Modified RNA agents with reduced off-target effect

Abstract

To provide dsRNA molecules which are advantageous for inhibition of target gene expression while having reduced off-target gene silencing effects.SOLUTION: The present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The antisense strand of the dsRNA molecule comprises at least one thermally destabilizing nucleotide occurring at a seed region; the dsRNA comprises at least four 2'-fluoro modifications; and the sense strand of the dsRNA molecule comprises a ligand, where the ligand is an ASGPR ligand.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 425,907, filed November 23, 2016, U.S. Provisional Patent Application No. 62 / 548,589, filed August 22, 2017, and U.S. Provisional Patent Application No. 62 / 561,514, filed September 21, 2017, the contents of all of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to the RNAi double-stranded agent that has specific motifs that are advantageous for inhibiting target gene expression by reducing undesired off-target effects, and the RNAi composition that is suitable for therapeutic use.In addition, the present invention provides the method for inhibiting target gene expression by administering these RNAi double-stranded agents, for example, for the treatment of various diseases. [Background technology]

[0003] RNA interference, or "RNAi," is a term first coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Non-Patent Document 1; Non-Patent Document 2). Short dsRNAs induce gene-specific posttranscriptional silencing in many organisms, including vertebrates, providing a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to possess short RNAs (approximately 22 nucleotides) derived from its double-stranded RNA trigger, but the protein component of this activity remained unknown.

[0004] One of the off-target effects of siRNA is the miRNA-like effect, in which Argonaute proteins, the core effectors in RNA interference, process artificially introduced siRNAs as miRNAs (microRNAs) to induce RNA interference (Non-Patent Document 3). For gene suppression, miRNAs recognize many target genes through base pairing between their seed region (positions 2–9 from the 5′ end) and the target mRNA. The off-target effects caused by siRNAs result from base complementarity between the seed region of the RISC-loaded antisense strand of the siRNA and one or more mRNAs. The miRNA-like off-target effect in siRNAs has been reported in several studies and can be so severe that it can affect the expression of multiple genes depending on the sequence of the seed region and account for up to 30% of positive hits in phenotypic screening based on siRNAs. In addition, it has been reported that miRNAs silence target genes through complementary pairing within their 3′-terminal regions (3′ complementary pairing) when the interaction between the seed region and the target becomes weak, implying that the miRNA-like off-target effect is likely mediated by such a mechanism. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Fire et al. (1998) Nature 391,806-811 [Non-patent document 2] Elbashir et al.(2001)Genes Dev.15,188-200 [Non-patent document 3] Lam et al.(2015)Molecular Therapy Nucleic Acids(2015)4,e252 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, efforts are ongoing to eliminate or reduce the miRNA-like off-target effects of siRNAs by adjusting the design of siRNAs through the judicious application of chemical modifications without compromising the gene silencing efficacy of siRNA gene therapy.The present invention is directed to these effects. [Means for solving the problem]

[0007] The present invention provides effective nucleotide or chemical motifs for dsRNA molecules that are advantageous for inhibiting target gene expression while reducing off-target gene silencing effects, and RNAi compositions suitable for therapeutic use.

[0008] In particular, the inventors have discovered that dsRNA molecules targeting can be more effective at mediating RNA interference than parent dsRNA molecules lacking the destabilizing modification if the antisense strand contains at least one thermally destabilizing modification of the duplex within the seed region (i.e., positions 2-9 of the 5' end, counting from the 5' end of the antisense strand) and the dsRNA molecule has a melting temperature within the range of about 40°C to about 80°C.

[0009] Thus, in one aspect, the present invention provides a dsRNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at positions 2 to 9 of the 5' end of the antisense strand, counting from the 5' end), and wherein the dsRNA has the following characteristics: (i) a melting temperature (T m(ii) the antisense comprises two, three, four, five, or six 2'-fluoro modifications; (iii) the antisense comprises one, two, three, or four phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated to a ligand; (v) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (vi) the sense strand comprises one, two, three, or four phosphorothioate internucleotide linkages; (vii) the dsRNA comprises at least four 2'-fluoro modifications; (viii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (ix) a blunt end at the 5' end of the antisense strand.

[0010] In some embodiments, the present invention provides a dsRNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., positions 2-9, preferably 3-8, counting from the 5' end of the antisense strand), and the dsRNA molecule has the following characteristics: (i) a melting temperature (T m (ii) the antisense strand contains 6, 7, 8, 9, 10, 11, or 12 2'-OMe modifications; (iii) the antisense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated to a ligand; (v) the sense strand contains 6, 7, 8, 9, 10, 11, or 12 2'-OMe modifications; (vi) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (vii) the dsRNA contains at least 1, 2, 3, 4, or 5 2'-deoxy modifications; (viii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (ix) a blunt end at the 5' end of the antisense strand.

[0011] In some embodiments, the dsRNA has a melting temperature at the lower end of the range from about 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C, and at the upper end of the range from about 70°C, 75°C, or 80°C. In some embodiments, the dsRNA has a melting temperature within the range of about 55°C to about 70°C. In some embodiments, the dsRNA has a melting temperature within the range of about 57°C to about 67°C. In certain embodiments, the dsRNA has a melting temperature within the range of about 60°C to about 67°C. In some further embodiments, the dsRNA has a melting temperature within the range of about 62°C to about 66°C.

[0012] The inventors have also discovered that dsRNA molecules having a melting temperature of at least 60° C. are more effective in vivo and in vitro. Thus, in some embodiments, the dsRNA has a melting temperature of at least 60° C.

[0013] The inventors have also discovered that for a dsRNA molecule to be more effective in vivo, at least 40-50% of the antisense strand must be present in, for example, mouse liver at day 7 in vivo after administration.

[0014] In another aspect, the present invention further provides a method for delivering a dsRNA molecule of the present invention to a specific target in a subject by subcutaneous or intravenous administration.The present invention further provides a dsRNA molecule of the present invention intended for use in a method for delivering said agent to a specific target in a subject by subcutaneous or intravenous administration.

