects, the antisense strand includes 5′-(E)-VP modified nucleotide at the first nucleotide from 5′ end of the antisense strand. In some embodiments, the antisense strand includes a 5′-(E)-VP-2′-OMe modified nucleotide at the first nucleotide from 5′ end of the antisense strand.

[0339] In some embodiments, an antisense strand of the dsRNA as described herein includes one 5′-(E)-VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, and one or more of 2′-OMe modified nucleotides. In some embodiments, an antisense strand of the dsRNA as described herein consists of one 5′-(E)-VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, and one or more of 2′-OMe modified nucleotides.PAT059650-WO-PCT

[0340] In some embodiments, an antisense strand of the dsRNA as described herein includes one 5′-(E)-VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, one GNA, and one or more of 2′-OMe modified nucleotides. In some embodiments, an antisense strand of the dsRNA as described herein consists of one 5′-(E)- VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, one GNA, and one or more of 2′-OMe modified nucleotides.

[0341] In some embodiments, an antisense strand of the dsRNA as described herein includes one 5′-(E)-VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, one TNA, and one or more of 2′-OMe modified nucleotides. In some embodiments, an antisense strand of the dsRNA as described herein consists of one 5′-(E)- VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, one TNA, and one or more of 2′-OMe modified nucleotides.

[0342] In some embodiments, an antisense strand of the dsRNA as described herein includes one 5′-(E)-VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, one 2′-deoxy modified nucleotides, and one or more of 2′-OMe modified nucleotides. In some embodiments, an antisense strand of the dsRNA as described herein consists of one 5′-(E)-VP-2′-OMe modified nucleotide, one or more of 2′-F modified nucleotides, one 2′-deoxy modified nucleotides, and one or more of 2′-OMe modified nucleotides.

[0343] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; and (ii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0344] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.PAT059650-WO-PCT

[0345] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; GNA at 5th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0346] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; GNA at 6th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0347] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; GNA at 7th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0348] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand;PAT059650-WO-PCT (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 3rd nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0349] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 5th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0350] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 6thnucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0351] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 7th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.PAT059650-WO-PCT

[0352] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; a 2′-deoxy modification at 5th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0353] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; a 2′-deoxy modification at 6thnucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0354] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; a 2′-deoxy modification at 7th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0355] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand;PAT059650-WO-PCT (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; GNA at 3rd and 5th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0356] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; GNA at 3rd and 6th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0357] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; GNA at 3rd and 7th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0358] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 3rd and 5th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.PAT059650-WO-PCT

[0359] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 3rd and 6th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0360] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; TNA at 3rd and 7th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0361] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; a 2′-deoxy modification at 3rd and 5th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0362] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand;PAT059650-WO-PCT (ii) 2′-F modifications at 2nd, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; a 2′-deoxy modification at 3rd and 6th nucleotide from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0363] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 2nd, 6th, 14th, and / or 16th nucleotides from the 5′ end of the antisense strand; a 2′-deoxy modification at 3rd and 7th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0364] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) 2′-F modifications at 14th nucleotide from the 5′ end of the antisense strand; a 2′- deoxy modification at 2nd, 5th, 7th and 12th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (ii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.

[0365] In some embodiments, the antisense strand having 23 nucleotides in length includes: (i) a 5′-(E)-VP-2′-OMe modification at the first nucleotide from 5′ end of the antisense strand; (ii) 2′-F modifications at 14thnucleotide from the 5′ end of the antisense strand; a 2′- deoxy modification at 2nd, 5th, 7th and 12th nucleotides from the 5′ end of the antisense strand; and 2′-OMe modifications in the remaining nucleotides; and (iii) 3′-PS modifications at the 1st, 2nd, 21st, and / or 22nd nucleotides from 5′ end of the antisense strand.PAT059650-WO-PCT

[0366] Exemplary modification patterns of antisense strands are shown in Table B. Table B 23-mer AS 5′-VP 2′F modified GNA TNA 2′-deoxy 2′-OMe 3′-PS modification modified nucleotide position position modified modified linkage pattern nucleotide nucleotide nucleotide position position position AS23-1 - 2, 6, 14, 161, 3, 4, 5, 7, 8,1, 2, 21, 22 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-2 1 2, 6, 14, 161, 3, 4, 5, 7, 8,1, 2, 21, 22 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-3 1 2, 6, 14, 16 51, 3, 4, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-4 1 2, 14, 16 61, 3, 4, 5, 7, 8,1, 2, 21, 22 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-5 1 2, 6, 14, 16 7 1, 3, 4, 5, 8, 9, 1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-6 1 2, 6, 14, 16 31, 4, 5, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-7 1 2, 6, 14, 16 51, 3, 4, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-8 1 2, 14, 16 61, 3, 4, 5, 7, 8,1, 2, 21, 22 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-9 1 2, 6, 14, 16 71, 3, 4, 5, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-10 1 2, 6, 14, 16 51, 3, 4, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-11 1 2, 14, 16 61, 3, 4, 5, 7, 8,1, 2, 21, 22 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-12 1 2, 6, 14, 16 7 1, 3, 4, 5, 8, 9, 1, 2, 21, 22 10, 11, 12, 13,PAT059650-WO-PCT 15, 17, 18, 19, 20, 21, 22, 23 AS23-13 1 2, 6, 14, 16 3, 5 1, 4, 7, 8, 9, 1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-14 1 2, 14, 16 3, 61, 4, 5, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-15 1 2, 6, 14, 16 3, 71, 4, 5, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-16 1 2, 6, 14, 16 3, 51, 4, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-17 1 2, 6, 14, 16 3, 6 1, 4, 5, 7, 8, 9, 1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-18 1 2, 14, 16 3, 71, 4, 5, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-19 1 2, 6, 14, 16 3, 51, 7, 8, 9, 10,1, 2, 21, 22 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-20 1 2, 14, 16 3, 61, 4, 5, 7, 8, 9,1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-21 1 2, 6, 14, 16 3, 7 1, 4, 5, 8, 9, 1, 2, 21, 22 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23 AS23-22 - 14 2, 5, 7, 1, 3, 4, 6, 8, 9, 1, 2, 21, 22 12 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23 AS23-23 1 14 2, 5, 7, 1, 3, 4, 6, 8, 9, 1, 2, 21, 22 12 10, 11, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23

[0367] In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-1. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-2. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-3. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-4. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-5. In some embodiments, the antisense strandPAT059650-WO-PCT having 23 nucleotides in length has the modification pattern of AS23-6. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-7. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-8. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-9. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-10. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-11. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-12. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-13. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-14. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-15. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-16. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-17. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-18. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-19. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-20. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-21. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-22. In some embodiments, the antisense strand having 23 nucleotides in length has the modification pattern of AS23-23.

[0368] In an aspect, the dsRNAi agent having the nucleotides modification patterns as described herein can improve half-life relative to a reference dsRNAi agent that does not contain such nucleotides modification patterns. In some embodiments, the dsRNAi agent having the nucleotides modification patterns as described herein improved half-life by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 7.0 fold, 8.0 fold, 9.0 fold, 10 fold, or more relative to a reference dsRNAi agent that does not contain such nucleotides modification patterns. In some embodiments, the dsRNAi agent having the nucleotides modification patterns as described herein improved half-life by about 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 7PAT059650-WO-PCT fold, or 10 fold, relative to a reference dsRNAi agent that does not contain such nucleotides modification patterns. COMPOUNDS

[0369] In an aspect, the disclosure provides a compound (e.g., dsRNAi agent) including a double stranded RNA.

[0370] In certain aspects, the dsRNAi agent for inhibiting expression of a target gene includes: (i) a sense strand comprising monomers of X1 to X23 and having the Formula (I): 5′-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22- X23-3′ (I), or a pharmaceutically acceptable salt thereof, and (ii) an antisense strand comprising monomers of X1′ to X25′ and having the Formula (II): 5′-X1′-X2′-X3′-X4′-X5′-X6′-X7′-X8′-X9′-X10′-X11′-X12′-X13′-X14′-X15′-X16′-X17′-X18′-X19′-X20′-X21′- X22′-X23′-X24′-X25′-3′ (II), or a pharmaceutically acceptable salt thereof, wherein: each X1and X1′independently has a structure of Formula (A), a pharmaceutically acceptable salt thereof,is absent or glycol nucleic acid (GNA), or has a structure of Formula (B),PAT059650-WO-PCT a pharmaceutically acceptable salt thereof, and has a structure of Formula (C),,: in each occurrence, B is independently absent or a nucleobase; in each occurrence, each R1, R2, R3, and R4is independently hydrogen, halogen, -OR11, -SR11, -NR12R13, -C(O)R14, -OC(O)R14, -C(O)NR12R13, -NR15C(O)H, - NR15C(O)OH, -NR15C(O)NR12R13, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl, wherein each R11, R12, R13, R14, and R15is independently hydrogen, or substituted or unsubstituted C1-C10 alkyl; or R2and R3combine to form a bond, or are joined together with the atoms to which they are attached to form a substituted heterocycloalkyl, and optionally R1and R4are joined together with the atoms to which they are attached to form a substituted cycloalkyl or substituted heterocycloalkyl, in each occurrence, R5is hydrogen, -OH, a detectable moiety, a first ligand, or a functional group capable of forming a covalent bond with a first ligand; in each occurrence, R6is hydrogen, a detectable moiety, -P(O)(OH)-OH, -P(O)(SH)- OH, a second ligand, or a functional group capable of forming a covalent bond with a second ligand; in each occurrence, L1is independently absent, -O-, -O-L2-, -P(O)(OH)-O-L2-, - P(O)(SH)-O-L2-, -P(O)(OH)-L3-, or -P(O)(SH)-L3-, wherein each L2is independently a substituted or unsubstituted C1-C4 alkylene and each L3is independently a substituted or unsubstituted C2-C4 alkenylene, andPAT059650-WO-PCT in each occurrence, is an attachment point to the adjacent monomers, provided that: (i) in at leastof X1 and X23, R2and R3combine to form a bond and R1is -O- CH2CH2-OCH3; (ii) in X3 to X21, when R2and R3combine to form a bond, (a) R1is not -O-CH2CH2-OCH3, (b) at least one of R1is not hydrogen, and (c) in no more than six nucleotides, R1is -F; and (iii) no more than eight monomers from X3 to X21 are absent and no more than ten monomers from X3′ to X23′ are absent.

[0371] In some embodiments, in X2, R2and R3combine to form a bond. In some embodiments, in X2, R2and R3are joined together with the atoms to which they are attached to form a 6 membered heterocycloalkyl. In some embodiments, in X2, R2and R3are joined together with the atoms to which they are attached to form a morpholine ring.

[0372] In some embodiments, in X22, R2and R3combine to form a bond. In some embodiments, in X22, R2and R3are joined together with the atoms to which they are attached to form a 6 membered heterocycloalkyl. In some embodiments, in X22, R2and R3are joined together with the atoms to which they are attached to form a morpholine ring. In some embodiments, in X2and X22, R2and R3combine to form a bond.

[0373] In some embodiments, in X3 to X21, R1is not -O-CH2CH2-OCH3. In some embodiments, in X3to X21, when R2and R3combine to form a bond, R1is not -O-CH2CH2- OCH3.

[0374] In some embodiments, in X3to X21, at least one of R1is hydrogen. In some embodiments, in X3 to X21 of the sense strand, at least one of R1is not hydrogen.

[0375] In some embodiments, in X3to X21, at least one of R1is halogen. In some embodiments, in X3 to X21, at least one of R1is -F. In some embodiments, in X3 to X21, in no more than six monomers, R1is -F.

[0376] In some embodiments, in X2to X22, at least one of R1is -OR11and R11is hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X2 to X22, at least one of R1is -OR11and R11is hydrogen. In some embodiments, in X2to X22, at least one of R1is -OR11and R11is substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X2to X22, at least one of R1is -OR11and R11is unsubstituted C1-C4alkyl. In somePAT059650-WO-PCT embodiments, in X2 to X22, at least one of R1is -OR11and R11is unsubstituted methyl, ethyl, propyl, or isopropyl.

[0377] In some embodiments, in X2 to X22, at least one of R1is independently -OR11and R11is substituted C1-C4alkyl. In some embodiments, in X2to X22, at least one of R1is independently -OR11and R11is alkoxyl-substituted C1-C4 alkyl. In some embodiments, R11is methoxy (-OCH3) substituted C1-C4alkyl. In some embodiments, R11is methoxyethyl (-CH2- CH2-OCH3). In some embodiments, R11is methoxyethyl (-CH2-OCH3). In some embodiments, R1is -O-CH2CH2-OCH3. In some embodiments, R1is -O-CH2-OCH3.

[0378] In some embodiments, in X2 to X22, at least one of R1is independently -NR12R13and each R12and R13is independently hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X2 to X22, at least one of R1is independently -NR12R13and each R12andR13is independently hydrogen. In some embodiments, in X2 to X22, at least one of R1is independently -NR12R13and each R12and R13is independently unsubstituted C1-C4 alkyl. In some embodiments, each R12and R13is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0379] In some embodiments, in X2 to X22, at least one of R1is independently -C(O)R14and R14is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X2to X22, at least one of R1is independently -C(O)R14and R14is hydrogen. In some embodiments, in X2to X22, at least one of R1is independently -C(O)R14and R14is unsubstituted C1-C4 alkyl. In some embodiments, each R14 isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0380] In some embodiments, in X2 to X22, at least one of R1is independently - OC(O)R14and R14is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X2 to X22, at least one of R1is independently - OC(O)R14and R14is hydrogen. In some embodiments, in X2to X22, at least one of R1is independently - OC(O)R14and R14is unsubstituted C1-C4 alkyl. In some embodiments, each R14 isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0381] In some embodiments, in X2 to X22, at least one of R1is independently - C(O)NR12R13and each R12and R13is independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X2 to X22, at least one of R1is independently - C(O)NR12R13and each R12and R13is independently hydrogen. In some embodiments, in X2to X22, at least one of R1is independently - C(O)NR12R13and each R12and R13isPAT059650-WO-PCT independently unsubstituted C1-C4 alkyl. In some embodiments, each R12and R13is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0382] In some embodiments, in X2 to X22, at least one of R1is independently -- NR15C(O)R14and each R15and R14is independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X2 to X22, at least one of R1is independently - NR15C(O)H and R15is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X2 to X22, at least one of R1is independently - NR15C(O)CH3 and R15is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X2to X22, at least one of R1is independently - NR15C(O)H and R15is unsubstituted C1-C4 alkyl. In some embodiments, each R15is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0383] In some embodiments, in X2 to X22, at least one of R1is independently - NR15C(O)OH and R15is hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X2 to X22, at least one of R1is independently - NR15C(O)OH and R15is hydrogen. In some embodiments, in X2 to X22, at least one of R1is independently - NR15C(O)OH and R15is unsubstituted C1-C4alkyl. In some embodiments, each R15is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0384] In some embodiments, in X2to X22, at least one of R1is independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, in X2 to X22, at least one of R1is independently substituted or unsubstituted C1-C4alkyl. In some embodiments, in X2to X22, at least one of R1is independently substituted C1-C4 alkyl. In some embodiments, in X2 to X22, at least one of R1is independently unsubstituted C1-C4alkyl. In some embodiments, in X2 to X22, at least one of R1is independently substituted or unsubstituted methyl. In some embodiments, in X2to X22, at least one of R1is independently substituted or unsubstituted ethyl.