[0015] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows some exemplary destabilizing modifications of the present invention. [Figure 2] Figure 2 shows the positional effect of a single (S)-glycol nucleic acid (GNA) modification in the antisense strand on in vitro conjugate activity. Single substitutions with (S)-GNA are well tolerated in the antisense seed region (positions 5-8 of the antisense strand) or its opposite positions, but are not tolerated at sensitive positions (positions 1 and 2 of the antisense strand and positions 11 and 12 of the sense strand). [Figure 3] FIG. 3 shows that exemplary dsRNAs according to the invention had equivalence to the parent dsRNA for the target activity. [Figure 4] FIG. 4 shows that exemplary dsRNAs of the invention had no off-target activity at high doses in rat hepatocytes. [Figure 5] FIG. 5 shows that exemplary dsRNAs of the invention have knockdown of equivalent genes (GO1 and TTR). [Figure 6] Figure 6 shows that exemplary dsRNAs of the invention mitigate endogenous off-target effects. As demonstrated, both dsRNAs significantly reduced the number of genes down- or up-regulated by their respective parent dsRNAs. [Figure 7] FIG. 7 shows that exemplary dsRNAs according to the invention are as potent as the parent dsRNAs. [Figure 8] FIG. 8 shows that exemplary dsRNAs according to the invention have comparable liver accumulation to the parent dsRNA. [Figure 9] FIG. 9 shows that exemplary dsRNAs according to the invention have comparable on-target activity to the parent dsRNA. [Figure 10] FIG. 10 shows clinicopathological parameters of exemplary dsRNA administered at various concentrations. [Figure 11] FIG. 11 shows normalized body weight gain and liver / body weight ratios in response to administration of exemplary dsRNAs of the invention. [Figure 12]Figure 12 shows the correlation between ΔTm and on- and off-target activity through specific positions of different sequences. Blue data points = on-target activity; red data points = off-target activity. [Figure 13] FIG. 13 shows the effect of melting temperature of dsRNA duplexes on in vitro and in vivo activity. [Figure 14] FIG. 14 shows that exemplary dsRNAs in accordance with the invention had comparable potency to the parent dsRNA but reduced off-target activity in vitro (PMH). [Figure 15] FIG. 15 shows that the exemplary dsRNAs had comparable potency in vivo (rodents) to the parent dsRNA. [Figure 16] FIG. 16 shows that liver toxicity is reduced in rats with exemplary dsRNAs of the invention (ESC+) versus parental dsRNA (ESC). [Figure 17] FIG. 17 shows that exemplary dsRNAs according to the invention had a 6-8 fold improvement in therapeutic index in vivo (rats) relative to the parent dsRNA. [Figure 18] FIG. 18 shows that exemplary dsRNAs in accordance with the invention had comparable IC50s for on-target activity in the COS luciferase system, but much lower off-target activity at comparable concentrations to the parent dsRNA, AD-61444. [Figure 19] FIG. 19 shows that exemplary dsRNAs in accordance with the invention had comparable IC50s for on-target activity in the COS luciferase system, but much lower off-target activity at comparable concentrations to the parent dsRNA, AD-77407. [Figure 20] 20 shows that substitutions with GNA and 2'-F at exemplary positions according to the invention have no adverse effect on in vivo activity compared to the parent dsRNA. The sequences of the dsRNAs are listed in Table 9. [Figure 21]Figure 21 shows that exemplary dsRNAs according to the invention reduced off-target effects. RNAseq from Hep3B cells transfected with 10 nm siRNA (16 hour treatment). [Figure 22] FIG. 22 shows that exemplary dsRNAs according to the invention had comparable single-dose activity compared to the parent dsRNA at various doses in non-human primates. [Figure 23] Figure 23 shows the study design and exemplary dsRNAs in the study in an in vivo mouse study. The sequences of the dsRNA sequences are listed in Table 9. [Figure 24] FIG. 24 shows that exemplary dsRNAs in accordance with the invention had comparable IC50s for on-target activity relative to the parent dsRNAs in the COS luciferase system, but had little off-target activity at comparable concentrations. [Figure 25] FIG. 25 shows that exemplary dsRNAs in accordance with the invention had comparable IC50s for on-target activity relative to the parent dsRNAs in the COS luciferase system, but had little off-target activity at comparable concentrations. [Figure 26] FIG. 26 shows that exemplary dsRNAs according to the invention have equivalent gene knockdown in the liver as the parent dsRNA, despite reduced accumulation in the liver. [Figure 27] FIG. 27 shows that exemplary dsRNAs according to the invention have comparable gene knockdown in the liver as the parental dsRNA, despite reduced accumulation in the liver. [Figure 28] FIG. 28 is a schematic representation of the structures of (S)-GNA and (R)-GNA. [Figure 29] Figure 29 shows the backbone-base tilt (ηB) and helical twist values ​​for A- and B-form RNA / DNA and (S)-GNA. The values ​​for (R)-GNA are extrapolated from the (S)-GNA values ​​by using simple inversion. [Figure 30] FIG. 30 is a schematic representation of the thermal regulation of siRNA conjugate duplexes using GNA. [Figure 31] Figure 31 is a schematic representation of the structure of hAgo2 adapted from PDB file 4W5O and generated using PyMOL. [Figure 32A] Figure 32A shows the position-specific metabolic stability of an exemplary dsRNA in vivo. [Figure 32B] Figure 32B shows the effect of metabolic stability on the resulting pharmacodynamics. [Figure 32C] FIG. 32C shows that thermostabilization of a GNA opposite the sense strand improves the metabolic stability and potency of exemplary dsRNAs. [Figure 33] FIG. 33 is a line graph showing thermal melting (Tm) analysis of exemplary siRNA duplexes containing (S)-GNA. [Figure 34] Figure 34A shows the crystal structure analysis of an RNA duplex modified with both GNA-T stereoisomers. Figure 34B shows the crystal structure analysis of an RNA duplex modified with both GNA-T stereoisomers. Figure 34C shows the crystal structure analysis of an RNA duplex modified with both GNA-T stereoisomers. Figure 34D shows the crystal structure analysis of an RNA duplex modified with both GNA-T stereoisomers. Figure 34E shows the crystal structure analysis of an RNA duplex modified with both GNA-T stereoisomers. Figure 34F shows the crystal structure analysis of an RNA duplex modified with both GNA-T stereoisomers. [Figure 35] FIG. 35 shows the structures of isocytidine and isoguanosine nucleotides and their potential to form perfectly complementary base pairs to "rotated" GNA-C or GNA-G. [Figure 36] Figure 36 shows the positional effect of single (S)-GNA base pair substitutions on in vitro silencing. The base pair at the indicated position in the guide strand was replaced with the corresponding GNA base pair. [Figure 37]Figure 37A is a bar graph showing TTR knockdown in mice with (S)-GNA-modified siRNA duplexes administered at 2.5 mg / kg. Figure 37A shows TTR mRNA levels measured in the liver. Error bars represent standard deviation from each cohort (n=3). Only statistically significant comparisons are shown on the graph; all others are non-significant, with the exception of all comparisons to PBS, which were all significant. G=guide strand, P=passenger strand. Figure 37B is a bar graph showing TTR knockdown in mice with (S)-GNA-modified siRNA duplexes administered at 2.5 mg / kg. Figure 37B shows TTR protein levels measured in serum. Error bars represent standard deviation from each cohort (n=3). Only statistically significant comparisons are shown on the graph; all others are non-significant, with the exception of all comparisons to PBS, which were all significant. G=guide strand, P=passenger strand. [Figure 38A] Figure 38A shows the structures of nucleotide analogs used at the 5' end of siRNA to prevent 5'-phosphorylation and thereby reduce RISC loading. [Figure 38B] This shows that blocking RISC loading reduces liver toxicity. Figure 38B is a bar graph showing liver exposure in parental (RNAi active) and capped (RNAi inactive) GalNAc-siRNA in rat and mouse toxicity studies, as assessed by stem-loop RT-qPCR for the antisense strand (AS) at autopsy (nx). Vertical dashed lines demarcate the boundaries of separately performed studies. [Figure 38C] Figure 38C shows serum alanine aminotransferase (ALT) levels measured at autopsy. Differences between group means were evaluated for statistical significance using one-way analysis of variance in GraphPad Prism 7. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 38D] Blocking RISC loading reduces hepatotoxicity. Figure 38D shows H&E staining of liver sections collected at necropsy. In rats, hepatotoxic siRNA (designated here as siRNA-1) exhibited hepatocellular degeneration (bracketed area), increased sinusoidal cells (#) due to Kupffer cell hyperplasia and / or leukocyte infiltration, single-cell necrosis (*), increased mitosis (^), and hepatocellular vacuolization (arrow). In mice, hepatotoxic siRNA (designated here as siRNA-7) was associated with a lower incidence and severity of single-cell necrosis and other findings commonly seen in rats. Capped RNAi inactive siRNA had minimal or no histological findings in both species. Cytoplasmic clearing observed in mice was consistent with glycogen due to incomplete fasting and was not considered test article-related. [Figure 39A] Figure 39A shows the effect of antisense strand 5'-modification on RNAi activity and toxic liver enzyme elevation with GalNAc-siRNA in a rat toxicity study. Figure 39A is a bar graph showing hepatic RISC loading of GalNAc-siRNA in the presence or absence of a 5'-cap, as assessed by stem-loop RT-qPCR on the antisense strand (AS) at necropsy (nx). [Figure 39B] Figure 39B shows the effect of antisense strand 5'-modification on RNAi activity and toxic liver enzyme elevation with GalNAc-siRNA in rat toxicity studies. Figure 39B is a bar graph showing the knockdown of liver mRNA in the presence or absence of 5'-cap, assessed at autopsy by RT-qPCR for target mRNA and normalized to housekeeping mRNA (18S rRNA), compared to saline control groups. [Figure 39C]Figure 39C shows the effect of antisense strand 5'-modification on RNAi activity and toxic liver enzyme elevation with GalNAc-siRNA in rat toxicity studies. Figure 39C shows serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBILI) levels measured at autopsy in RISC loading blockade studies. Q2d, every other day administration; iB, inverted abasic; Mo, morpholino; H, 5'-deoxy. [Figure 40] Figure 40A shows the effect of sense strand 5'-modification on the hepatotoxicity of exemplary toxic GalNAc-siRNAs in rat toxicity studies. Figure 40A is a bar graph showing liver exposure to toxic GalNAc-siRNAs in the presence or absence of modifications to the 5' end of the sense strand (SS) in rat toxicity studies, as assessed by stem-loop RT-qPCR on the antisense strand (AS) at necropsy (nx). Figure 40B shows the effect of sense strand 5'-modification on the hepatotoxicity of exemplary toxic GalNAc-siRNAs in rat toxicity studies. Figure 40B shows serum alanine aminotransferase (ALT) levels measured at necropsy. Figure 40C shows the effect of sense strand 5'-modification on the hepatotoxicity of exemplary toxic GalNAc-siRNAs in rat toxicity studies. Figure 40C is an image showing H&E staining of liver sections collected at necropsy. Toxic siRNAs exhibited microscopic findings consisting of hepatocellular degeneration (brackets), single-cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and / or infiltrating leukocytes (#), and hepatocellular vacuolization (arrows). Addition of a sense strand cap had no effect on the incidence or severity of these findings. Q2d, every other day administration; iB, inverted abasic; Mo, morpholino. [Figure 41]Figure 41A shows the effect of 5'-modifications on the hepatotoxicity of exemplary non-toxic GalNAc-siRNAs in rat toxicity studies. Figure 41A is a bar graph showing liver exposure to non-toxic GalNAc-siRNAs in the presence or absence of 5'-terminal modifications on both the sense and antisense strands in rat toxicity studies, as assessed by stem-loop RT-qPCR on the antisense strand (AS) at necropsy (nx). Figure 41B shows the effect of 5'-modifications on the hepatotoxicity of exemplary non-toxic GalNAc-siRNAs in rat toxicity studies. Figure 41B shows serum alanine aminotransferase (ALT) levels measured at necropsy. Figure 41C shows the effect of 5'-modifications on the hepatotoxicity of exemplary non-toxic GalNAc-siRNAs in rat toxicity studies. Figure 41C is an image showing H&E staining of liver sections collected at necropsy. Administration of known non-toxic siRNAs in the presence or absence of a 5'-cap resulted in minimal hepatocellular vacuolization (arrows) in both cases. Q2d, alternate day administration; iB, inverted abasic; Mo, morpholino. [Figure 42A] Figure 442A shows that altering siRNA chemical modification does not alleviate hepatotoxicity. Figure 442A shows the chemical modification patterns of high 2'F and low 2'F GalNAc-siRNAs with the same PS content and sequence. [Figure 42B] Figure 42B is a bar graph showing liver exposure in rat and mouse toxicity studies, assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). [Figure 42C] Figure 42C is a bar graph showing RISC loading in the liver as assessed by stem-loop RT-qPCR for antisense at necropsy, showing that altering siRNA chemical modifications does not alleviate hepatotoxicity. [Figure 42D]This shows that altering siRNA chemical modifications does not alleviate hepatotoxicity. Figure 42D shows serum alanine aminotransferase (ALT) levels measured at autopsy. Differences between group means were evaluated for statistical significance using one-way analysis of variance in GraphPad Prism 7. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 42E] This shows that altering the siRNA chemical modification does not alleviate hepatotoxicity. Figure 42E shows images of H&E staining of liver sections collected at necropsy. In rats, both the high 2'F and low 2'F siRNA-6 compounds were associated with hepatocellular degeneration (brackets), single-cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and / or infiltrating leukocytes (#), and hepatocellular vacuolization (arrows). In mice, findings consisted of single-cell necrosis in both chemical modification patterns. [Figure 43] Figure 43 is a bar graph showing the in vivo potency of the high 2'F version (48% 2'F and 52% 2'OMe) and low 2'F version (21% 2'F and 79% 2'OMe) of siRNA-6. After a single subcutaneous injection of 3 mg / kg into C57BL / 6 female mice, liver on-target mRNA knockdown was assessed by RT-qPCR for target mRNA on days 14 and 28 compared to saline control, normalized to housekeeping mRNA (GAPDH). [Figure 44A] Figure 44A shows that restoring RISC activity with antisense strand loading reduces liver toxicity. Figure 44A shows the study design showing the prevention and treatment of GalNAc-siRNA toxicity in rats using REVERSIR™. [Figure 44B] Figure 44B is a bar graph showing liver exposure to GalNAc-siRNA in a prophylactic (siRNA-1 and siRNA-4) or treatment (siRNA-5) toxicity study in rats, as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). [Figure 44C] Figure 44C shows that restoring RISC activity loaded with the antisense strand reduces liver toxicity. Figure 44C is a bar graph showing RISC loading in the liver in the presence or absence of REVERSIR treatment, as assessed by stem-loop RT-qPCR for the antisense strand at autopsy. [Figure 44D] This shows that restoring RISC activity with antisense strand loading reduces liver toxicity. Figure 44D shows serum glutamate dehydrogenase (GLDH) levels measured at autopsy. Differences between group means were evaluated for statistical significance using one-way analysis of variance in GraphPad Prism 7. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 44E] This demonstrates that restoring RISC activity with antisense strand loading reduces liver toxicity. Figure 44E shows an image depicting H&E staining of liver sections collected at autopsy. Known toxic siRNA administered alone or with scrambled control (Ctr) REVERSIR™ was associated with hepatocellular degeneration (brackets), single-cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and / or infiltrating leukocytes (#), increased mitosis (^), bile duct hyperplasia with fibrosis (+), and hepatocellular vacuolization (arrows). Co-administration of complementary REVERSIR™ reduced the severity of these findings and often limited their distribution. [Figure 45] Figure 45 is a bar graph showing the effect of REVERSIR™ compounds on RNAi activity in a rat toxicity study. Liver on-target mRNA knockdown using siRNA-1 and siRNA-5 compared to saline control groups was assessed by RT-qPCR for target mRNA at necropsy (nx) and normalized to housekeeping mRNA (18S rRNA). On-target serum protein levels with siRNA-4 compared to saline control groups were assessed by ELISA at necropsy. Q2d, every other day dosing; qw, weekly dosing. [Figure 46A] Figure 46A shows the chemical structures of seeds swapping between hepatotoxic and non-hepatotoxic GalNAc-siRNA. [Figure 46B] Figure 46B is a bar graph showing liver exposure to parental and seed-exchanged GalNAc-siRNAs in a rat toxicity study, as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). [Figure 46C] Figure 46C is a bar graph showing RISC loading in the liver as assessed by stem-loop RT-qPCR on the antisense strand at autopsy, showing that exchanging the seed region reduces hepatotoxicity. [Figure 46D] Figure 46D shows that replacing the seed region reduces hepatotoxicity. Serum alanine aminotransferase (ALT) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one-way analysis of variance in GraphPad Prism 7. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 46E] This demonstrates that exchanging seed regions reduces hepatotoxicity. Figure 46E shows an image showing H&E staining of liver sections collected at autopsy. Toxic siRNA exhibited hepatocellular degeneration (bracket), single-cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and / or infiltrating leukocytes (#), and hepatocellular vacuolization (arrows), whereas non-toxic siRNA exhibited only minimal vacuolization. Non-toxic seeds in toxic scaffolds were comparable to full-length non-toxic siRNA, and toxic seeds in non-toxic scaffolds exhibited single-cell necrosis, increased sinusoidal cells, and vacuolization, but at a lower severity grade than the full-length toxic compound. [Figure 47A]Figure 47A shows that siRNA off-targets are enriched for seed complementarity in vitro and in vivo. Figure 47A is a volcano plot showing global gene expression changes in rat hepatocytes 24 hours after transfection with 10 nM of GalNAc-siRNA of four different sequences. [Figure 47B] Figure 47B shows that siRNA off-targets are enriched for seed complementarity in vitro and in vivo. Figure 47B is a volcano plot showing global gene expression changes in rat liver 24 hours after subcutaneous administration of GalNAc-siRNA at 50 mg / kg. Two parent GalNAc-siRNAs and their RNAi-inactive versions blocked with an inverted abasic (iB) cap are shown. Blue points, adjusted P value ≤ 0.05; red points, adjusted P value > 0.05; N = 3 animals per group. Adjusted P values ​​for fold change were calculated with DESeq2 using a Wald test with multiple testing correction. P values ​​for seed enrichment were calculated using Fisher's exact test. Variances were statistically similar between groups compared. [Figure 48]Figure 48A shows that destabilizing seed-mediated base pairing minimizes off-target effects and reduces hepatotoxicity. Figure 48A shows a heat-destabilizing glycol nucleic acid (GNA) modification at position 7 of the antisense strand of a toxic exemplary siRNA-5. Figure 48B shows that destabilizing seed-mediated base pairing minimizes off-target effects and reduces hepatotoxicity. Figure 48B is a volcano plot showing global gene expression changes in rat hepatocytes 24 hours after transfection of 10 nM of parental or GNA-modified GalNAc-siRNA. N=3 technical replicates. Figure 48C shows that destabilizing seed-mediated base pairing minimizes off-target effects and reduces hepatotoxicity. Figure 48C is a bar graph showing liver exposure to parental and seed-modified siRNA-5 in a rat toxicity study, assessed by stem-loop reverse transcription quantitative PCR (RT-qPCR) on the antisense strand (AS) at necropsy (nx). Figure 48D shows that destabilizing seed-mediated base pairing minimizes off-target effects and reduces hepatotoxicity. Figure 48D is a bar graph showing RISC loading in the liver, as assessed by stem-loop RT-qPCR on the antisense strand at autopsy. Figure 48E shows that destabilizing seed-mediated base pairing minimizes off-target effects and reduces hepatotoxicity. Figure 48E shows serum glutamate dehydrogenase (GLDH) levels measured at autopsy. Differences between group means were evaluated for statistical significance using one-way ANOVA in GraphPad Prism 7. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; **, p<0.0001. Figure 48F shows that destabilizing seed-mediated base pairing minimizes off-target effects and reduces hepatotoxicity. Figure 48F is an image showing H&E staining of liver sections collected at necropsy. The toxic parental siRNA-5 had fibrosis (circles), hepatocellular degeneration (brackets), single-cell necrosis (*), increased mitosis (^), increased sinusoidal cells consistent with Kupffer cell hyperplasia and / or infiltrating leukocytes (#), and hepatocellular vacuolization (arrows), whereas the non-toxic siRNA had only minimal vacuolization.Seed GNA-modified siRNA-5 had degeneration, single-cell necrosis, increased mitosis, and vacuolization, but at a lower incidence and severity grade than parental siRNA-5. N = 4 animals / group; qw, weekly administration; GNA, glycol nucleic acid. [Figure 49] Figure 49A shows the effect of an exemplary heat-stabilized GNA seed modification on on-target activity. Figure 49A is a bar graph showing rat hepatocyte mRNA knockdown assessed by reverse transcription quantitative PCR (RT-qPCR) on target mRNA at 24 hours post-transfection with 10 nM compared to mock transfection, normalized to housekeeping mRNA (18S rRNA). Figure 49B shows the effect of an exemplary heat-stabilized GNA seed modification on on-target activity. Figure 49B is a bar graph showing liver mRNA knockdown assessed at necropsy by RT-qPCR on target mRNA and normalized to housekeeping mRNA (18S rRNA) compared to saline control. Qw, weekly administration; GNA, glycol nucleic acid. [Figure 50] 1 shows IC50 curves for exemplary dsRNAs targeting TTR in rat hepatocytes. [Figure 51] 1 shows IC50 curves for exemplary dsRNAs targeting Factor IX (F9) in mouse hepatocytes. [Figure 52] Figure 52 shows that the exemplary dsRNA of the present invention for TTR reduces endogenous off-target effect.Figure 52 shows that the position-specific reduction of off-target effect in the dsRNA targeting TTR in vitro.As shown, dsRNA significantly reduces the number of genes that are down-regulated or up-regulated by their respective parental dsRNA. [Figure 53] Figure 53 shows that the exemplary dsRNA of the present invention for F9 reduces endogenous off-target effect.Figure 53 shows that the position-specific reduction of off-target effect in the dsRNA targeting F9 in vitro.As shown, dsRNA significantly reduces the number of genes that are down-regulated or up-regulated by their respective parental dsRNA. [Figure 54] Figure 54 is a line graph showing knockdown of target TTR by exemplary dsRNAs with thermodestabilizing modifications Mod3, 6, 7, and 10. As can be seen, all modifications are able to maintain activity similar to the parent. [Figure 55] Figure 55 is a bar graph showing knockdown of target GO1 by exemplary dsRNAs with thermodestabilizing modifications Mod3, 5, 6, 7, 10, and 12. As can be seen, all modifications are able to maintain activity similar to the parent. DETAILED DESCRIPTION OF THE INVENTION