[0385] In some embodiments, in X2to X22, at least one of R1is independently substituted or unsubstituted 2 to 6 membered heteroalkyl. In some embodiments, in X2 to X22, at least one of R1is independently substituted or unsubstituted 2 to 5 membered heteroalkyl. In some embodiments, in X2 to X22, at least one of R1is independently substituted or unsubstituted 2 to 4 membered heteroalkyl. In some embodiments, in X2to X22, at least one of R1is independently substituted or unsubstituted 2 to 3 membered heteroalkyl. In some embodiments, in X2to X22, at least one of R1is independently -O-CH3. In some embodiments, in X2 to X22, at least one of R1is independently -O-CH2-CH3.PAT059650-WO-PCT

[0386] In some embodiments, in X2, R1is -O-CH2CH2-OCH3. In some embodiments, in X22, R1is -O-CH2CH2-OCH3.In some embodiments, in X2and X22, R1is -O-CH2CH2- OCH3. In some embodiments, in X2 and X22, R2and R3combine to form a bond and R1is -O- CH2CH2-OCH3. In some embodiments, in X1, X2, X22, and X23, R2and R3combine to form a bond and R1is -O-CH2CH2-OCH3.

[0387] In some embodiments, in X3to X21, at least one of R1is -OR11and R11is hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X3 to X21, at least one of R1is -OR11and R11is hydrogen. In some embodiments, in X3to X21, at least one of R1is -OR11and R11is substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X3to X21, at least one of R1is -OR11and R11is unsubstituted C1-C4alkyl. In some embodiments, in X3 to X21, at least one of R1is -OR11and R11is unsubstituted methyl, ethyl, propyl, or isopropyl.

[0388] In some embodiments, in X3 to X21, at least one of R1is independently -OR11and R11is substituted C1-C4 alkyl. In some embodiments, in X3 to X21, at least one of R1is independently -OR11and R11is alkoxyl-substituted C1-C4alkyl. In some embodiments, R11is methoxy (-OCH3) substituted C1-C4 alkyl. In some embodiments, R11is methoxyethyl (-CH2- CH2-OCH3). In some embodiments, R11is methoxyethyl (-CH2-OCH3). In some embodiments, R1is -O-CH2CH2-OCH3. In some embodiments, R1is -O-CH2-OCH3.

[0389] In some embodiments, in X3to X21, at least one of R1is independently -NR12R13and each R12and R13is independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X3to X21, at least one of R1is independently -NR12R13and each R12and R13is independently hydrogen. In some embodiments, in X3 to X21, at least one of R1is independently -NR12R13and each R12and R13is independently unsubstituted C1-C4alkyl. In some embodiments, each R12and R13is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0390] In some embodiments, in X3 to X21, at least one of R1is independently -C(O)R14and R14is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X3to X21, at least one of R1is independently -C(O)R14and R14is hydrogen. In some embodiments, in X3to X21, at least one of R1is independently -C(O)R14and R14is unsubstituted C1-C4 alkyl. In some embodiments, each R14 isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0391] In some embodiments, in X3 to X21, at least one of R1is independently - OC(O)R14and R14is hydrogen or substituted or unsubstituted C1-C4 alkyl. In somePAT059650-WO-PCT embodiments, in X3 to X21, at least one of R1is independently - OC(O)R14and R14is hydrogen. In some embodiments, in X3to X21, at least one of R1is independently - OC(O)R14and R14is unsubstituted C1-C4 alkyl. In some embodiments, each R14 isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0392] In some embodiments, in X3 to X21, at least one of R1is independently - C(O)NR12R13and each R12and R13is independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X3 to X21, at least one of R1is independently - C(O)NR12R13and each R12and R13is independently hydrogen. In some embodiments, in X3to X21, at least one of R1is independently - C(O)NR12R13and each R12and R13is independently unsubstituted C1-C4alkyl. In some embodiments, each R12and R13is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0393] In some embodiments, in X3 to X21, at least one of R1is independently -- NR15C(O)R14and each R15and R14is independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X3 to X21, at least one of R1is independently - NR15C(O)H and R15is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X3 to X21, at least one of R1is independently - NR15C(O)CH3 and R15is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X3to X21, at least one of R1is independently - NR15C(O)H and R15is unsubstituted C1-C4 alkyl. In some embodiments, each R15is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0394] In some embodiments, in X3 to X21, at least one of R1is independently - NR15C(O)OH and R15is hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in X3 to X21, at least one of R1is independently - NR15C(O)OH and R15is hydrogen. In some embodiments, in X3to X21, at least one of R1is independently - NR15C(O)OH and R15is unsubstituted C1-C4 alkyl. In some embodiments, each R15is independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0395] In some embodiments, in X3 to X21, at least one of R1is independently substituted or unsubstituted C1-C6alkyl. In some embodiments, in X3to X21, at least one of R1is independently substituted or unsubstituted C1-C4 alkyl. In some embodiments, in X3 to X21, at least one of R1is independently substituted C1-C4alkyl. In some embodiments, in X3to X21, at least one of R1is independently unsubstituted C1-C4 alkyl. In some embodiments, in X3to X21, at least one of R1is independently substituted or unsubstituted methyl. In some embodiments, in X3 to X21, at least one of R1is independently substituted or unsubstituted ethyl.PAT059650-WO-PCT

[0396] In some embodiments, in X3 to X21, at least one of R1is independently substituted or unsubstituted 2 to 6 membered heteroalkyl. In some embodiments, in X3to X21, at least one of R1is independently substituted or unsubstituted 2 to 5 membered heteroalkyl. In some embodiments, in X3to X21, at least one of R1is independently substituted or unsubstituted 2 to 4 membered heteroalkyl. In some embodiments, in X3 to X21, at least one of R1is independently substituted or unsubstituted 2 to 3 membered heteroalkyl. In some embodiments, in X3 to X21, at least one of R1is independently -O-CH3. In some embodiments, in X3to X21, at least one of R1is independently -O-CH2-CH3.

[0397] In some embodiments, in X3 to X21, when R2and R3combine to form a bond, R1is not -O-CH2CH2-OCH3.In some embodiments, in X3to X21,when R2and R3combine to form a bond, at least one of R1is not hydrogen. In some embodiments, in X3 to X21, when R2and R3combine to form a bond, in no more than six monomers, R1is -F.

[0398] In some embodiments, in X1, L1is -P(O)(OH)-O-L2-, or -P(O)(SH)-O-L2-. In some embodiments, L2is unsubstituted C1-C4 alkylene. In some embodiments, in X1, L1is - P(O)(OH)-O-L2-and L2is unsubstituted C1-C2alkylene. In some embodiments, in X1, L1is - P(O)(SH)-O-L2and L2is unsubstituted methylene. In some embodiments, in X1, L1is - P(O)2-O-CH2- and R5is OH. In some embodiments, in X1, L1is -P(O)(SH)-O-CH2- and R5is OH.

[0399] In some embodiments, in X1, L1is -O-, or -O-L2-, or -L2-O-. In some embodiments, L2is unsubstituted C1-C4 alkylene. In some embodiments, in X1, L1is -O-L2- and L2is unsubstituted C1-C2alkylene. In some embodiments, in X1, L1is -O-L2- and L2is unsubstituted methylene. In some embodiments, in X1, L1is -O-CH2- and R5is hydrogen. In some embodiments, R5is the functional group capable of forming a covalent bond with the first ligand and the functional group includes -P(O)(SH)-O- or -P(O)(OH)-O-.

[0400] In some embodiments, X1has a structure of ,wherein W1is -OH or -SH; and B is a nucleobase.PAT059650-WO-PCT

[0401] In some embodiments, W1is -OH. In some embodiments, W1is -SH.

[0402] In some embodiments, X1has a structure of a pharmaceutically acceptable salt thereof,

[0403] in X23, L1is -P(O)2-O-L2- and L2is unsubstituted methylene. In some embodiments, in X23, L1is -P(O)(SH)-O-L2- and L2is unsubstituted methylene.

[0404] In some embodiments, X23has a structure of , or a pharmaceutically acceptable salt thereof,or -SH.

[0405] In some embodiments, B is a nucleobase. In some embodiments, B is absent.

[0406] In some embodiments, W3is -OH. In some embodiments, W3is -SH.

[0407] In some embodiments, R6is hydrogen. In some embodiments, R6is -P(O)(OH)- OH. In some embodiments, R6is -P(O)(SH)-OH. In some embodiments, R6is a second ligand. In some embodiments, R6is the functional group capable of forming a covalent bond with the second ligand and the functional group includes -P(O)(SH)-O- or -P(O)(OH)-O-.

[0408] In some embodiments, X2has a structure ofPAT059650-WO-PCT L1B O a pharmaceutically acceptable salt thereof,

[0409] has a structure of a pharmaceutically acceptable salt thereof,

[0410] In some embodiments, in X2and X22, L1is -P(O)(OH)-O-L2- or -P(O)(SH)-O-L2-. In some embodiments, L2is unsubstituted C1-C4 alkylene. In some embodiments, in X2 and X22, L1is -P(O)(OH)-O-L2- and L2is unsubstituted C1-C2alkylene. In some embodiments, in X2 and X22, L1is - P(O)(SH)-O-L2- and L2is unsubstituted methylene.

[0411] In some embodiments, X2and X22independently has a structure of a pharmaceutically acceptable salt thereof,is as described above.

[0412] In some embodiments, in X2, L1is -P(O)2-O-L2- and L2is unsubstituted methylene. In some embodiments, in X2, L1is -P(O)(SH)-O-L2- and L2is unsubstituted methylene. In some embodiments, in X22, L1is -P(O)2-O-L2- and L2is unsubstituted methylene. In some embodiments, in X22, L1is -P(O)(SH)-O-L2- and L2is unsubstitutedPAT059650-WO-PCT methylene. In some embodiments, in X2, L1is -P(O)(SH)-O-CH2- and in X22, L1is -P(O)2-O- CH2-. In some embodiments, in X2, L1is -P(O)(SH)-O-CH2- and in X22, L1is -P(O)(SH)-O- CH2-.

[0413] In some embodiments, X2has a or a pharmaceutically acceptable salt thereof. Ina pharmaceutically acceptable salt thereof.nucleobase. In some embodiments, B is absent.

[0415] In some embodiments, in X1′, R2and R3combine to form a bond. In some embodiments, in X1′, R2and R3are joined together with the atoms to which they are attached to form a 6 membered heterocycloalkyl. In some embodiments, in X1′, R2and R3are joined together with the atoms to which they are attached to form a morpholine ring.

[0416] In some embodiments, in X1′, L1is -P(O)(OH)-L3-, or -P(O)(SH)-L3-. In some embodiments, L3is unsubstituted C2-C4 alkenylene. In some embodiments, in X1′, L1is - P(O)(OH)-L3-, or -P(O)(SH)-L3- and L3is unsubstituted C2 alkenylene (vinyl). In some embodiments, in X1′, L1is -P(O)(OH)-L3-, or -P(O)(SH)-L3- and L3is unsubstituted C3alkenylene. In some embodiments, in X1′, L1is -P(O)(OH)-L3-, or -P(O)(SH)-L3- and L3is unsubstituted C4alkenylene.PAT059650-WO-PCT

[0417] In some , or itspharmaceutically acceptable salt form. In some orL1embodiments, or its pharmaceutically acceptable saltform, and R5 is -OH. In some

[0418] In some embodiments, X1′ has a,wherein B is a nucleobase.

[0419] In some embodiments, in X1′, L1is independently -P(O)(OH)-O-L2- or - P(O)(SH)-O-L2-. In some embodiments, in X1′, L2is unsubstituted C1-C4alkylene. In some embodiments, in X1′, L1is independently -P(O)(OH)-O-L2- or -P(O)(SH)-O-L2-, and L2is unsubstituted C1-C2alkylene. In some embodiments, in X1′, L1is independently -P(O)(OH)- O-L2- or -P(O)(SH)-O-L2- and L2is unsubstituted methylene. In some embodiments, in X1′,PAT059650-WO-PCT L1is -P(O)(OH)-O-CH2- or -P(O)(SH)-O-CH2-, and R5is -OH. In some embodiments, in X1′, R2and R3combine to form a bond, L1is -P(O)(OH)-O-CH2- or - P(O)(SH)-O-CH2-, and R5is -OH.

[0420] In some embodiments, X1′has a structure of ,

[0421] In some embodiments, in X1′, L1is -O- or -O-L2-. In some embodiments, L2is unsubstituted C1-C4 alkylene. In some embodiments, in X1′, L1is -O-L2- and L2is unsubstituted C1-C2alkylene. In some embodiments, in X1′, L1is -O-L2- and L2is unsubstituted methylene. In some embodiments, in X1′, L1is -O-CH2- and R5is hydrogen. In some embodiments, in X1′, R2and R3combine to form a bond, L1is -O-CH2- and R5is hydrogen.

[0422] In some embodiments, X1′ has a structure of , or a pharmaceutically acceptable salt thereof

[0423] In some embodiments, in Formula (I), no more than eight monomers from X3 to X21 are absent. In some embodiments, in Formula (I), no more than seven monomers from X3 to X21are absent. In some embodiments, in Formula (I), no more than six monomers from X3to X21 are absent. In some embodiments, in Formula (I), no more than five monomers from X3to X21are absent. In some embodiments, in Formula (I), no more than four monomers from X3 to X21 are absent. In some embodiments, in Formula (I), no more than three monomers from X3to X21are absent. In some embodiments, in Formula (I), no more thanPAT059650-WO-PCT two monomers from X3 to X21 are absent. In some embodiments, in Formula (I), no more than one nucleotide from X3to X21are absent.