[0017] The inventor has found that the off-target effect of RNA molecules can be reduced or inhibited by incorporating thermolabile nucleotides at specific positions in the antisense strand of dsRNA.By using these thermolabile modifications at specific positions in antisense strand, dsRNA molecules can reduce off-target gene silencing, while retaining the gene silencing activity similar to that of parent dsRNA.In addition, the number of off-target genes that are down-regulated or up-regulated by the dsRNA molecules that contain these thermolabile modifications is also reduced compared with parent dsRNA.

[0018] Thus, in one aspect, the present invention provides double-stranded RNAi (dsRNA) agent that can inhibit the expression of target gene.Generally, the dsRNA molecule of the present invention shows high on-target gene silencing, while reducing or minimizing off-target gene silencing and / or toxicity.Although not limited, the dsRNA molecule of the present invention can be substituted for the dsRNA molecule, and can be used in the gene silencing technology based on RNA interference, including but not limited to in vitro or in vivo application.

[0019] Generally, the dsRNA molecule comprises a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand). Each strand of the dsRNA molecule can be in the range of 12 to 40 nucleotides in length. For example, each strand can be 14 to 40 nucleotides in length, 17 to 37 nucleotides in length, 25 to 37 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. Without limitation, the sense and antisense strands can be of equal or unequal length.

[0020] In some embodiments, the antisense strand is 18 to 35 nucleotides in length. In some embodiments, the antisense strand is 21 to 25, 19 to 25, 19 to 21, or 21 to 23 nucleotides in length. In certain embodiments, the antisense strand is 23 nucleotides in length. Like the antisense strand, the sense strand is, in some embodiments, 18 to 35 nucleotides in length. In some embodiments, the sense strand is 21 to 25, 19 to 25, 19 to 21, or 21 to 23 nucleotides in length. In certain embodiments, the antisense strand is 21 nucleotides in length.

[0021] The inventor has also found that for dsRNA molecules to be more effective in vivo, antisense strand must have some metabolic stability.In other words, for dsRNA molecules to be more effective in vivo, some amount of antisense strand may need to exist in vivo after administration for a while.Therefore, in some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo after in vivo administration for example in mouse liver for 5 days.In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo after in vivo administration for example in mouse liver for 6 days. In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration on day 7, for example, in mouse liver.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration on day 8, for example, in mouse liver.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration on day 9, for example, in mouse liver.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration for example in mouse liver on the 10th day.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration for example in mouse liver on the 11th day.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration for example in mouse liver on the 12th day. In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration on the 13th day, for example, in mouse liver.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration on the 14th day, for example, in mouse liver.In some embodiments, at least 40% of the antisense strand of dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, is present in vivo after in vivo administration on the 15th day, for example, in mouse liver.

[0022] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, between positions 2 and 9, counting from the 5' end), and the dsRNA has a melting temperature (T) of about 40°C to about 80°C. m ), and the dsRNA optionally further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following features: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA has a T of about 40°C to about 80°C. m is optional.

[0023] In some embodiments, the dsRNA molecule has a double-stranded region 12 to 40 nucleotide pairs in length, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 of the 5' end of the antisense strand, counting from the 5' end), and the dsRNA has a T of about 40°C to about 80°C. mand the dsRNA optionally further has at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following features: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and vii) a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA has a T of about 40°C to about 80°C. m is optional.

[0024] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), and the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0025] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA has a melting temperature of about 40°C to about 80°C (e.g., 40°C, 50°C, 60°C, 70°C, or 80°C). m is optional.

[0026] In certain embodiments, the thermodestabilizing modifications of the duplex are present at positions 5, 6, 7, or 8 of the antisense strand, counting from the 5' end of the antisense strand.

[0027] In certain embodiments, the thermodestabilizing modification of the duplex is at position 5 of the antisense strand, counting from the 5' end of the antisense strand.

[0028] In certain embodiments, the thermodestabilizing modification of the duplex is at position 6 of the antisense strand, counting from the 5' end of the antisense strand.

[0029] In certain embodiments, the thermodestabilizing modification of the duplex is at position 7 of the antisense strand, counting from the 5' end of the antisense strand.

[0030] In certain embodiments, the thermodestabilizing modification of the duplex is at position 8 of the antisense strand, counting from the 5' end of the antisense strand.

[0031] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 of the 5' end of the antisense strand, counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the antisense strand has the following characteristics: (i) 2, 3, 4, 5, or 6 2'-fluoro modifications; and (ii) 1, 2, 3, or 4 phosphorothioate internucleotide linkages and The sense strand has the following characteristics: (i) a ligand conjugated to the sense strand; (ii) 2, 3, 4, or 5 2'-fluoro modifications; and (iii) 1, 2, 3, or 4 phosphorothioate internucleotide linkages In some embodiments, the T m is optional.

[0032] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and a ligand is conjugated to the sense strand, and the dsRNA has a melting temperature of about 40°C to about 80°C.

[0033] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, a ligand is conjugated to the sense strand, and the dsRNA comprises at least four 2'-fluoro modifications. In some embodiments, the dsRNA molecule is capable of withstanding a T of about 40°C to about 80°C. m is optional.

[0034] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, the sense strand comprises a ligand, and the dsRNA has a melting temperature of about 40°C to about 80°C. In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, the T of about 40°C to about 80°C. m is optional.

[0035] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4-8 from the 5' end, and the sense strand comprises a ligand, wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, and the dsRNA has a melting temperature of about 40°C to about 80°C. In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, the T of about 40°C to about 80°C is m is optional.

[0036] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the antisense strand further comprises at least two of the following features: (i) the thermally destabilizing modification of the duplex is located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro modifications; or (iii) a phosphorothioate internucleotide linkage between positions 1 and 2 of the nucleotide (counting from the 5' end), and the antisense strand has a length of 18 to 35 nucleotides. In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, the T m is optional.

[0037] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the sense strand has at least one of the following characteristics: (i) the ligand is attached to one end of the sense strand; (ii) the sense strand comprises at least two 2'-fluoro modifications; and (iii) the sense strand and antisense strand exhibit sufficient complementarity to form a double-stranded region spanning at least 19 nucleotide positions, wherein the thermodestabilizing modification of the duplex is located within the double-stranded region. In some embodiments, the T m is optional.

[0038] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, and wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the thermally destabilizing modification of the duplex is [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). In some embodiments, the T m is optional.

[0039] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4-8 from the 5' end, wherein the sense strand comprises a ligand, and wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). In some embodiments, the T m is optional.

[0040] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, the antisense strand comprises at least one thermodestabilizing modification of the duplex located at position 7 from the 5' end of the antisense strand, the sense strand comprises a ligand, and the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0041] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at position 7 from the 5' end, wherein the sense strand comprises a ligand, and wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). In some embodiments, the T m is optional.

[0042] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and the sense strand comprises a ligand, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the ligand comprises one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. In some embodiments, the T of about 40°C to about 80°C. m is optional.

[0043] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, wherein the dsRNA optionally has a melting temperature of about 40°C to about 80°C, and wherein the ligand has the structure: [ka] In some embodiments, the ASGPR ligand has a T of about 40° C. to about 80° C. m is t-like.

[0044] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand comprises at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand comprises 3, 4 , 5, or 6 2'-fluoro modifications, and 2, 3, 4, or 5 phosphorothioate internucleotide linkages; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0045] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand comprises at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally, a host molecule between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3. and (iii) the dsRNA has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0046] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand comprises at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand comprises at least one heat-labile nucleotide at positions 2, 6, 8, 9, 14, or 16, or at positions 2, 6, 14, or 16, or at positions 2, 1 and wherein the antisense strand comprises 2'-fluoro modifications at positions 4 and 16; and wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0047] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand comprises at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand comprises 2'-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or at positions 2, 6, 14, or 16, or at positions 2, 14, and 16. and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22, between nucleotide positions 22 and 23, between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region of 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0048] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand comprises at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3; and wherein the antisense strand is conjugated to at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand). , 14, or 16, or 2, 6, 14, or 16, or 2, 14, and 16; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0049] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, wherein the antisense strand comprises at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorotypic modifications; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region of 18, 19, 20, 21, 22, 23, 24, or 24 nucleotide pairs in length; and (viii) the dsRNA comprises a blunt end at the 5' end of the sense strand (e.g., 1, 2, 3, 4, 5, 6, 7, or 8). In certain embodiments, the sense strand is 19, 20, 21, or 22 nucleotides in length, and the antisense strand is 20, 21, or 22 nucleotides in length. In some embodiments, the T m is optional.

[0050] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand comprises at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3; and wherein the antisense strand comprises at positions 2, 6, 8, 9, 14, or 16, or and wherein the antisense strand comprises a 2'-fluoro modification at positions 2, 6, 14, or 16, or at positions 2, 14, and 16; and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22, between nucleotide positions 22 and 23, between nucleotide positions 1 and 2, or between nucleotide positions 2 and 3; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, the dsRNA has a melting temperature of about 40°C to about 80°C. m is optional.

[0051] In some embodiments, one end of the dsRNA is blunt and the other end has an overhang, wherein the antisense strand comprises at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; The sense strand further comprises at least one (e.g., 1, 2, 3, 4, or 7) of the following: (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length. In some embodiments, an overhang is present on the 3' end of the antisense strand, and a blunt end is present at the 5' end of the antisense strand. In certain embodiments, the overhang is 2, 3, or 4 nucleotides in length. In some embodiments, the dsRNA is heated to a temperature of about 40°C to about 80°C. m is optional.

[0052] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length, wherein one end of the dsRNA is blunt and the other end has an overhang, and wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications (e.g., 1, 2, 3, 5, or all 6), and optionally, a 2-nucleotide overhang is present on the 3' end of the antisense strand and a blunt end is present at the 5' end of the antisense strand. In some embodiments, the overhang is present on the 3' end of the antisense strand and a blunt end is present at the 5' end of the antisense strand. In some embodiments, the dsRNA is heated to a temperature of about 40°C to about 80°C. m is optional.

[0053] In some embodiments, the dsRNA molecules of the invention may also have two blunt ends at either end of the dsRNA duplex.

[0054] In some embodiments, the dsRNA has blunt ends at both ends of the duplex, wherein the antisense strand comprises at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3 (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length. In some embodiments, the dsRNA is heated to a T of about 40°C to about 80°C. m is optional.

[0055] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length and has blunt ends at both ends of the duplex, wherein one end of the dsRNA is blunt and the other end has an overhang, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has any of the following characteristics: (i) a (ii) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications. In some embodiments, the dsRNA is heated to a T of about 40°C to about 80°C. m is optional.

[0056] In some embodiments, a dsRNA molecule of the invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt-ended while the other end comprises a 2-nt overhang, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense strand comprises a 2-nt sense strand and a 23-nt antisense strand; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a blunt end at the 5' end of the antisense strand. Preferably, a 2-nt overhang is present at the 3' end of the antisense strand. In some embodiments, the T m is optional.

[0057] In some embodiments, in the dsRNA molecule of the present invention comprising a sense and an antisense strand, the sense strand is 25 to 30 nucleotide residues in length, wherein positions 1 to 23 of the sense strand, starting from the 5'-terminal nucleotide (position 1), comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, wherein, starting from the 3'-terminal nucleotide, at least 8 ribonucleotides within its positions are paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5'-end of the antisense strand comprises 10 to 30 consecutive nucleotides that are not paired with the sense strand, thereby forming a 10 to 30 consecutive nucleotides. wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; and wherein, when the double-stranded nucleic acid is introduced into a mammalian cell, the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression; and wherein the antisense strand contains at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 of the 5'-end of the antisense strand), and wherein the dsRNA has a melting temperature of about 40°C to about 80°C.For example, the heat-destabilizing nucleotide is located between positions 14 and 17 opposite or complementary to the 5'-end of the sense strand, and the dsRNA optionally further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following characteristics: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a double-stranded region 12 to 30 nucleotide pairs in length. In some embodiments, the T is between about 40°C and about 80°C. m is optional.

[0058] In some embodiments, a dsRNA molecule of the present invention comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides, and the antisense strand having a length of at most 30 nucleotides comprises, together with the sense strand, a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, and the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3' end, wherein in the double-stranded region having a length of at least 25 nucleotides, when the dsRNA molecule is introduced into a mammalian cell, the antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the antisense strand to reduce expression of a target gene, and wherein Dicer cleavage of the dsRNA preferentially yields siRNA comprising the 3' end of the antisense strand, thereby reducing expression of the target gene in the mammal, The antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotides; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA has a double-stranded region 12 to 29 nucleotide pairs in length. In some embodiments, the dsRNA has a T of about 40°C to about 80°C. m is optional.

[0059] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 3, 4, or 5 phosphodiesterase (PDS) modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA is heated to a T of about 40°C to about 80°C. m is optional.

[0060] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand includes one, two, three, four, or five 2'-fluoro modifications; (v) the sense strand includes one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA includes at least four 2'-fluoro modifications; (vi) the dsRNA includes a double-stranded region between 12 and 40 nucleotide pairs in length; (vii) the dsRNA includes a double-stranded region between 12 and 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA is heated to a T of about 40°C to about 80°C. m is optional.

[0061] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the dsRNA optionally has the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phospho- (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA is heated to a T of about 40°C to about 80°C. m is optional.

[0062] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA Optionally, the dsRNA further comprises at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA is heated to a T of about 40°C to about 80°C. m is optional.

[0063] In one aspect, the present invention provides a dsRNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at positions 2-9 of the 5' end, counting from the 5' end of the antisense strand), and wherein the dsRNA has the following characteristics: (i) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2′-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) a blunt end at the 5' end of the antisense strand The present invention provides dsRNA molecules further comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8) of the following:

[0064] In certain embodiments, the thermodestabilizing modified antisense strand of the duplex is at position 7, counting from the 5' end of the antisense strand.

[0065] In some embodiments, the thermodestabilizing modifications of the duplex are present at positions 2, 3, 4, 5, 6, 8, or 9 of the antisense strand, counting from the 5' end of the antisense strand.

[0066] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., positions 2-9 of the 5' end, counting from the 5' end of the antisense strand), and the antisense strand has the following characteristics: (iii) 2, 3, 4, 5, or 6 2'-fluoro modifications; and (iv) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages and The sense strand has the following characteristics: (iv) a ligand conjugated to the sense strand; (v) 2, 3, 4, or 5 2'-fluoro modifications; and (vi) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages Includes one, two or three of the following:

[0067] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and a ligand is conjugated to the sense strand.

[0068] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, a ligand is conjugated to the sense strand, and the dsRNA comprises at least four 2'-fluoro modifications.

[0069] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and the sense strand comprises a ligand. In some further embodiments, the ligand is an ASGPR ligand.

[0070] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4-8 from the 5' end, and wherein the sense strand comprises a ligand, and wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications. In some further embodiments thereof, the ligand is an ASGPR ligand.

[0071] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro residues, the antisense strand comprises at least one double-stranded thermolabile modification within the first nine nucleotide positions counting from the 5' end, and the sense strand comprises a ligand, and the antisense strand further comprises at least two of the following features: (i) the double-stranded thermolabile modification is located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro residues; or (iii) a phosphorothioate internucleotide linkage between positions 1 and 2 (counting from the 5' end) of the nucleotide, and the antisense strand has a length of 18 to 35 nucleotides. In some further embodiments, the ligand is an ASGPR ligand.

[0072] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and wherein the sense strand has at least one of the following characteristics: (i) the ligand is attached to one end of the sense strand; (ii) the sense strand comprises at least two 2'-fluoro modifications; and (iii) the sense strand and the antisense strand exhibit sufficient complementarity to form a double-stranded region spanning at least 19 nucleotide positions, wherein the thermally destabilizing modification of the duplex is located within the double-stranded region.

[0073] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and wherein the thermally destabilizing modification of the duplex is [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0074] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, and wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located within 4-8 positions from the 5' end, wherein the sense strand comprises a ligand, and each of the sense and antisense strands comprises at least two 2'-fluoro modifications, and the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0075] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at position 7 from the 5' end of the antisense strand, and wherein the sense strand comprises a ligand.