[0424] In some embodiments, in Formula (I), one to eight monomers from X3 to X21 are absent. In some embodiments, in Formula (I), one to six monomers from X3to X21are absent. In some embodiments, in Formula (I), one nucleotide from X3 to X21 are absent. In some embodiments, in Formula (I), two monomers from X3to X21are absent. In some embodiments, in Formula (I), three monomers from X3 to X21 are absent. In some embodiments, in Formula (I), four monomers from X3to X21are absent. In some embodiments, in Formula (I), five monomers from X3 to X21 are absent. In some embodiments, in Formula (I), six monomers from X3to X21are absent. In some embodiments, in Formula (I), seven monomers from X3 to X21 are absent. In some embodiments, in Formula (I), eight monomers from X3 to X21 are absent.

[0425] In some embodiments, in Formula (II), no more than ten monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), no more than nine monomers from X3′to X23′are absent. In some embodiments, in Formula (II), no more than eight monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), no more than seven monomers from X3′to X23′are absent. In some embodiments, in Formula (II), no more than six monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), no more than five monomers from X3′to X23′are absent. In some embodiments, in Formula (II), no more than four monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), no more than three monomers from X3′to X23′are absent. In some embodiments, in Formula (II), no more than two monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), no more than one nucleotide from X3′to X23′are absent.

[0426] In some embodiments, in Formula (II), one to ten monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), one to eight monomers from X3′to X23′are absent. In some embodiments, in Formula (II), one nucleotide from X3′ to X23′ are absent. In some embodiments, in Formula (II), two monomers from X3′to X23′are absent. In some embodiments, in Formula (II), three monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), four monomers from X3′to X23′are absent. In some embodiments, in Formula (II), five monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), six monomers from X3′to X23′are absent. In some embodiments, in Formula (II), seven monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), eight monomers from X3′ to X23′ are absent. In somePAT059650-WO-PCT embodiments, in Formula (II), nine monomers from X3′ to X23′ are absent. In some embodiments, in Formula (II), ten monomers from X3′to X23′are absent.

[0427] In some embodiments, X3 to X21 include Xn, Xn+1, Xn+2, Xn+3, and Xn+4 when n is an integer from 3 to 17. In some embodiments, X3to X21include Xn, Xn+1, Xn+2, Xn+3, and Xn+4when n is an integer from 5 to 10.

[0428] In some embodiments, in two monomers of Xn, Xn+2, Xn+3, and Xn+4(n is an integer from 3 to 17), R1is -F and Xn+1 is present. In some embodiments, in three monomers of Xn, Xn+2, Xn+3, and Xn+4(n is an integer from 3 to 17), R1is -F and Xn+1is present. In some embodiments, in Xn, Xn+2, Xn+3, and Xn+4 (n is an integer from 3 to 17), R1is -F and Xn+1is present. In some embodiments, n is an integer from 3 to 10. In some embodiments, n is an integer from 3 to 7. In some embodiments, n is an integer from 3 to 5. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0429] In some embodiments, in two, three or four monomers of X3, X5, X6 and X7, R2and R3combine to form a bond and R1is -F and X4 is present. In some embodiments, in two monomers of X3, X5, X6and X7, R2and R3combine to form a bond and R1is -F and X4is present. In some embodiments, in three monomers of X3, X5, X6 and X7, R2and R3combine to form a bond and R1is -F and X4is present. In some embodiments, in X3, X5, X6and X7, R2and R3combine to form a bond and R1is -F and X4 is present.

[0430] In some embodiments, in two, three or four monomers of X4, X6, X7and X8, R2and R3combine to form a bond and R1is -F and X5 is present. In some embodiments, in two monomers of X4, X6, X7and X8, R2and R3combine to form a bond and R1is -F and X5is present. In some embodiments, in three monomers of X4, X6, X7 and X8, R2and R3combine to form a bond and R1is -F and X5is present. In some embodiments, in X4, X6, X7and X8, R2and R3combine to form a bond and R1is -F and X5 is present.

[0431] In some embodiments, in two, three or four monomers of X5, X7, X8and X9, R2and R3combine to form a bond and R1is -F, and X6 is present. In some embodiments, in two monomers of X5, X7, X8and X9, R2and R3combine to form a bond and R1is -F, and X6is present. In some embodiments, in three monomers of X5, X7, X8 and X9, R2and R3combine to form a bond and R1is -F, and X6is present. In some embodiments, in X5, X7, X8and X9, R2and R3combine to form a bond and R1is -F, and X6 is present.

[0432] In some embodiments, in two, three or four monomers of X6, X8, X9and X10, R2and R3combine to form a bond and R1is -F, and X7 is present. In some embodiments, in two monomers of X6, X8, X9 and X10, R2and R3combine to form a bond and R1is -F, and X7 isPAT059650-WO-PCT present. In some embodiments, in three monomers of X6, X8, X9 and X10, R2and R3combine to form a bond and R1is -F, and X7is present. In some embodiments, in X6, X8, X9and X10, R2and R3combine to form a bond and R1is -F, and X7 is present.

[0433] In some embodiments, in two, three or four monomers of X7, X9, X10and X11, R2and R3combine to form a bond and R1is -F, and X8 is present. In some embodiments, in two monomers of X7, X9, X10and X11, R2and R3combine to form a bond and R1is -F, and X8is present. In some embodiments, in three monomers of X7, X9, X10 and X11, R2and R3combine to form a bond and R1is -F, and X8is present. In some embodiments, in X7, X9, X10and X11, R2and R3combine to form a bond and R1is -F, and X8 is present.

[0434] In some embodiments, in two, three or four monomers of X8, X10, X11and X12, R2and R3combine to form a bond and R1is -F, and X9 is present. In some embodiments, in two monomers of X8, X10, X11 and X12, R2and R3combine to form a bond and R1is -F, and X9 is present. In some embodiments, in three monomers of X8, X10, X11 and X12, R2and R3combine to form a bond and R1is -F, and X9 is present. In some embodiments, in X8, X10, X11and X12, R2and R3combine to form a bond and R1is -F, and X9is present.

[0435] In some embodiments, in two, three or four monomers of X9, X11, X12 and X13, R2and R3combine to form a bond and R1is -F, and X10is present. In some embodiments, in two monomers of X9, X11, X12 and X13, R2and R3combine to form a bond and R1is -F, and X10is present. In some embodiments, in three monomers of X9, X11, X12and X13, R2and R3combine to form a bond and R1is -F, and X10 is present. In some embodiments, in X9, X11, X12and X13, R2and R3combine to form a bond and R1is -F, and X10is present.

[0436] In some embodiments, in two, three or four monomers of X10, X12, X13 and X14, R2and R3combine to form a bond and R1is -F, and X11is present. In some embodiments, in two monomers of X10, X12, X13 and X14, R2and R3combine to form a bond and R1is -F, and X11is present. In some embodiments, in three monomers of X10, X12, X13and X14, R2and R3combine to form a bond and R1is -F, and X11 is present. In some embodiments, in X10, X12, X13and X14, R2and R3combine to form a bond and R1is -F, and X11is present.

[0437] In some embodiments, in two, three or four monomers of X11, X13, X14 and X15, R2and R3combine to form a bond and R1is -F, and X12is present. In some embodiments, in two monomers of X11, X13, X14 and X15, R2and R3combine to form a bond and R1is -F, and X12is present. In some embodiments, in three monomers of X11, X13, X14and X15,R2and R3combine to form a bond and R1is -F, and X12 is present. In some embodiments, in X11, X13, X14 and X15, R2and R3combine to form a bond and R1is -F, and X12 is present.PAT059650-WO-PCT

[0438] In some embodiments, in two, three or four monomers of X12, X14, X15 and X16, R2and R3combine to form a bond and R1is -F, and X13is present. In some embodiments, in two monomers of X12, X14, X15 and X16, R2and R3combine to form a bond and R1is -F, and X13is present. In some embodiments, in three monomers of X12, X14, X15and X16, R2and R3combine to form a bond and R1is -F, and X13 is present. In some embodiments, in X12, X14, X15and X16, R2and R3combine to form a bond and R1is -F, and X13is present.

[0439] In some embodiments, in two, three or four monomers of X13, X15, X16 and X17, R2and R3combine to form a bond and R1is -F, and X14is present. In some embodiments, in two monomers of X13, X15, X16 and X17, R2and R3combine to form a bond and R1is -F, and X14is present. In some embodiments, in three monomers of X13, X15, X16and X17, R2and R3combine to form a bond and R1is -F, and X14 is present. In some embodiments, in X13, X15, X16 and X17, R2and R3combine to form a bond and R1is -F, and X14 is present.

[0440] In some embodiments, in two, three or four monomers of X14, X16, X17 and X18, R2and R3combine to form a bond and R1is -F, and X15 is present. In some embodiments, in two monomers of X14, X16, X17and X18, R2and R3combine to form a bond and R1is -F, and X15 is present. In some embodiments, in three monomers of X14, X16, X17 and X18, R2and R3combine to form a bond and R1is -F, and X15is present. In some embodiments, in X14, X16, X17 and X18, R2and R3combine to form a bond and R1is -F, and X15 is present.

[0441] In some embodiments, in two, three or four monomers of X15, X17, X18and X19, R2and R3combine to form a bond and R1is -F, and X16 is present. In some embodiments, in two monomers of X15, X17, X18and X19, R2and R3combine to form a bond and R1is -F, and X16 is present. In some embodiments, in three monomers of X15, X17, X18 and X19, R2and R3combine to form a bond and R1is -F, and X16is present. In some embodiments, in X15, X17, X18 and X19, R2and R3combine to form a bond and R1is -F, and X16 is present.

[0442] In some embodiments, in two, three or four monomers of X16, X18, X19and X20, R2and R3combine to form a bond and R1is -F, and X17 is present. In some embodiments, in two monomers of X16, X18, X19and X20, R2and R3combine to form a bond and R1is -F, and X17 is present. In some embodiments, in three monomers of X16, X18, X19 and X20, R2and R3combine to form a bond and R1is -F, and X17is present. In some embodiments, in X16, X18, X19 and X20, R2and R3combine to form a bond and R1is -F, and X17 is present.

[0443] In some embodiments, in two, three or four monomers of X17, X19, X20and X21, R2and R3combine to form a bond and R1is -F, and X18 is present. In some embodiments, in two monomers of X17, X19, X20 and X21, R2and R3combine to form a bond and R1is -F, andPAT059650-WO-PCT X18 is present. In some embodiments, in three monomers of X17, X19, X20 and X21, R2and R3combine to form a bond and R1is -F, and X18is present. In some embodiments, in X17, X19, X20 and X21, R2and R3combine to form a bond and R1is -F, and X18 is present.

[0444] In some embodiments, in two, four or six monomers from X3′to X23′, R1is -F. In some embodiments, in two monomers from X3′ to X23′, R1is -F. In some embodiments, in four monomers from X3′to X23′, R1is -F. In some embodiments, in six monomers from X3′to X23′, R1is -F. In some embodiments, in more than six monomers from X3′ to X23′, R1is -F.

[0445] In some embodiments, in two, four or six monomers from X3′to X23′, R2and R3combine to form a bond and R1is -F. In some embodiments, in two monomers from X3′ to X23′, R2and R3combine to form a bond and R1is -F. In some embodiments, in four monomers from X3′ to X23′, R2and R3combine to form a bond and R1is -F. In some embodiments, in six monomers from X3′ to X23′, R2and R3combine to form a bond and R1is -F. In some embodiments, in more than six monomers from X3′ to X23′, R2and R3combine to form a bond and R1is -F.

[0446] In some embodiments, in X3to X21,R1is independently hydrogen, -F, -OH, - OCH3, -OCH2CH3, -OCH2CH2-OCH3, -CH3, -CH2CH3, C(O)NH, -C(O)OH, -C(O)NH2, and - NH2. In some embodiments, in X3to X21, each R1is independently hydrogen, -F, -OH, - OCH3, -OCH2CH3, -OCH2CH2-OCH3, -CH3, -CH2CH3, -C(O)NH2, and -NH2. In some embodiments, in at least one nucleotide of X3to X21, R1is hydrogen. In some embodiments, in at least one nucleotide of X3 to X21, R1is -F. In some embodiments, in at least one nucleotide of X3to X21, R1is -OH. In some embodiments, in at least one nucleotide of X3to X21, R1is -OCH3. In some embodiments, in at least one nucleotide of X3 to X21, R1is - OCH2CH2-OCH3.

[0447] In some embodiments, in any one of X3 to X21, R1 and R4 may be joined together with the atoms to which they are attached to form a substituted cycloalkyl. In some embodiments, in any one of X3 to X21, R1 and R4 may be joined together with the atoms to which they are attached to form a substituted cyclohexyl.

[0448] In some embodiments, any one of X3 to X21, R1and R4are joined together with the atoms to which they are attached to form a substituted heterocycloalkyl. In some embodiments, any one of X3 to X21, R1and R4are joined together with the atoms to which they are attached to form a substituted 5 to 6 membered heterocycloalkyl. In some embodiments, any one of X3 to X21, R1and R4are joined together with the atoms to which they are attached to form a substituted 6 membered heterocycloalkyl.PAT059650-WO-PCT

[0449] In some embodiments, in X2 to X23, L1is independently -P(O)(OH)-O-CH2-, or - P(O)(SH)-O-CH2-. In some embodiments, in X2,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X3, L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X2and X3,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X4,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X5, L1is independently - P(O)(SH)-O-CH2-. In some embodiments, in X4and X5, L1is independently -P(O)(SH)-O- CH2-. In some embodiments, in X2, X3, X4 and X5, L1is independently -P(O)(SH)-O-CH2-.

[0450] In some embodiments, in X23,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X22, L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X22 and X23,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X21,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X20, L1is independently - P(O)(SH)-O-CH2-. In some embodiments, in X20 and X21, L1is independently -P(O)(SH)-O- CH2-. In some embodiments, in X20, X21, X22 and X23, L1is independently -P(O)(SH)-O-CH2- .

[0451] In some embodiments, in X2,X3,X22and X23,L1is independently -P(O)(SH)-O- CH2-. In some embodiments, in X2, X3, X4, X5, X20, X21, X22 and X23, L1is independently - P(O)(SH)-O-CH2-.

[0452] In some embodiments, in X2′ to X25′, L1is independently -P(O)(OH)-O-CH2-, or - P(O)(SH)-O-CH2-. In some embodiments, in X2′,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X3′, L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X2′and X3′,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X4′,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X5′, L1is independently - P(O)(SH)-O-CH2-. In some embodiments, in X4′and X5′, L1is independently - P(O)(SH)-O- CH2-. In some embodiments, in X2′, X3′, X4′ and X5′, L1is independently -P(O)(SH)-O-CH2-.

[0453] In some embodiments, in X25′,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X24′, L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X24′ and X25′,L1is independently -P(O)(SH)-O-CH2-. In some embodiments, in X23′,L1is independently - P(O)(SH)-O-CH2-. In some embodiments, in X22′, L1is independently - P(O)(SH)-O-CH2-. In some embodiments, in X22′and X23′,L1is independently -P(O)(SH)-O- CH2-. In some embodiments, in X22′, X23′, X24′ and X25′, L1is independently -P(O)(SH)-O- CH2-.PAT059650-WO-PCT

[0454] In some embodiments, in X2′, X3′, X24′ and X25′, L1is independently -P(O)(SH)-O- CH2-. In some embodiments, in X2′,X3′,X4′,X5′,X22′, X23′, X24′and X25′,L1is independently - P(O)(SH)-O-CH2-.

[0455] In some embodiments, one nucleotide from X2′to X25′contain GNA. In some embodiments, two monomers from X2′ to X25′ contain GNA. In some embodiments, three monomers from X2′to X25′contain GNA. In some embodiments, X4′contains GNA. In some embodiments, X5′ contains GNA. In some embodiments, X6′ contains GNA. In some embodiments, X7′contains GNA.

[0456] In certain aspects, the dsRNAi agent for inhibiting expression of a target gene includes: (i) a sense strand comprising monomers of N1 to N23 and having the Formula (III): 5′-N1-N2-N3-N4-N5-N6-N7-N8-N9-N10-N11-N12-N13-N14-N15-N16-N17-N18-N19-N20-N21-N22- N23-3′ (III), wherein: N1 has a structure of Formula (A-1), a pharmaceutically acceptable salt thereof,a structure of Formula (B-1), a pharmaceutically acceptable salt thereof, andof Formula (B-1),PAT059650-WO-PCT a pharmaceutically acceptable salt thereof, and Formula (C),a pharmaceutically acceptable salt thereof,(ii) an monomers of N1′ to N25′ and having the Formula (IV): 5′-N1′-N2′-N3′-N4′-N5′-N6′-N7′-N8′-N9′-N10′-N11′-N12′-N13′-N14′-N15′-N16′-N17′-N18′-N19′-N20′-N21′- N22′-N23′-N24′-N25′-3′ (IV), or a pharmaceutically acceptable salt thereof, wherein: N1′ has a structure of Formula (A-1), a pharmaceutically acceptable salt thereof,a structure of Formula (B-1), a pharmaceutically acceptable salt thereof, andPAT059650-WO-PCT each N3′ and N23′ is absent or glycol nucleic acid (GNA), or has a structure Formula (B-1), Formula (B-2),, , 1), (B-2), and (C-1):in each occurrence, B is independently absent or a nucleobase; in each occurrence, each R21, R22, R23, and R24is independently hydrogen, halogen, - ORA, -SRA, -NRBRC, -C(O)RD, -OC(O)RD, -C(O)NRBRC, -NREC(O)RD, - NREC(O)ORD, -NREC(O)NRBRC, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl, wherein each RA, RB, RC, RD, and RFis independently hydrogen, or C1-C10alkyl optionally substituted with halogen, -OH, -O-C1-4alkyl, or -O-C1-4 haloalkyl; in each occurrence, R25is hydrogen, -OH, a detectable moiety, a first ligand, or a functional group capable of forming a covalent bond with a first ligand; in each occurrence, R26is hydrogen, a detectable moiety, -P(O)(OH)-OH, -P(O)(SH)- OH, a second ligand, or a functional group capable of forming a covalent bond with a second ligand; in each occurrence, L21is independently absent, -O-, -O-L22-, -P(O)(OH)-O-L22-, - P(O)(SH)-O-L22-, -P(O)(OH)-L23-, or -P(O)(SH)-L23-, wherein each L22is independently a substituted or unsubstituted C1-C4alkylene and each L23is independently a substituted or unsubstituted C2-C4 alkenylene, andPAT059650-WO-PCT in each occurrence, is an attachment point to the adjacent monomers, provided that:(i) in at least N1 and N23, R22is -O-CH2CH2-OCH3, (ii) in N1′, R22is not -O-CH2CH2-OCH3,(iii) in N3 to N21, (a) R22is not -O-CH2CH2-OCH3, (b) at least one of R22is not hydrogen, and (c) in no more than six nucleotides, R22is -F, and (iv) no more than eight monomers from N3 to N21 are absent and no more than ten monomers from N3′ to N23′ are absent.

[0457] In some embodiments, N1has a structure of , or a pharmaceutically acceptable salt thereof. Insome embodiments, N23 has a apharmaceutically acceptable saltof , or a pharmaceutically acceptable salt thereof and N23has aPAT059650-WO-PCT apharmaceutically acceptable salt thereof. Bis a described herein.

[0458] , or -O-L22-. In some embodiments, L22is unsubstituted C1-C4alkylene. In some embodiments, in N1, L21is -O-L22- and L22is unsubstituted C1-C2 alkylene. In some embodiments, in N1, L21is -O-L22- and L22is unsubstituted methylene. In some embodiments, in N1, L21is -O-CH2- and R25is hydrogen. In some embodiments, R25is the functional group capable of forming a covalent bond with the first ligand and the functional group includes -P(O)(SH)-O- or -P(O)(OH)-O-.

[0459] In some embodiments, in N1, L21is -P(O)(OH)-O-L22-, or -P(O)(SH)-O-L22-. In some embodiments, L22is unsubstituted C1-C4alkylene. In some embodiments, in N1, L21is - P(O)(OH)-O-L22-and L22is unsubstituted C1-C2 alkylene. In some embodiments, in N1, L21is -P(O)(SH)-O-L22and L22is unsubstituted methylene. In some embodiments, in N1, L21is - P(O)2-O-CH2- and R25is OH. In some embodiments, in N1, L21is -P(O)(SH)-O-CH2- and R25is OH.

[0460] In some embodiments, N1 has a structure ofsome embodiments, W1is -OH. In some embodiments, W1is -SH.

[0461] In some embodiments, in N1, R21is hydrogen. In some embodiments, in N1, R24is hydrogen. In some embodiments, in N1, R21and R24are hydrogen.

[0462] In some embodiments, N1has a structure ofPAT059650-WO-PCT a pharmaceutically

[0463] - 2- unsubstituted methylene. In some embodiments, in N23, L21is -P(O)(SH)-O-L22- and L22is unsubstituted methylene.

[0464] In some embodiments, in N23, R21is hydrogen. In some embodiments, in N1, R24is hydrogen. In some embodiments, in N23, R21and R24are hydrogen.

[0465] In some embodiments, N23has a structure of

[0466] In some embodiments, R26is hydrogen. In some embodiments, R26is - P(O)(OH)-OH. In some embodiments, R26is -P(O)(SH)-OH. In some embodiments, R26is a second ligand. In some embodiments, R26is the functional group capable of forming a covalent bond with the second ligand and the functional group includes -P(O)(SH)-O- or - P(O)(OH)-O-.

[0467] In some embodiments, N2has a structure of a pharmaceutically acceptable salt thereof. B is aN22 has a structure ofPAT059650-WO-PCT a pharmaceutically acceptable salt thereof. B is a

[0468] and N22 independently has a structure of

[0469] a pharmaceutically acceptable salt thereof. B is a and L21are as described herein.

[0470] In some embodiments, in N2and N22, L21is -P(O)(OH)-O-L22- or -P(O)(SH)-O- L22-. In some embodiments, L22is unsubstituted C1-C4 alkylene. In some embodiments, in N2and N22, L21is -P(O)(OH)-O-L22- and L22is unsubstituted C1-C2alkylene. In some embodiments, in N2 and N22, L21is - P(O)(SH)-O-L22- and L22is unsubstituted methylene.

[0471] In some embodiments, in N2and N22, R21is hydrogen. In some embodiments, in N2and N22, R24is hydrogen. In some embodiments, in N2 and N22, R21and R24are hydrogen.

[0472] In some embodiments, N2and N22independently has a structure of a pharmaceutically acceptable salt thereof. B is aas described herein.

[0473] In some embodiments, in N2, L21is -P(O)2-O-L22- and L22is unsubstituted methylene. In some embodiments, in N2, L21is -P(O)(SH)-O-L22- and L22is unsubstituted methylene. In some embodiments, in N22, L21is -P(O)2-O-L22- and L22is unsubstituted methylene. In some embodiments, in N22, L21is -P(O)(SH)-O-L22- and L22is unsubstituted methylene. In some embodiments, in N2, L21is -P(O)(SH)-O-CH2- and in N22, L21is -P(O)2-PAT059650-WO-PCT O-CH2-. In some embodiments, in N2, L21is -P(O)(SH)-O-CH2- and in N22, L21is - P(O)(SH)-O-CH2-. O POO

[0474] , whereina pharmaceutically acceptable salt thereof. In someembodiments, N2has a a pharmaceutically acceptable salt thereof. InO O , wherein W2is -OH or -SH. In some embodiments, N22has a a pharmaceutically acceptable salt thereof.PAT059650-WO-PCT In some embodiments, N22 has a a pharmaceutically acceptable salt

[0475] In some embodiments, N2 has a a pharmaceutically acceptable salt thereof. Ina pharmaceutically acceptable salt thereof. B is a

[0476] In some embodiments, in N1′, L21is -P(O)(OH)-L23-, or -P(O)(SH)-L23-. In some embodiments, L23is unsubstituted C2-C4alkenylene. In some embodiments, in N1′, L21is - P(O)(OH)-L23-, or -P(O)(SH)-L23- and L23is unsubstituted C2 alkenylene (vinyl). In some embodiments, in N1′, L21is -P(O)(OH)-L23-, or -P(O)(SH)-L23- and L23is unsubstituted C3alkenylene. In some embodiments, in N1′, L21is -P(O)(OH)-L23-, or -P(O)(SH)-L23- and L23is unsubstituted C4 alkenylene.

[0477] In some , or itspharmaceutically acceptable salt form. In some orPAT059650-WO-PCT or its salt form. In some in L21or its pharmaceutically acceptable saltform, and R25 is -OH. In some

[0478] In some embodiments, N1′has a, and R21, R22 24, and R are as described herein.

[0480] In some embodiments, in N1′, L21is independently -P(O)(OH)-O-L22- or -P(O)(SH)- O-L22-. In some embodiments, in N1′, L22is unsubstituted C1-C4alkylene. In some embodiments, in N1′, L21is independently -P(O)(OH)-O-L22- or -P(O)(SH)-O-L22-, and L22is unsubstituted C1-C2alkylene. In some embodiments, in N1′, L21is independently -P(O)(OH)- O-L22- or -P(O)(SH)-O-L22- and L22is unsubstituted methylene. In some embodiments, in N1′, L21is -P(O)(OH)-O-CH2- or -P(O)(SH)-O-CH2-, and R25is -OH. In some embodiments, in N1′, L21is -P(O)(OH)-O-CH2- or - P(O)(SH)-O-CH2-, and R25is -OH.

[0481] In some embodiments, N1′has a structure ofPAT059650-WO-PCT ,

[0482] - - . embodiments, L22is unsubstituted C1-C4alkylene. In some embodiments, in N1′, L21is -O-L22- and L22is unsubstituted C1-C2 alkylene. In some embodiments, in N1′, L21is -O L22- and L22is unsubstituted methylene. In some embodiments, in N1′, L21is -O-CH2- and R25is hydrogen. In some embodiments, in N1′, L21is -O-CH2- and R25is hydrogen.

[0483] In some embodiments, N1′ has a a pharmaceutically acceptable salt thereof. B is a

[0484] In some embodiments, in N1′, R21is hydrogen. In some embodiments, in N1′, R24is hydrogen. In some embodiments, in N1′, R21and R24are hydrogen.

[0485] In some embodiments, N1′has a structure of , or a pharmaceutically acceptable saltN1′ has a structure ofPAT059650-WO-PCT , or a pharmaceutically acceptable salt thereof. B is asome in N1′, R22is -ORAand RAis unsubstituted C1-C4alkyl. In some embodiments, in N1′, R22is independently -ORAand RAis methyl. In some embodiments, in N1′, R22is independently -ORAand RAis ethyl. In some embodiments, in N1′, R22is -OCH3 (OMe).

[0487] In some embodiments, in N1′, when R22is -ORA, RAis not substituted C1-C4alkyl. In some embodiments, in N1′, R22is not -O-CH2CH2-OCH3.

[0488] In some embodiments, in Formula (III), no more than eight monomers from N3to N21are absent. In some embodiments, in Formula (III), no more than seven monomers from N3 to N21 are absent. In some embodiments, in Formula (III), no more than six monomers from N3to N21are absent. In some embodiments, in Formula (III), no more than five monomers from N3 to N21 are absent. In some embodiments, in Formula (III), no more than four monomers from N3to N21are absent. In some embodiments, in Formula (III), no more than three monomers from N3 to N21 are absent. In some embodiments, in Formula (III), no more than two monomers from N3to N21are absent. In some embodiments, in Formula (III), no more than one nucleotide from N3 to N21 are absent.

[0489] In some embodiments, in Formula (III), one to eight monomers from N3to N21are absent. In some embodiments, in Formula (III), one to six monomers from N3 to N21 are absent. In some embodiments, in Formula (III), one nucleotide from N3to N21are absent. In some embodiments, in Formula (III), two monomers from N3 to N21 are absent. In someembodiments, in Formula (III), three monomers from N3to N21are absent. In some embodiments, in Formula (III), four monomers from N3 to N21 are absent. In some embodiments, in Formula (III), five monomers from N3to N21are absent. In some embodiments, in Formula (III), six monomers from N3 to N21 are absent. In some embodiments, in Formula (III), seven monomers from N3to N21are absent. In some embodiments, in Formula (III), eight monomers from N3 to N21 are absent.PAT059650-WO-PCT

[0490] In some embodiments, in Formula (IV)), no more than ten monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), no more than nine monomers from N3′to N23′ are absent. In some embodiments, in Formula (IV)), no more than eight monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), no more than seven monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), no more than six monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), no more than five monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), no more than four monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), no more than three monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), no more than two monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), no more than one nucleotide from N3′ to N23′ are absent.

[0491] In some embodiments, in Formula (IV)), one to ten monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), one to eight monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), one nucleotide from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), two monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), three monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), four monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), five monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), six monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), seven monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), eight monomers from N3′to N23′are absent. In some embodiments, in Formula (IV)), nine monomers from N3′ to N23′ are absent. In some embodiments, in Formula (IV)), ten monomers from N3′to N23′are absent.

[0492] In some embodiments, in N3 to N21, if present, R21is hydrogen. In some embodiments, in N3to N21,if present, R24is hydrogen. In some embodiments, in N3to N21,if present, R21and R24are hydrogen.