[0076] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at position 7 from the 5' end, wherein the sense strand comprises a ligand, and each of the sense and antisense strands comprises at least two 2'-fluoro modifications, and the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0077] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, wherein the ligand comprises one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

[0078] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoros, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, wherein the ligand has the structure: [ka] It is an ASGPR ligand.

[0079] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; wherein the antisense strand comprises 3, 4, 5, or 6 2'-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0080] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally between nucleotides 1 and 2, and between nucleotides 3 and 4. and (iii) the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand.

[0081] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand has at least one heat-labile nucleotide at positions 2, 6, 8, 9, 14, or 16, or at positions 2, 6, 14, or 16. or 16, or at positions 2, 14, and 16; and the antisense comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22, and between nucleotides 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand.

[0082] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand has at least one heat-labile nucleotide at positions 2, 6, 8, 9, 14, or 16, or at positions 2, 6, 14, or 16, or at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at positions 2, 3, 4, 5, 6, 7, 8, 9, 15, or 16, or at positions 2, 3, 4, 5, 6, 7, 8, 9, 16, or 17, or at positions 2, 3, 4, 5, 6, 7, 8, 9, 17, or 18, or at positions 2, 4, 5, 6, 7, 8, 9, 18, or 19, or at positions 3, 4, 5, 6, 7, 8, 9, 19, or 20, or at positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or at positions 2, 3, 4, 5, 7, 8, 9, 14, and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand.

[0083] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; the antisense strand comprises a 2'-fluoro modification at positions 2, 6, 8, 9, 14, or 16, or at positions 2, 6, 14, or 16, or at positions 2, 14, and 16; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22 and between nucleotides 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand.

[0084] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; and wherein the antisense strand has at least one heat-labile nucleotide in length of 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand.

[0085] In certain embodiments, the dsRNA molecules of the invention comprise: (a)(i) 21 nucleotides long; (ii) an ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker attached to the 3' end; and (iii) 2′-F modifications at positions 7, 10, and 11 (counting from the 5′ end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) double-stranded heat-destabilizing modifications at positions 5, 6, and 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0086] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 6, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0087] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0088] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0089] In another specific embodiment, the dsRNA molecule of the invention comprises: (i) 2'-F modifications at positions 2, 14, and 16 (counting from the 5' end); and (2) A heat-destabilizing modification at position 6 or 7 of the duplex (counting from the 5' end). The antisense strand comprises the following:

[0090] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (ii) a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5′ end); and an antisense strand having:

[0091] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) ASGPR ligand attached to the 3′ end, comprising three GalNAc derivatives linked via a trivalent branched linker; (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0092] In some embodiments, the dsRNA molecule further comprises at least one ASGPR ligand. For example, the ASGPR ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker, such as [ka] Examples include:

[0093] In one example, the ASGPR ligand is attached to the 3' end of the sense strand.

[0094] In some cases, 2'-fluoro modifications in the seed region of the antisense strand, for example, positions 2-9, particularly positions 3-9, may have minimal effect on the in vitro potency of the dsRNA, but may adversely affect the in vivo activity of the dsRNA. In particular, the inventors have discovered that the in vivo activity of such dsRNA can be restored to a level comparable to that of the parent dsRNA by removing some or all of the 2'-fluoro modifications from the seed region of the antisense strand, i.e., positions 2-9, particularly positions 3-9, counting from the 5' end.

[0095] Thus, in some embodiments, the present invention provides a dsRNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, between positions 2 and 9, counting from the 5' end), and the dsRNA has the following characteristics: (i) a melting temperature (T) of about 40°C to about 80°C; m (ii) the antisense strand contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro modifications; (iii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated to a ligand; (v) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (vi) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages. (vii) the dsRNA comprises at least four 2'-fluoro modifications; (viii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; (ix) a blunt end at the 5' end of the antisense strand; (x) the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications.

[0096] In some embodiments, the present invention provides a dsRNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, between positions 2 and 9, counting from the 5' end), and the dsRNA has the following characteristics: (i) a melting temperature (T m(ii) the antisense contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro modifications; (iii) the antisense contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated to a ligand; (v) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (vi) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vii) the dsRNA contains at least four (viii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; (ix) a blunt end at the 5' end of the antisense strand; and (x) the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, wherein the 2'-fluoro modifications are not present at positions 3 to 9 (counting from the 5' end) of the antisense strand.

[0097] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, from positions 2 to 9, counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the antisense strand has one of the following characteristics: (i) 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'- and (ii) one or both of 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and the sense strand comprises one, two, or three of the following features: (i) a ligand conjugated to the sense strand; (ii) 2, 3, 4, or 5 2'-fluoro modifications; (iii) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (iv) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0098] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, from positions 2 to 9, counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the antisense strand comprises (i) 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluorouracils. and (ii) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and wherein the sense strand further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and wherein the sense strand optionally comprises one, two, or three of the following features: (i) a ligand conjugated to the sense strand; (ii) 2, 3, 4, or 5 2'-fluoro modifications; (iii) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (iv) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0099] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, from positions 2 to 9, counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the antisense strand has 1, 2, 3, or 4 nucleotides. and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and the sense strand optionally comprises a ligand conjugated to the sense strand, 2, 3, 4, or 5 2'-fluoro modifications; and / or 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0100] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at positions 2 to 9 of the 5' end of the antisense strand, counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the antisense strand comprises 1, 2, 3, or 4 phosphorothioate nucleotides. and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, with the proviso that no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end), and the sense strand optionally comprises a ligand conjugated to the sense strand, 2, 3, 4, or 5 2'-fluoro modifications; and / or 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0101] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, a ligand is conjugated to the sense strand, and the dsRNA comprises at least four 2'-fluoro modifications and no 2'-fluoro modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end).

[0102] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and there are no 2'-fluoro modifications at positions 3 to 9 of the antisense strand (counting from the 5' end). In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0103] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4 to 8 from the 5' end, wherein the sense strand comprises a ligand, wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and there are no 2'-fluoro modifications at positions 3 to 9 of the antisense strand (counting from the 5' end). In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0104] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and wherein the dsRNA is capable of being heated at about 40°C to about 8 The antisense strand has a melting temperature of about 0°C, and wherein the antisense strand further comprises at least two of the following features: (i) a duplex heat-destabilizing modification is located at positions 4-8 of the antisense strand; (ii) at least two 2'-fluoro modifications; (iii) a phosphorothioate internucleotide linkage between nucleotides 1 and 2 (counting from the 5' end); and (iv) the antisense strand has a length of 18-35 nucleotides, and there are no 2'-fluoro modifications at positions 3-9 of the antisense strand (counting from the 5' end). In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0105] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the sense strand has the following characteristics: The antisense strand has at least one of the following characteristics: (i) a ligand is attached to one end of the sense strand; (ii) the sense strand contains at least two 2'-fluoro modifications; (iii) the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; and (iv) the sense strand and antisense strand exhibit sufficient complementarity to form a double-stranded region spanning at least 19 nucleotide positions, wherein the thermodestabilizing modifications of the duplex are located within the double-stranded region, and positions 3-9 of the antisense strand (counting from the 5' end) are free of 2'-fluoro modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0106] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4 to 8, counting from the 5' end, wherein the sense strand comprises a ligand, optionally at least one LNA modification, and each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the dsRNA has a melting temperature of from about 40°C to about 80°C, and wherein the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0107] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 5, 6, or 7, counting from the 5' end of the antisense strand, and wherein there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end) of the antisense strand, wherein the sense strand comprises a ligand, optionally at least one LNA modification, and wherein the dsRNA has a melting temperature of about 40°C to about 80°C.