[0493] In some embodiments, in at least one of N3to N21, R22is hydrogen. In some embodiments, in N3 to N21, at least one of R22is not hydrogen.

[0494] In some embodiments, in at least one of N3to N21, R22is halogen. In some embodiments, in two, three, or four monomers selected from N3 to N21, R22is -F. In some embodiments, in no more than six monomers selected from N3to N21, R22is -F.

[0495] In some embodiments, in at least one of N3 to N21, each R22is -ORAand RAis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least onePAT059650-WO-PCT of N3 to N21, R22is -ORAand RAis hydrogen. In some embodiments, in at least one of N3 to N21, when R22is -ORA, RAis not hydrogen. In some embodiments, in at least one of N3to N21, each R22is -ORAand RAis substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3to N21, each R22is -ORAand RAis unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3 to N21, each R22is -ORAand RAis unsubstituted methyl, ethyl, propyl, or isopropyl.

[0496] In some embodiments, in at least one of N3 to N21, each R22is independently -ORAand RAis substituted C1-C4alkyl. In some embodiments, in at least one of N3to N21, each R22is independently -ORAand RAis alkoxyl-substituted C1-C4 alkyl. In some embodiments, RAis methoxy (-OCH3) substituted C1-C4alkyl. In some embodiments, R22is -O-CH2-OCH3.

[0497] In some embodiments, in N3 to N21, if present, R22is not -O-CH2CH2-OCH3.

[0498] In some embodiments, in at least one of N3 to N21, R22is independently -NRBRCand each RBand RCis independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3 to N21, R22is independently -NRBRCand each RBand RCis independently hydrogen. In some embodiments, in at least one of N3to N21, each R22is independently -NRBRCand each RBand RCis independently hydrogen or unsubstituted C1-C4alkyl. In some embodiments, each RBand RCis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0499] In some embodiments, in at least one of N3to N21, each R22is independently - C(O)RDand RDis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3to N21, each R22is independently -C(O)RDand RDis hydrogen. In some embodiments, in at least one of N3 to N21, each R22is independently - C(O)RDand RDis unsubstituted C1-C4alkyl. In some embodiments, each RD isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0500] In some embodiments, in at least one of N3to N21, each R22is independently - OC(O)RDand RDis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3to N21, each R22is independently - OC(O)RDand RDis hydrogen. In some embodiments, in at least one of N3 to N21, each R22is independently - OC(O) RDand RDis unsubstituted C1-C4alkyl. In some embodiments, each RD isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0501] In some embodiments, in at least one of N3to N21, each R22is independently - C(O)NRBRCand each RBand RCis independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3 to N21, each R22is independently -PAT059650-WO-PCT C(O)NRBRCand each RBand RCis independently hydrogen. In some embodiments, in at least one of N3to N21, each R22is independently - C(O)NRBRCand each RBand RCis independently unsubstituted C1-C4 alkyl. In some embodiments, each RBand RCis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0502] In some embodiments, in at least one of N3 to N21, each R22is independently – NREC(O)RDand each REand RDis independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3 to N21, each R22is independently - NREC(O)H and REis hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3 to N21, each R22is independently - NREC(O)CH3 and REis hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3 to N21, each R22is independently - NREC(O)H and REis unsubstituted C1-C4 alkyl. In some embodiments, each REis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0503] In some embodiments, in at least one of N3 to N21, each R22is independently - NREC(O)OH and REis hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3 to N21, each R22is independently - NREC(O)OH and REis hydrogen. In some embodiments, in at least one of N3to N21, each R22is independently - NREC(O)OH and REis unsubstituted C1-C4 alkyl. In some embodiments, each REis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0504] In some embodiments, in at least one of N3 to N21, each R22is independently substituted or unsubstituted C1-C6alkyl. In some embodiments, in at least one of N3to N21, each R22is independently substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3to N21, each R22is independently substituted C1-C4alkyl. In some embodiments, in at least one of N3 to N21, each R22is independently unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3to N21, each R22is independently substituted or unsubstituted methyl. In some embodiments, in N3 to N21, at least one of R22is independently substituted or unsubstituted ethyl.

[0505] In some embodiments, in at least one of N3 to N21, each R22is independently substituted or unsubstituted 2 to 6 membered heteroalkyl. In some embodiments, in at least one of N3 to N21, each R22is independently substituted or unsubstituted 2 to 5 membered heteroalkyl. In some embodiments, in at least one of N3to N21, each R22is independently substituted or unsubstituted 2 to 4 membered heteroalkyl. In some embodiments, in at least one of N3 to N21, each R22is independently substituted or unsubstituted 2 to 3 memberedPAT059650-WO-PCT heteroalkyl. In some embodiments, in at least one of N3 to N21, each R22is independently -O- CH3. In some embodiments, in at least one of N3to N21, each R22is independently -O-CH2- CH3. In some embodiments, in N3 to N21, if present, R22is not -O-CH2CH2-OCH3.

[0506] In some embodiments, in N3to N21,if present, R22is independently hydrogen, -F, - OH, and -OCH3; and R21and R24are hydrogen. In some embodiments, in N3 to N21, if present, R22is independently hydrogen, -F, - and -OCH3; and R21and R24are hydrogen.

[0507] In some embodiments, in two monomers of N3 to N21, R22is -F. In some embodiments, in three monomers of N3to N21, R22is -F. In some embodiments, in four monomers of N3 to N21, R22is -F.

[0508] In some embodiments, N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4when n is an integer from 3 to 17. In some embodiments, N3 to N21 include Nn, Nn+1, Nn+2, Nn+3, and Nn+4 when n is an integer from 5 to 10.

[0509] In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4 in the sense strand of formula (III) are present wherein n is an integer from 3 to 17, and in two, three or four monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4in the sense strand of formula (III) are present wherein n is an integer from 3 to 17, and in two monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4 are present wherein n is an integer from 3 to 17, and in three monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4are present wherein n is an integer from 3 to 17, and in four monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F.

[0510] In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4 are present wherein n is an integer from 5 to 10, and in two, three or four monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is - F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4 are present wherein n is an integer from 5 to 10, and in two monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4 are present wherein n is an integer from 5 to 10, and in three monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4 are present wherein n is an integer from 5 to 10, and in four monomers of Nn, Nn+2, Nn+3, and Nn+4, R22is -F. In some embodiments, Nn, Nn+1, Nn+2, Nn+3, and Nn+4are present wherein n is an integer from 5 to 10, and in three monomers of Nn+2, Nn+3, and Nn+4, R22is -F.

[0511] In some embodiments, when N3, N4, N5, N6 and N7 are present, in two, three or four monomers of N3, N5, N6 and N7, R22is -F. In some embodiments, when N3, N4, N5, N6 andPAT059650-WO-PCT N7 are present, in two monomers of N3, N5, N6 and N7, R22is -F. In some embodiments, when N3, N4, N5, N6and N7are present, in three monomers of N3, N5, N6and N7, R22is -F. In some embodiments, when N3, N4, N5, N6 and N7 are present, in N3, N5, N6 and N7, R22is - F. In some embodiments, when N3, N4, N5, N6and N7are present, in N5, N6and N7, R22is -F.

[0512] In some embodiments, when N4, N5, N6, N7 and N8 are present, in two, three or four monomers of N4, N6, N7and N8, R22is -F. In some embodiments, when N4, N5, N6, N7and N8 are present, in two monomers of N4, N6, N7 and N8, R22is -F. In some embodiments, when N4, N5, N6, N7and N8are present, in three monomers of N4, N6, N7and N8, R22is -F. In some embodiments, when N4, N5, N6, N7 and N8 are present, in N4, N6, N7 and N8, R22is - F. In some embodiments, when N4, N5, N6, N7and N8are present, in N6, N7and N8, R22is -F.

[0513] In some embodiments, when N5, N6, N7, N8 and N9 are present, in two, three or four monomers of N5, N7, N8 and N9, R22is -F. In some embodiments, when N5, N6, N7, N8 and N9 are present, in two monomers of N5, N7, N8 and N9, R22is -F. In some embodiments, when N5, N6, N7, N8 and N9 are present, in three monomers of N5, N7, N8 and N9, R22is -F. In some embodiments, when N5, N6, N7, N8and N9are present, in N5, N7, N8and N9, R22is - F. In some embodiments, when N5, N6, N7, N8 and N9 are present, in N7, N8 and N9, R22is -F.

[0514] In some embodiments, when N6, N7, N8, N9and N10are present, in two, three or four monomers of N6, N8, N9 and N10, R22is -F. In some embodiments, when N6, N7, N8, N9 and N10are present, in two monomers of N6, N8, N9and N10, R22is -F. In some embodiments, when N6, N7, N8, N9 and N10 are present, in three monomers of N6, N8, N9 and N10, R22is -F. In some embodiments, when N6, N7, N8, N9and N10are present, in N6, N8, N9and N10, R22is -F. In some embodiments, when N6, N7, N8, N9 and N10 are present, in N8, N9 and N10, R22is -F.

[0515] In some embodiments, when N7, N8, N9, N10 and N11 are present, in two, three or four monomers of N7, N9, N10and N11, R22is -F. In some embodiments, when N7, N8, N9, N10 and N11 are present, in two monomers of N7, N9, N10 and N11, R22is -F. In some embodiments, when N7, N8, N9, N10and N11are present, in three monomers of N7, N9, N10and N11, R22is -F. In some embodiments, when N7, N8, N9, N10 and N11 are present, in N7, N9, N10and N11, R22is -F. In some embodiments, when N7, N8, N9, N10and N11are present, in N9, N10 and N11, R22is -F.

[0516] In some embodiments, when N8, N9, N10, N11and N12are present, in two, three or four monomers of N8, N10, N11 and N12, R22is -F. In some embodiments, when N8, N9, N10, N11 and N12 are present, in two monomers of N8, N10, N11 and N12, R22is -F. In somePAT059650-WO-PCT embodiments, when N8, N9, N10, N11 and N12 are present, in three monomers of N8, N10, N11 and N12, R22is -F. In some embodiments, when N8, N9, N10, N11and N12are present, in N8, N10, N11 and N12, R22is -F. In some embodiments, when N8, N9, N10, N11 and N12 are present, in N10, N11and N12, R22is -F.

[0517] In some embodiments, when N9, N10, N11, N12 and N13 are present, in two, three or four monomers of N9, N11, N12and N13, R22is -F. In some embodiments, when N9, N10, N11, N12 and N13 are present, in two monomers of N9, N11, N12 and N13, R22is -F. In some embodiments, when N9, N10, N11, N12and N13are present, in three monomers of N9, N11, N12and N13, R22is -F. In some embodiments, when N9, N10, N11, N12 and N13 are present, in N9, N11, N12and N13, R22is -F. In some embodiments, when N9, N10, N11, N12and N13are present, in N11, N12 and N13, R22is -F.

[0518] In some embodiments, when N10, N11, N12, N13 and N14 are present, in two, three or four monomers of N10, N12, N13 and N14, R22is -F. In some embodiments, when N10, N11, N12, N13 and N14 are present, in two monomers of N10, N12, N13 and N14, R22is -F. In some embodiments, in three monomers of N10, N12, N13and N14, R22is -F, and N11is present. In some embodiments, in N10, N12, N13 and N14, R22is -F, and N11 is present. In some embodiments, in N12, N13and N14, R22is -F, and N11is present.

[0519] In some embodiments, when N11, N12, N13, N14 and N15 are present, in two, three or four monomers of N11, N13, N14and N15, R22is -F. In some embodiments, when N11, N12, N13, N14 and N15 are present, in two monomers of N11, N13, N14 and N15, R22is -F. In some embodiments, when N11, N12, N13, N14and N15are present, in three monomers of N11, N13, N14 and N15, R22is -F. In some embodiments, when N11, N12, N13, N14 and N15 are present, in N11, N13, N14and N15,R22is -F, and N12is present. In some embodiments, when N11, N12, N13, N14 and N15 are present, in N13, N14 and N15, R22is -F, and N12 is present.

[0520] In some embodiments, when N12, N13, N14, N15and N16are present, in two, three or four monomers of N12, N14, N15 and N16, R22is -F. In some embodiments, when N12, N13, N14, N15and N16are present, in two monomers of N12, N14, N15and N16, R22is -F. In some embodiments, when N12, N13, N14, N15 and N16 are present, in three monomers of N12, N14, N15and N16, R22is -F. In some embodiments, when N12, N13, N14, N15and N16are present, in N12, N14, N15 and N16, R22is -F. In some embodiments, when N12, N13, N14, N15 and N16 are present, in N14, N15and N16, R22is -F.

[0521] In some embodiments, when N13, N14, N15, N16 and N17 are present, in two, three or four monomers of N13, N15, N16 and N17, R22is -F. In some embodiments, when N13, N14,PAT059650-WO-PCT N15, N16 and N17 are present, in two monomers of N13, N15, N16 and N17, R22is -F. In some embodiments, when N13, N14, N15, N16and N17are present, in three monomers of N13, N15, N16 and N17, R22is -F. In some embodiments, when N13, N14, N15, N16 and N17 are present, in N13, N15, N16and N17, R22is -F. In some embodiments, when N13, N14, N15, N16and N17are present, in N15, N16 and N17, R22is -F

[0522] In some embodiments, when N14, N15, N16, N17and N18are present, in two, three or four monomers of N14, N16, N17 and N18, R22is -F. In some embodiments, when N14, N15, N16, N17and N18are present, in two monomers of N14, N16, N17and N18, R22is -F. In some embodiments, when N14, N15, N16, N17 and N18 are present, in three monomers of N14, N16, N17and N18, R22is -F. In some embodiments, when N14, N15, N16, N17and N18are present, in N14, N16, N17 and N18, R22is -F. In some embodiments, when N14, N15, N16, N17 and N18 are present, in N16, N17 and N18, R22is -F.

[0523] In some embodiments, when N15, N16, N17, N18 and N19 are present, in two, three or four monomers of N15, N17, N18 and N19, R22is -F. In some embodiments, when N15, N16, N17, N18and N19are present, in two monomers of N15, N17, N18and N19, R22is -F. In some embodiments, when N15, N16, N17, N18 and N19 are present, in three monomers of N15, N17, N18and N19, R22is -F. In some embodiments, when N15, N16, N17, N18and N19are present, in N15, N17, N18 and N19, R22is -F. In some embodiments, when N15, N16, N17, N18 and N19 are present, in N17, N18and N19, R22is -F.

[0524] In some embodiments, when N16, N17, N18, N19 and N20 are present, in two, three or four monomers of N16, N18, N19and N20, R22is -F. In some embodiments, when N16, N17, N18, N19 and N20 are present, in two monomers of N16, N18, N19 and N20, R22is -F. In some embodiments, when N16, N17, N18, N19and N20are present, in three monomers of N16, N18, N19 and N20, R22is -F. In some embodiments, when N16, N17, N18, N19 and N20 are present, in N16, N18, N19and N20, R22is -F. In some embodiments, when N16, N17, N18, N19and N20are present, in N18, N19 and N20, R22is -F.

[0525] In some embodiments, when N17, N18, N19, N20and N21are present, in two, three or four monomers of N17, N19, N20 and N21, R22is -F. In some embodiments, when N17, N18, N19, N20and N21are present, in two monomers of N17, N19, N20and N21, R22is -F. In some embodiments, when N17, N18, N19, N20 and N21 are present, in three monomers of N17, N19, N20and N21, R22is -F. In some embodiments, when N17, N18, N19, N20and N21are present, in N17, N19, N20 and N21, R22is -F. In some embodiments, when N17, N18, N19, N20 and N21 are present, in N19, N20 and N21, R22is -F.PAT059650-WO-PCT

[0526] In some embodiments, when Nn, Nn+1, Nn+2, Nn+3, and Nn+4 in the sense strand are present wherein n is an integer from 3 to 17, in Nn, Nn+2, and Nn+3,R22is -F, and Nn+4, R22is hydrogen. In some embodiments, when Nn, Nn+1, Nn+2, Nn+3, and Nn+4 in the sense strand are present wherein n is an integer from 5 to 10, (i) in Nn, Nn+2, and Nn+3,R22is -F, and (ii) in Nn+4, R22is hydrogen.

[0527] In some embodiments, when N3, N4, N5, N6and N7are present, (i) in N3, N5, and N6, R22is -F; and (ii) in N7, R22is -H.

[0528] In some embodiments, when N4, N5, N6, N7and N8are present, (i) in N4, N6, and N7, R22is -F; and (ii) in N8, R22is -H.

[0529] In some embodiments, when N5, N6, N7, N8and N9are present, (i) in N5, N7, and N8, R22is -F; and (ii) in N9, R22is -H.

[0530] In some embodiments, when N6, N7, N8, N9 and N10 are present, (i) in N6, N8, and N9, R22is -F; and (ii) in N10, R22is -H.

[0531] In some embodiments, when N7, N8, N9, N10 and N11 are present, (i) in N7, N9, and N10, R22is -F; and (ii) in N11, R22is -H.

[0532] In some embodiments, when N8, N9, N10, N11 and N12 are present, (i) in N8, N10, and N11, R22is -F; and (ii) in N12, R22is -H.

[0533] In some embodiments, when N9, N10, N11, N12 and N13 are present, (i) in N9, N11, and N12, R22is -F; and (ii) in N13, R22is -H.

[0534] In some embodiments, when N10, N11, N12, N13 and N14 are present, (i) in N10, N12, and N13, R22is -F; and (ii) in N14, R22is -H.

[0535] In some embodiments, when N11, N12, N13, N14 and N15 are present, (i) in N11, N13, and N14, R22is -F; and (ii) in N15, R22is -H.

[0536] In some embodiments, when N12, N13, N14, N15 and N16 are present, (i) in N12, N14, and N15, R22is -F; and (ii) in N16, R22is -H.

[0537] In some embodiments, when N13, N14, N15, N16 and N17 are present, (i) in N13, N15, and N16, R22is -F; and (ii) in N17, R22is -H.

[0538] In some embodiments, when N14, N15, N16, N17 and N18 are present, (i) in N14, N16, and N17, R22is -F; and (ii) in N18, R22is -H.

[0539] In some embodiments, when N15, N16, N17, N18 and N19 are present, (i) in N15, N17, and N18, R22is -F; and (ii) in N19, R22is -H.

[0540] In some embodiments, when N16, N17, N18, N19 and N20 are present, (i) in N16, N18, and N19, R22is -F; and (ii) in N20, R22is -H.PAT059650-WO-PCT

[0541] In some embodiments, when N17, N18, N19, N20 and N21 are present, (i) in N17, N19, and N20, R22is -F; and (ii) in N21, R22is -H.

[0542] In some embodiments, in N2′, R22is -ORAand RAis unsubstituted C1-C4 alkyl. In some embodiments, in N2′, R22is independently -ORAand RAis methyl. In some embodiments, in N2′, R22is independently -ORAand RAis ethyl. In some embodiments, in N1′, R22is -OCH3(OMe). In some embodiments, in N2′, when R22is -ORA, RAis not substituted C1-C4 alkyl. In some embodiments, in N2′, R22is not -O-CH2CH2-OCH3.

[0543] In some embodiments, in N2′, R22is -F. In some embodiments, in N2′, R21is hydrogen, R22is -F, and R24is hydrogen.

[0544] In some embodiments, in N2′, R22is -OCH3. In some embodiments, in N2′, R21is hydrogen, R22is -OCH3, and R24is hydrogen.

[0545] In some embodiments, in N24′, R22is -ORAand RAis unsubstituted C1-C4 alkyl. In some embodiments, in N24′, R22is independently -ORAand RAis methyl. In some embodiments, in N24′, R22is independently -ORAand RAis ethyl. In some embodiments, in N24′, R22is -OCH3(OMe). In some embodiments, in N24′, when R22is -ORA, RAis not substituted C1-C4 alkyl. In some embodiments, in N24′, R22is not -O-CH2CH2-OCH3.

[0546] In some embodiments, in N24′, R22is -F. In some embodiments, in N24′, R21is hydrogen, R22is -F, and R24is hydrogen.

[0547] In some embodiments, in N24′, R22is -OCH3. In some embodiments, in N24′, R21is hydrogen, R22is -OCH3, and R24is hydrogen.

[0548] In some embodiments, at least one of N3′to N23′in the antisense strand of Formula (IV) is a glycol nucleic acid (GNA). In some embodiments, one or two of N3′ to N23′ in the antisense strand of Formula (IV) each is a glycol nucleic acid (GNA). In some embodiments, only one of N3′ to N23′ in the antisense strand of Formula (IV) is a glycol nucleic acid (GNA). In some embodiments, two of N3′to N23′in the antisense strand of Formula (IV) is a glycol nucleic acid (GNA).

[0549] In some embodiments, at least one of X3′to X23′in the antisense strand of Formula (II) has a structure of Formula (B-2),PAT059650-WO-PCT

[0550] a pharmaceutically acceptable salt thereof. R21, R22, R23,

[0551] of N3′to N23′independently have a structure of Formula (B-2), or a pharmaceutically acceptable salt thereof. In some embodiments, only one of N3′to N23′has the structure of Formula (B-2) and R21is hydrogen. In some embodiments, only one of N3′ to N23′ has the structure of Formula (B-2) and R23is hydrogen. In some embodiments, only one of N3′to N23′has the structure of Formula (B-2) and R24is hydrogen. In some embodiments, only one of N3′ to N23′ has the structure of Formula (B-2) and R21, R23, and R24are hydrogen.

[0552] In some embodiments, two of N3′ to N23′ independently have a structure of Formula (B-2), or a pharmaceutically acceptable salt thereof. In some embodiments, two of N3′to N23′have the structure of Formula (B-2) and R21is hydrogen. In some embodiments, two of N3′ to N23′have the structure of Formula (B-2) and R23is hydrogen. In some embodiments, two of N3′ to N23′ have the structure of Formula (B-2) and R24is hydrogen. In some embodiments, two of N3′ to N23′ have the structure of Formula (B-2) and R21, R23, and R24are hydrogen.

[0553] In some embodiments, at least one of N3′to N23′in the antisense strand of Formula (IV) have a structure of Formula (B-1), a pharmaceutically acceptable salt thereof. R21, R22,

[0554] In some embodiments, one or two of N3′ to N23′ independently have a structure of Formula (B-1), or a pharmaceutically acceptable salt thereof. In some embodiments, only one of N3′ to N23′ has the structure of Formula (B-1) and R21is hydrogen. In some embodiments, only one of N3′ to N23′ has the structure of Formula (B-1) and R22is hydrogen.PAT059650-WO-PCT In some embodiments, only one of N3′ to N23′ has the structure of Formula (B-1) and R24is hydrogen. In some embodiments, only one of N3′to N23′has the structure of Formula (B-1) and R21, R22, and R24are hydrogen.

[0555] In some embodiments, in any one of N3′to N23′having a structure of Formula (B-1), if present, R21is hydrogen. In some embodiments, in N3′ to N23′ having a structure of Formula (B-1), if present, R24is hydrogen. In some embodiments, in any one of N3′to N23′having a structure of Formula (B-1), if present, R21and R24are hydrogen.

[0556] In some embodiments, in at least one of N3′to N23′having a structure of Formula (B- 1), if present, R22is hydrogen. In some embodiments, in N3′ to N23′ having a structure of Formula (B-1), if present, at least one of R22is not hydrogen.

[0557] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, R22is halogen. In some embodiments, in two, three, or four monomers selected from N2′ to N25′, R22is -F. In some embodiments, in no more than six monomers selected from N2′ to N25′, R22is -F.

[0558] In some embodiments, in at least one of N3′to N23′having a structure of Formula (B- 1), if present, each R22is -ORAand RAis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N2′to N25′having a structure of Formula (B-1),if present, R22is -ORAand RAis hydrogen. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, when R22is -ORA, RAis not hydrogen. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is -ORAand RAis substituted or unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is -ORAand RAis unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is -ORAand RAis unsubstituted methyl, ethyl, propyl, or isopropyl.

[0559] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently -ORAand RAis substituted C1-C4alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently -ORAand RAis alkoxyl-substituted C1-C4alkyl. In some embodiments, RAis methoxy (-OCH3) substituted C1-C4 alkyl. In some embodiments, R22is - O-CH2-OCH3.

[0560] In some embodiments, in N3′ to N23′ having a structure of Formula (B-1), if present, R22is not -O-CH2CH2-OCH3.PAT059650-WO-PCT

[0561] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, R22is independently -NRBRCand each RBand RCis independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, R22is independently -NRBRCand each RBand RCis independently hydrogen. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently -NRBRCand each RBand RCis independently hydrogen or unsubstituted C1-C4 alkyl. In some embodiments, each RBand RCis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0562] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently -C(O)RDand RDis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently -C(O)RDand RDis hydrogen. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently -C(O)RDand RDis unsubstituted C1-C4 alkyl. In some embodiments, each RD isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0563] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently -OC(O)RDand RDis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently - OC(O)RDand RDis hydrogen. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently - OC(O)RDand RDis unsubstituted C1-C4alkyl. In some embodiments, each RD isindependently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0564] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently -C(O)NRBRCand each RBand RCis independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently - C(O)NRBRCand each RBand RCis independently hydrogen. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently - C(O)NRBRCand each RBand RCis independently unsubstituted C1-C4 alkyl. In some embodiments, each RBand RCis independently unsubstituted methyl, ethyl, propyl, or isopropyl.PAT059650-WO-PCT

[0565] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently –NREC(O)RDand each REand RDis independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently - NREC(O)H and REis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently - NREC(O)CH3 and REis hydrogen or substituted or unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently - NREC(O)H and REis unsubstituted C1-C4 alkyl. In some embodiments, each REis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0566] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently -NREC(O)OH and REis hydrogen or substituted or unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently - NREC(O)OH and REis hydrogen. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently - NREC(O)OH and REis unsubstituted C1-C4alkyl. In some embodiments, each REis independently unsubstituted methyl, ethyl, propyl, or isopropyl.

[0567] In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B- 1), if present, each R22is independently substituted or unsubstituted C1-C6alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently substituted or unsubstituted C1-C4alkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of Formula (B-1), if present, each R22is independently substituted C1-C4alkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently unsubstituted C1-C4 alkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently substituted or unsubstituted methyl. In some embodiments, in N2to N22, at least one of R22is independently substituted or unsubstituted ethyl.

[0568] In some embodiments, in at least one of N3′to N23′having a structure of Formula (B- 1), if present, each R22is independently substituted or unsubstituted 2 to 6 membered heteroalkyl. In some embodiments, in at least one of N3′ to N23′ having a structure of FormulaPAT059650-WO-PCT (B-1), if present, each R22is independently substituted or unsubstituted 2 to 5 membered heteroalkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently substituted or unsubstituted 2 to 4 membered heteroalkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently substituted or unsubstituted 2 to 3 membered heteroalkyl. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently -O-CH3. In some embodiments, in at least one of N3′to N23′having a structure of Formula (B-1), if present, each R22is independently -O-CH2- CH3. In some embodiments, in N3′ to N23′ having a structure of Formula (B-1), if present, R22is not -O-CH2CH2-OCH3.

[0569] In some embodiments, in any one of N3′ to N23′ having a structure of Formula (B-1), if present, R22is independently hydrogen, -F, -OH, and -OCH3; and R21and R24are hydrogen. In some embodiments, in any one N3′ to N23′ having a structure of Formula (B-1), R22is independently hydrogen, -F, - and -OCH3; and R21and R24are hydrogen.

[0570] In some embodiments, in two monomers of N3′to N23′having a structure of Formula (B-1), if present, R22is -F. In some embodiments, in three monomers of N3′ to N23′ having a structure of Formula (B-1), if present, R22is -F. In some embodiments, in four monomers of N3′ to N23′ having a structure of Formula (B-1), if present, R22is -F.

[0571] In some embodiments, in two, four or six monomers from N3′to N23′, R22is -F. In some embodiments, in two monomers from N3′ to N23′, R22is -F. In some embodiments, in four monomers from N3′to N23′, R22is -F. In some embodiments, in six monomers from N3′to N23′, R22is -F. In some embodiments, in more than six monomers from N3′ to N23′, R22is - F.

[0572] In some embodiments, in two, four or six monomers from N3′ to N23′, R22is -F. In some embodiments, in two monomers from N3′to N23′, R22is -F. In some embodiments, in four monomers from N3′ to N23′, R22is -F. In some embodiments, in six monomers from N3′ to N23′, R22is -F. In some embodiments, in more than six monomers from N3′to N23′, R22is - F.

[0573] In some embodiments, in one monomer from N3′to N23′, R22is -F and two, three or four monomers from N3′ to N23′, R22is -H. In some embodiments, in one monomer from N3′ to N23′, R22is -F and two monomers from N3′to N23′, R22is -H. In some embodiments, in one monomer from N3′ to N23′, R22is -F and three monomers from N3′ to N23′, R22is -H.PAT059650-WO-PCT

[0574] In some embodiments, in N2 to N23 of the sense strand of Formula (III), L21is independently -P(O)(OH)-O-CH2-, or -P(O)(SH)-O-CH2-.

[0575] In some embodiments, in N2, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N3,if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N2 and N3, if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N4,if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N5, if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N4and N5, if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N2, N3, N4 and N5, if present, L21is independently -P(O)(SH)-O-CH2-.

[0576] In some embodiments, in N23,L21is independently -P(O)(OH)-O-CH2-. In some embodiments, in N22, L21is independently - P(O)(OH)-O-CH2-. In some embodiments, in N22 and N23, L21is independently - P(O)(OH)-O-CH2-. In some embodiments, in N21, if present, L21is independently - P(O)(OH)-O-CH2-. In some embodiments, in N20, if present, L21is independently -P(O)(OH)-O-CH2-. In some embodiments, in N20 and N21, if present, L21is independently -P(O)(OH)-O-CH2. In some embodiments, in N20, N21, N22and N23,if present, L21is independently - P(O)(OH)-O-CH2-.

[0577] In some embodiments, in N23,L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N22, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N22 and N23, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N21,if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N20, if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N20and N21,if present, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N20, N21, N22 and N23, if present, L21is independently -P(O)(SH)-O-CH2-.

[0578] In some embodiments, in N2, N3, N22 and N23, if present, L21is independently - P(O)(SH)-O-CH2-. In some embodiments, in N2,N3,N4,N5,N20, N21, N22and N23,if present, L21is independently -P(O)(SH)-O-CH2-.

[0579] In some embodiments, in N2′to N25′of the antisense of Formula (IV), if present, L21is independently -P(O)(OH)-O-CH2-, or -P(O)(SH)-O-CH2-.

[0580] In some embodiments, in N2′,L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N3′, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N2′ and N3′,L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N4′,L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N5′, L21is independently -PAT059650-WO-PCT P(O)(SH)-O-CH2-. In some embodiments, in N4′ and N5′, L21is independently - P(O)(SH)-O- CH2-. In some embodiments, in N2′, N3′, N4′and N5′,L21is independently -P(O)(SH)-O-CH2-.

[0581] In some embodiments, in N25′, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N24′,L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N24′and N25′, L21is independently -P(O)(SH)-O-CH2-. In some embodiments, in N23′, L21is independently - P(O)(SH)-O-CH2-. In some embodiments, in N22′,L21is independently - P(O)(SH)-O-CH2-. In some embodiments, in N22′ and N23′, L21is independently -P(O)(SH)- O-CH2-. In some embodiments, in N22′, N23′, N24′and N25′,L21is independently -P(O)(SH)-O- CH2-.

[0582] In some embodiments, in N2′,N3′,N24′and N25′,L21is independently -P(O)(SH)-O- CH2-. In some embodiments, in N2′, N3′, N4′, N5′, N22′, N23′, N24′ and N25′, L21is independently - P(O)(SH)-O-CH2-.

[0583] In certain aspects, a double stranded RNAi (dsRNAi) agent includes: (i) a sense strand comprising monomers (e.g., nucleotides) of Y1 to Y23 and having the Formula (I′): 5′-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15-Y16-Y17-Y18-Y19-Y20-Y21-Y22- Y23-3′ (I′) (ii) an antisense strand comprising monomers (e.g., nucleotides) of Y1′to Y25′and having the Formula (II′): 5′-Y1′-Y2′-Y3′-Y4′-Y5′-Y6′-Y7′-Y8′-Y9′-Y10′-Y11′-Y12′-Y13′-Y14′-Y15′-Y16′-Y17′-Y18′-Y19′- Y20′-Y21′-Y22′-Y23′-Y24′-Y25′-3′ (II′) wherein: each Y1, Y2, Y22, and Y23is independently a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, a 2′-aminoalkyl modified nucleotide, a locked RNA (LNA), or a glycol motif nucleotide (GNA), wherein each alkyl, amino, or amino alkyl may be substituted or unsubstituted; Y3 to Y21 and Y1′ to Y25′, are each, independently absent, a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, a 2′-PAT059650-WO-PCT aminoalkyl modified nucleotide, a locked RNA (LNA), or a glycol motif nucleotide (GNA), wherein each alkyl, amino, or amino alkyl may be substituted or unsubstituted; provided that: (i) at least one of Y1and Y23are 2′-O-methoxyethyl (MOE) modified nucleotides; (ii) Y1′ is not 2′-MOE modified nucleotide; (iii) in Y3to Y21, (a) none of Y3 to Y21 are 2′-MOE modified nucleotide, (b) at least one of Y3to Y21is not deoxynucleotide (DNA), and (c) no more than six monomers are 2′-F modified nucleotides; and (iv) no more than eight monomers from Y3to Y21are absent and no more than ten monomers from Y3′ to Y23′ are absent.

[0584] In some embodiments, in Formula (I′), Y1 is a 2′-MOE modified nucleotide. In some embodiments, Y23 is a 2′-MOE modified nucleotide. In some embodiments, Y1 and Y23 are 2′-MOE modified nucleotide.

[0585] In some embodiments, in Formula (I′), Y2 is a 2′-MOE modified nucleotide. In some embodiments, Y22is a 2′-MOE modified nucleotide. In some embodiments, Y2and Y22are 2′-MOE modified nucleotide. In some embodiments, Y1, Y2, Y22, and Y23 are 2′-MOE modified nucleotide.

[0586] In some embodiments, in Formula (II′), each Y1′, Y2′, Y24′ and Y25′ are not absent. In some embodiments, in Formula (II′), each Y1′, Y2′, Y24′and Y25′is independently a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C- alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, and a 2′-aminoalkyl modified nucleotide. In some embodiments, each Y3′ to Y23′ is independently absent, a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, a 2′-aminoalkyl modified nucleotide, a locked RNA (LNA), a threofuranosyl nucleotide (TNA), or a glycol motif nucleotide (GNA). Each alkyl, amino, or amino alkyl may be substituted or unsubstituted.

[0587] In some embodiments, the 2′-O-alkyl modified nucleotide includes the 2′-O-(C1- C6alkyl) modified nucleotide. In some embodiments, the 2′-O-alkyl modified nucleotide includes the 2′-O-(C1-C4 alkyl) modified nucleotide. In some embodiments, the 2′-O- alkyl modified nucleotide includes the 2′-O-(C1-C3 alkyl) modified nucleotide. In somePAT059650-WO-PCT embodiments, the 2′-O-alkyl modified nucleotide is 2′-O-CH3 ( or “2′-OMe”) modified nucleotide. In some embodiments, the 2′-O-alkyl modified nucleotide is 2′-O-CH2CH3(or “2′-OEt”) modified nucleotide.

[0588] In some embodiments, the 2′- alkyl modified nucleotide includes the 2′-(C1-C6alkyl) modified nucleotide. In some embodiments, the 2′- alkyl modified nucleotide includes the 2′-(C1-C4alkyl) modified nucleotide. In some embodiments, the 2′- alkyl modified nucleotide includes the 2′-(C1-C3 alkyl) modified nucleotide. In some embodiments, the 2′- alkyl modified nucleotide is 2′-CH3(2′-Me) modified nucleotide. In some embodiments, the 2′-alkyl modified nucleotide is 2′-CH2CH3 (2′-Et) modified nucleotide.

[0589] In some embodiments, at least one of Y1′to Y25′is independently absent, 2′-F modified nucleotide, 2′-O-methyl (OMe) modified nucleotide, 2′-MOE modified nucleotide, RNA, DNA, GNA, or LNA. In some embodiments, at least one of Y1′ to Y25′ is 2′-F modified nucleotide. In some embodiments, at least one of Y1′ to Y25′ is 2′-O-methyl (OMe) modified nucleotide. In some embodiments, at least one of Y1′ to Y25′ is 2′-MOE modified nucleotide. In some embodiments, Y1′to Y25′do not include 2′-MOE modified nucleotide. In some embodiments, at least one of Y1′ to Y25′ is RNA. In some embodiments, Y1′ to Y25′ do not include RNA. In some embodiments, at least one of Y1′to Y25′is DNA. In some embodiments, Y1′ to Y25′ do not include DNA. In some embodiments, at least one of Y1′ to Y25′is GNA. In some embodiments, Y1′to Y25′do not include GNA.

[0590] In some embodiments, Y1′ includes a 5′-(E)-vinyl phosphonate group. In some embodiments, Y1′includes a phosphate group. In some embodiments, Y1′includes a phosphorothioate (PS) group.

[0591] In some embodiments, in Formula (I′), no more than eight monomers from Y3to Y21 are absent. In some embodiments, in Formula (I′), no more than seven monomers from Y3to Y21are absent. In some embodiments, in Formula (I′), no more than six monomers from Y3 to Y21 are absent. In some embodiments, in Formula (I′), no more than five monomers from Y3to Y21are absent. In some embodiments, in Formula (I′), no more than four monomers from Y3 to Y21 are absent. In some embodiments, in Formula (I′), no more than three monomers from Y3to Y21are absent. In some embodiments, in Formula (I′), no more than two monomers from Y3 to Y21 are absent. In some embodiments, in Formula (I′), no more than one nucleotide from Y3to Y21are absent.

[0592] In some embodiments, in Formula (I′), one nucleotide from Y3 to Y21 are absent. In some embodiments, in Formula (I′), two monomers from Y3 to Y21 are absent. In somePAT059650-WO-PCT embodiments, in Formula (I′), three monomers from Y3 to Y21 are absent. In some embodiments, in Formula (I′), four monomers from Y3to Y21are absent. In some embodiments, in Formula (I′), five monomers from Y3 to Y21 are absent. In some embodiments, in Formula (I′), six monomers from Y3to Y21are absent. In some embodiments, in Formula (I′), seven monomers from Y3 to Y21 are absent. In some embodiments, in Formula (I′), eight monomers from Y3to Y21are absent.

[0593] In some embodiments, in Formula (II′), no more than ten monomers from Y3′ to Y23′are absent. In some embodiments, in Formula (II′), no more than nine monomers from Y3′ to Y23′ are absent. In some embodiments, in Formula (II′), no more than eight monomers from Y3′to Y23′are absent. In some embodiments, in Formula (II′), no more than seven monomers from Y3′ to Y23′ are absent. In some embodiments, in Formula (II′), no more than six monomers from Y3′ to Y23′ are absent. In some embodiments, in Formula (II′), no more than five monomers from Y3′ to Y23′ are absent. In some embodiments, in Formula (II′), no more than four monomers from Y3′ to Y23′ are absent. In some embodiments, in Formula (II′), no more than three monomers from Y3′to Y23′are absent. In some embodiments, in Formula (II′), no more than two monomers from Y3′ to Y23′ are absent. In some embodiments, in Formula (II′), no more than one nucleotide from Y3′to Y23′are absent. [0...

Claims

PAT059650-WO-PCT WHAT IS CLAIMED:

1. A double stranded RNAi (dsRNAi) agent for inhibiting expression of a target gene comprising: (i) a sense strand comprising monomers of N1to N23and having the Formula (III): 5′-N1-N2-N3-N4-N5-N6-N7-N8-N9-N10-N11-N12-N13-N14-N15-N16-N17-N18-N19-N20-N21-N22- N23-3′ (III), or a pharmaceutically acceptable salt thereof, wherein: N1 has a structure of Formula (A-1), a pharmaceutically acceptable salt thereof; a structure of Formula (B-1),a pharmaceutically acceptable salt thereof;of Formula (B-1), a pharmaceutically acceptable salt thereof; and,PAT059650-WO-PCT a pharmaceutically acceptable salt thereof; (ii)of N1′ to N25′ and having the Formula (IV): 5′-N1′-N2′-N3′-N4′-N5′-N6′-N7′-N8′-N9′-N10′-N11′-N12′-N13′-N14′-N15′-N16′-N17′-N18′-N19′-N20′-N21′- N22′-N23′-N24′-N25′-3′ (IV), or a pharmaceutically acceptable salt thereof, wherein: N1′ has a structure of Formula (A-1), a pharmaceutically acceptable salt thereof;a structure of Formula (B-1), a pharmaceutically acceptable salt thereof;acid (GNA), or has a structure Formula (B-1), Formula (B-2),PAT059650-WO-PCT, a pharmaceutically acceptable salt thereof; 2), and (C-1):occurrence, absent or a nucleobase; in each occurrence, each R21, R22, R23, and R24is independently hydrogen, halogen, - ORA, -SRA, -NRBRC, -C(O)RD, -OC(O)RD, -C(O)NRBRC, -NREC(O)RD, - NREC(O)ORD, -NREC(O)NRBRC, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl, wherein each RA, RB, RC, RD, and RFis independently hydrogen, or C1-C10alkyl optionally substituted with halogen, -OH, -O-C1-4alkyl, or -O-C1-4 haloalkyl; in each occurrence, R25is hydrogen, -OH, a detectable moiety, a first ligand, or a functional group capable of forming a covalent bond with a first ligand; in each occurrence, R26is hydrogen, a detectable moiety, -P(O)(OH)-OH, -P(O)(SH)- OH, a second ligand, or a functional group capable of forming a covalent bond with a second ligand; in each occurrence, L21is independently absent, -O-, -O-L22-, -P(O)(OH)-O-L22-, - P(O)(SH)-O-L22-, -P(O)(OH)-L23-, or -P(O)(SH)-L23-, wherein each L22is independently a substituted or unsubstituted C1-C4 alkylene and each L23is independently a substituted or unsubstituted C2-C4 alkenylene, and in each is an attachment point to the adjacent monomers,PAT059650-WO-PCT provided that: (i) in at least one of N1and N23, R22is -O-CH2CH2-OCH3, (ii) in N1′, R22is not -O-CH2CH2-OCH3, (iii) in N3to N21,(a) R22is not -O-CH2CH2-OCH3, (b) at least one of R22is not hydrogen, and (c) in no more than six nucleotides, R22is -F, and (iv) no more than eight monomers from N3to N21are absent and no more than ten monomers from N3′ to N23′ are absent.

2. The dsRNA agent of claim 1, wherein N1 has a structure of , or a pharmaceutically acceptable salt3. The dsRNA agent of claim 1 or 2, wherein N23has a structure of a pharmaceutically acceptable salt thereof.

4. The dsRNA agent of any one of claims 1 through 3, wherein:PAT059650-WO-PCT N1 has aacceptable saltN23 has a a pharmaceuticallyacceptable salt5. The dsRNA agent of any one of claims 1 through 4, wherein each N2and N22independently has a structure of a pharmaceutically acceptable salt thereof.

6. The dsRNA agent of any one of claims 1 through 5, wherein: N1 has a structure ofPAT059650-WO-PCT N2 and N22 independently has a structure of, or a pharmaceutically acceptable salt thereof; -SH or -OH.

7. The dsRNA agent of any one of claims 1 through 6,of claims 1 through 7, wherein N1′has a structure of, or a pharmaceutically acceptable salt thereof.

9. The dsRNAi agent of any one of claims 1 through 8, wherein one to six monomers from N3 to N21 are absent.PAT059650-WO-PCT 10. The dsRNAi agent of any one of claims 1 through 9, wherein two monomers from N3to N21 are absent.

11. The dsRNAi agent of any one of claims 1 through 10, wherein one to eight monomers from N3′to N23′are absent.

12. The dsRNAi agent of any one of claims 1 through 11, wherein two monomers from N3′ to N23′ are absent.

13. The dsRNAi agent of any one of claims 1 through 12, wherein N3 to N21 include Nn, Nn+1, Nn+2, Nn+3, and Nn+4 wherein n is an integer from 3 to 17, and in two, three or four monomers of Nn, Nn+2, Nn+3, and Nn+4 and n is an integer from 3 to 17, R22is -F.

14. The dsRNAi agent of any one of claims 1 through 13, wherein N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4 wherein n is an integer from 5 to 10, and in two, three or four monomers of Nn, Nn+2, Nn+3, and Nn+4when n is an integer from 5 to 10, R22is -F.

15. The dsRNAi agent of any one of claims 1 through 12, wherein N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4 wherein n is an integer from 3 to 17, wherein: in each monomer of Nn, Nn+2, and Nn+3, R22is -F and in Nn+4, R22is hydrogen.

16. The dsRNAi agent of any one of claims 1 through 12 and 15, wherein N3to N21include Nn, Nn+1, Nn+2, Nn+3, and Nn+4 wherein n is an integer from 5 to 10, wherein: in each monomer of Nn, Nn+2, and Nn+3, R22is -F and in Nn+4, R22is hydrogen.

17. The dsRNAi agent of any one of claims 1 through 16, wherein: (i) in only one monomer from N3′ to N23′, R22is -F, and in two to four monomer from N3′ to N23′, R22is -H; orPAT059650-WO-PCT (ii) in two to six monomers from N3′ to N23′, R22is -F.

18. The dsRNAi agent of any one of claims 1 through 17, wherein, in more than six monomers from N3′to N23′, R22is -F.

19. The dsRNAi agent of any one of claims 1 through 18, wherein in N3to N21, if present, R22is independently hydrogen, -F, -OH, -OCH3, -OCH2CH3, -CH3, -CH2CH3, - C(O)OH, -C(O)NH2, and -NH2.

20. The dsRNAi agent of any one of claims 1 through 19, wherein in N3to N21,if present, R22is independently hydrogen, -F, or -OCH3.

21. The dsRNAi agent of any one of claims 1 through 20, wherein in N3 to N21, if present, L21is independently -P(O)(OH)-O-CH2-, or -P(O)(SH)-O-CH2-.

22. The dsRNAi agent of any one of claims 1 through 21, wherein N2 and N3 are present and in N2and N3, L21is independently -P(O)(SH)-O-CH2-.

23. The dsRNAi agent of any one of claims 1 through 22, wherein N22and N23are present and in N22 and N23, L21is independently -P(O)2-O-CH2- or -P(O)(SH)-O-CH2-.

24. The dsRNAi agent of any one of claims 1 through 23, wherein, in two to six of N3 to N21, L21are independently -P(O)(SH)-O-CH<sub>2-.

25. The dsRNAi agent of any one of claims 1 through 24, wherein in N2′, R22is -F or - OCH3.

26. The dsRNAi agent of any one of claims 1 through 25, wherein one or two of N3′ to N23′are each a glycol nucleic acid (GNA).

27. The dsRNAi agent of any one of claims 1 through 25, wherein one or two of N3′to N23′ each independently have a structure of Formula (B-1),PAT059650-WO-PCT , wherein R22is hydrogen, or a pharmaceutically<img src='' class="img-anchor img-center" img-id="IMGF000304_0001" / >28. The dsRNAi agent of any one of claims 1 through 25, wherein one or two of N3′ to N23′in the antisense strand of Formula (IV) each have a structure of Formula (B-2), a pharmaceutically acceptable salt thereof.<img src='' class="img-anchor img-center" img-id="IMGF000304_0002" / >29. The dsRNAi agent of any one of claims 1 through 28, wherein in N2′to N25′, if present, L21is independently -P(O)(OH)-O-CH2-, or -P(O)(SH)-O-CH2-.

30. The dsRNAi agent of any one of claims 1 through 29, wherein N2′ and N3′ are present and in N2′and N3′,L21is independently -P(O)(SH)-O-CH2-.

31. The dsRNAi agent of any one of claims 1 through 30, wherein N24′and N25′are present and in N24′ and N25′, L21is independently -P(O)(SH)-O-CH2-.

32. The dsRNAi agent of any one of claims 1 through 31, in two to six of N3′ to N23′, L21are independently -P(O)(SH)-O-CH2-.

33. The dsRNAi agent of any one of claims 1 through 32, wherein R21, R23, R24are hydrogen.PAT059650-WO-PCT 34. A double stranded RNAi (dsRNAi) agent for inhibiting expression of a target gene, comprising: (i) a sense strand comprising monomers of Y1 to Y23 and having a Formula (I′); 5′-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14-Y15-Y16-Y17-Y18-Y19-Y20-Y21-Y22- Y23-3′ (I′) or a pharmaceutically acceptable salt thereof; and (ii) an antisense strand comprising monomers of Y1′to Y25′and having a Formula (II′): 5′-Y1′-Y2′-Y3′-Y4′-Y5′-Y6′-Y7′-Y8′-Y9′-Y10′-Y11′-Y12′-Y13′-Y14′-Y15′-Y16′-Y17′-Y18′-Y19′-Y20′-Y21′- Y22′-Y23′-Y24′-Y25′-3′ (II′), or a pharmaceutically acceptable salt thereof, wherein: each Y1, Y2, Y22, and Y23 is independently a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, a 2′-aminoalkyl modified nucleotide, a locked RNA (LNA), or a glycol motif nucleotide (GNA), wherein each alkyl, amino, or amino alkyl may be substituted or unsubstituted; Y3 to Y21 and Y1′ to Y25′, are each, independently absent, a ribonucleotide (RNA), a deoxynucleotide (DNA), a 2′-O-alkyl modified nucleotide, a 2′-C-alkyl modified nucleotide, a 2′-halo modified nucleotide, a 2′-amino modified nucleotide, a 2′- aminoalkyl modified nucleotide, a locked RNA (LNA), or a glycol motif nucleotide (GNA), wherein each alkyl, amino, or amino alkyl may be substituted or unsubstituted; provided that: (i) at least one of Y1 and Y23 are 2′-O-methoxyethyl (MOE) modified nucleotides; (ii) Y1′is not 2′-MOE modified nucleotide; (iii) in Y3 to Y21, (a) none of Y3to Y21are 2′-MOE modified nucleotide, (b) at least one of Y3 to Y21 is not deoxynucleotide (DNA), and (c) no more than six monomers are 2′-F modified nucleotides; and (iv) no more than eight monomers from Y3 to Y21 are absent and no more than ten monomers from Y3′to Y23′are absent.

35. The dsRNAi agent of claim 34, wherein Y1 and Y23 are 2′-MOE modified nucleotides.PAT059650-WO-PCT 36. The dsRNAi agent of claim 34 or 35, wherein Y2and Y22are 2′-MOE modified nucleotides.

37. The dsRNAi agent of any one of claims 34 through 36, wherein each Y3 to Y21 and Y1′ to Y25′is independently absent, a 2′-F modified nucleotide, a 2′-O-methyl (OMe) modified nucleotide, an abasic nucleotide, RNA, DNA, GNA, or LNA.

38. The dsRNAi agent of any one of claims 34 through 37, wherein Y1′ comprises a 5′- (E)-vinyl phosphonate group.

39. The dsRNAi agent of any one of claims 34 through 38, wherein one to six monomers from Y3 to Y21 are absent.

40. The dsRNAi agent of any one of claims 34 through 39, wherein two monomers from Y3 to Y21 are absent.

41. The dsRNAi agent of any one of claims 34 through 40, wherein one to eight monomers from Y3′to Y23′are absent.

42. The dsRNAi agent of any one of claims 34 through 41, wherein two monomers from Y3′ to Y23′ are absent.

43. The dsRNAi agent of any one of claims 34 through 42, wherein Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4wherein n is an integer from 3 to 17, and two, three or four monomers of Yn, Yn+2, Yn+3, and Yn+4 are 2′-F modified nucleotides.

44. The dsRNAi agent of any one of claims 34 through 43, wherein Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4wherein n is an integer from 5 to 10, and (i) Yn, Yn+2, Yn+3, and Yn+4 are 2′-F modified nucleotides, or (ii) Yn+2, Yn+3, and Yn+4are 2′-F modified nucleotides.PAT059650-WO-PCT 45. The dsRNAi agent of any one of claims 34 through 43, wherein Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4wherein n is an integer from 3 to 17, and Yn, Yn+2, and Yn+3 are 2′-F modified nucleotides, and Yn+4 is a DNA.

46. The dsRNAi agent of any one of claims 34 through 43 and 45, wherein Y3 to Y21 comprise Yn, Yn+1, Yn+2, Yn+3, and Yn+4wherein n is an integer from 5 to 10, Yn, Yn+2, and Yn+3 are 2′-F modified nucleotides, and Yn+4 is a DNA.

47. The dsRNAi agent of any one of claims 34 through 46, wherein one to six monomers from Y3′to Y23′are 2′-F modified nucleotides.

48. The dsRNAi agent of any one of claims 34 through 46, wherein more than six monomers from Y3′ to Y23′ are 2′-F modified nucleotides.

49. The dsRNAi agent of any one of claims 34 through 48, wherein one or two of Y3′to Y23′ are each a GNA.

50. The dsRNAi agent of any one of claims 34 through 48, wherein one to four of Y3′ to Y23′each independently have a DNA.

51. The dsRNAi agent of any one of claims 34 through 48, wherein one or two of Y3′to Y23′ each independently are each a TNA.

52. The dsRNAi agent of any one of claims 34 through 51, wherein Y1 and Y2 is connected via a phosphorothioate linkage.

53. The dsRNAi agent of any one of claims 34 through 52, wherein Y2and Y3is connected via a phosphorothioate linkage.

54. The dsRNAi agent of any one of claims 34 through 53, wherein Y21 and Y22 is connected via a phosphate or phosphorothioate linkage.PAT059650-WO-PCT 55. The dsRNAi agent of any one of claims 34 through 54, wherein Y22 and Y23 is connected via a phosphate or phosphorothioate linkage.

56. The dsRNAi agent of any one of claims 34 through 55, wherein at least one to six monomers between Y3 and Y21 are connected via a phosphorothioate linkage.

57. The dsRNAi agent of any one of claims 34 through 56, wherein Y1′ and Y2′ is connected via a phosphorothioate linkage.

58. The dsRNAi agent of any one of claims 34 through 57, wherein Y2′and Y3′is connected via a phosphorothioate linkage.

59. The dsRNAi agent of any one of claims 34 through 58, wherein Y23′ and Y24′ is connected via a phosphorothioate linkage.

60. The dsRNAi agent of any one of claims 34 through 59, wherein Y24′ and Y25′ is connected via a phosphorothioate linkage.

61. The dsRNAi agent of any one of claims 34 through 60, wherein at least one to six monomers between Y3′ and Y24′ are connected via a phosphorothioate linkage.

62. The dsRNAi agent of any one of claims 1 through 61, further comprising one or more ligands and each ligand comprises a GalNAc moiety.

63. The dsRNAi agent of any one of claims 1 through 61, wherein the first ligand and / or the second ligand comprise a GalNAc moiety.

64. The dsRNAi agent of claim 62 or 63, wherein the GalNAc moiety has a structure of:PAT059650-WO-PCT wherein: each LAis an independently a linker which may be same or different in each occurrence; LBis a linker; m is an integer from 1 to 3; and is an attachment point to the sense strand or antisense strand.

65. dsRNAi agent of claim 64, wherein the GalNAc moiety comprises the following structure of ,Each p1, p2, p3, q1, q2, r1, r2 and r3 is independently an integer from 0 to 12; Each m1, m2, and m3 is independently an integer from 1 to 3; and “*” is an attachment point to LB.PAT059650-WO-PCT 66. The dsRNAi agent of claim 62 or 63, wherein the GalNAc moiety has a structure of: ,each LC, LD, LE, LF, and LGis an independently a linker; LBis a linker as described above; is an attachment point to the sense strand or the antisense strand, or to a conjugate conjugated to the sense strand or the antisense strand.

67. The dsRNAi agent of claim 66, wherein the ligand has a structure of: ,PAT059650-WO-PCT ,each p11 and q11 is independently an integer from 0 to 12; each z1, z2, and z3 is independently an integer of 0 to 12; and is an attachment point to the sense strand or the antisense strand, or to a conjugate conjugated to the sense strand or the antisense strand.

68. The dsRNAi agent of any one of claims 63 through 67, wherein the GalNAc moiety comprises the following structure: ,PAT059650-WO-PCT ,<img src='' class="img-anchor img-center" img-id="IMGF000312_0001" / >69. The dsRNAi agent of any one of claims 1 through 68, wherein the dsRNAi agent is in a pharmaceutically acceptable salt form.

70. The dsRNAi agent of claim 69, wherein the pharmaceutically acceptable salt is a sodium salt.

71. The dsRNAi agent of any one of claims 1 through 70, wherein the dsRNAi agent has improved half-life relative to a reference dsRNAi agent that does not contain chemical modification in nucleotides.

72. A pharmaceutical composition comprising the dsRNAi agent of any one of claims 1 through 71.PAT059650-WO-PCT 73. A method of inhibiting expression of a target gene in a subject comprising: administering to the subject a therapeutically effective amount of the dsRNAi agent of any one of claims 1 through 71, or the pharmaceutical composition of claim 72.

74. A method of treating or preventing a disorder or disease associated with a target gene in a subject comprising: administering to the subject a therapeutically effective amount of the dsRNAi agent of any one of claims 1 through 71, or the pharmaceutical composition of claim 72, wherein the dsRNAi agent inhibits the expression of the target gene.

75. The method of claim 73 or 74, wherein the dsRNAi agent or the pharmaceutical composition is administered subcutaneously or intravenously.

76. The method of any one of claims 73 through 75, wherein the subject is a human.

77. A kit comprising the dsRNAi agent of any one of claims 1 through 71, or the pharmaceutical composition of claim 72.

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