[0108] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at position 5, 6, or 7, counting from the 5' end, wherein the sense strand comprises a ligand, optionally at least one LNA modification, and each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the dsRNA has a melting temperature of from about 40°C to about 80°C, and wherein the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0109] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, optionally at least one LNA modification, wherein no 2'-fluoro modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end), and wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the ligand comprises one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

[0110] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, optionally at least one LNA modification, wherein no 2'-fluoro modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the ligand has the structure: [ka] It is an ASGPR ligand.

[0111] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and optionally comprises at least one LNA modification, 3 or 4 2'-fluoro modifications, and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand (counting from the 5' end) ) contain 3, 4, 5, or 6 2'-fluoro modifications, provided that no 2'-modifications are present at positions 3-9 of the antisense strand, and contain 2, 3, 4, or 5 phosphorothioate internucleotide linkages; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12-25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0112] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally at least and one LNA modification; wherein the antisense strand contains three, four, five, or six 2'-fluoro modifications, provided that no 2'-modifications are present at positions 3-9 of the antisense strand (counted from the 5'-end), and two, three, four, or five phosphorothioate internucleotide linkages; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA contains a double-stranded region 12-25 nucleotide pairs in length; (ii) the dsRNA contains a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0113] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and optionally at least one LNA modification; and wherein the antisense strand is 2, 14, or 16 and the antisense strand comprises a 2'-fluoro modification at position 21; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0114] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand, contains 3 or 4 2'-fluoro modifications, contains at least one LNA modification, and optionally contains 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand is 2, 14, or 16 and the antisense strand comprises a 2'-fluoro modification at position 21; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further has at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0115] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and optionally at least one LNA modification; and wherein the antisense strand contains a 2'-fluoro modification at position 2, 14, or 16. and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0116] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages and at least one LNA modification; wherein the antisense strand contains a 2'-fluoro modification at position 2, 14, or 16; and The antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0117] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally comprising phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally at least and (iii) the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA comprises at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0118] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), at least one LNA modification, and optionally phosphorothioate nucleotides between nucleotides 1 and 2 and between nucleotides 2 and 3. and (iii) the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region of 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA comprises at least a two-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0119] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, wherein the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, with the proviso that no 2'-modifications are present at positions 3-9 of the antisense strand (counting from the 5' end). (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region that is 18, 19, 20, 21, 22, 23, 24, or 24 nucleotide pairs in length; (viii) the dsRNA comprises a blunt end at the 5' end of the sense strand; and (ix) the sense strand further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of 1, 2, 3, 4, 5, 6, 7, 8, 9 1, or 10 LNA modifications. In certain embodiments, the sense strand is 19, 20, or 21, or 22 nucleotides in length, and the antisense strand is 20, 21, or 22 nucleotides in length. In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0120] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, wherein the antisense strand has at least one heat-labile nucleotide and 1, 2, 3, or 4 phosphorothioate internucleotide linkages, wherein at least one heat-labile nucleotide is present in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has any of the following characteristics: (i) the antisense strand has at least one heat-labile nucleotide and 1, 2, 3, or 4 phosphorothioate internucleotide linkages (counting from the 5' end) of the antisense strand; (ii) the sense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, provided that there are no 2'-modifications at positions 3-9 of the antisense strand; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (iv) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 18, 19, 20, 21, 22, 23, 24, or 24 nucleotide pairs in length; and (vii) the dsRNA contains a blunt end at the 5' end of the sense strand (e.g., 1, 2, 3, 4, 5, 6, or 7). In certain embodiments, the sense strand is 19, 20, 21, or 22 nucleotides in length, and the antisense strand is 20, 21, or 22 nucleotides in length. In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0121] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; wherein the antisense strand comprises a 2'-fluoro modification at position 2, 9, 14, or 16, or at position 2, 14, or 16; and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, or between nucleotides 2 and 3; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0122] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LAN modifications, and optionally phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; The sense strand comprises a 2'-fluoro modification at positions 2, 9, 14, or 16, or at positions 2, 14, or 16; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, or between nucleotides 2 and 3; wherein the dsRNA has a melting temperature of about 40°C to about 80°C; and wherein the dsRNA optionally further comprises at least one (e.g., one, two, or all three) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0123] In some embodiments, one end of the dsRNA is blunt and the other end has an overhang, wherein the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end); (ii) the antisense contains The dsRNA further comprises at least one of the following (e.g., 1, 2, 3, 4, or 8): (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (vii) the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, the overhang is located at the 3' end of the antisense strand, and the blunt end is located at the 5' end of the antisense strand. In certain embodiments, the overhang is 2, 3, or 4 nucleotides in length. In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0124] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length, wherein one end of the dsRNA is blunt-ended and the other end has an overhang, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); The sense strand further comprises at least one (e.g., 1, 2, 3, 5, 6, or 7) of the following: (i) the sense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally, a 2-nucleotide overhang is present on the 3'-end of the antisense strand, and a blunt end is present on the 5'-end of the antisense strand. In some embodiments, the overhang is present on the 3'-end of the antisense strand, and a blunt end is present on the 5'-end of the antisense strand. In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0125] In some embodiments, the dsRNA molecules of the invention may also have two blunt ends at either end of the dsRNA duplex.

[0126] In some embodiments, the dsRNA has blunt ends at both ends of the duplex, wherein the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense strand contains 1, 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the sense strand contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0127] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length and has blunt ends at both ends of the duplex, wherein one end of the dsRNA is blunt and the other end has an overhang, and wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein (ii) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein (iii) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein (iv) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein (v ... (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the sense strand contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0128] In some embodiments, a dsRNA molecule of the invention comprises a 21 nucleotide (nt) sense strand and a 23 nucleotide (nt) antisense strand, wherein the antisense strand has at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt while the other end comprises a 2 nt overhang, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand is 2, (ii) the antisense strand comprises 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3 to 9 (counting from the 5' end of the antisense strand); (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. and (viii) the sense strand further comprises at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, or 8) of the following: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0129] In some embodiments, in the dsRNA molecule of the present invention comprising a sense and an antisense strand, the sense strand is 25 to 30 nucleotide residues in length, wherein positions 1 to 23 of the sense strand, starting from the 5'-terminal nucleotide (position 1), comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, wherein, starting from the 3'-terminal nucleotide, at least 8 ribonucleotides within its positions are paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5' end of the antisense strand comprises 10 to 30 consecutive nucleotides that are not paired with the sense strand, thereby forming a duplex. forming a 30-nucleotide single-stranded 5' overhang; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; and wherein, when the double-stranded nucleic acid is introduced into a mammalian cell, the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression; and wherein the antisense strand has at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 of the 5'-end of the antisense strand), and the dsRNA has a melting temperature of about 40°C to about 80°C.For example, the heat-destabilizing nucleotide is present between positions 14-17 opposite or complementary to positions 14-17 at the 5' end of the sense strand, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, where positions 3-9 (counting from the 5' end of the antisense strand) are absent of 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand. (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 30 nucleotide pairs in length; and the sense strand comprises one, two, three, four, five, six, seven, or eight LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0130] In some embodiments, a dsRNA molecule of the present invention comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides, and the antisense strand having a length of at most 30 nucleotides comprises, together with the sense strand, a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, and the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3' end, wherein in the double-stranded region having a length of at least 25 nucleotides, when the dsRNA molecule is introduced into a mammalian cell, the antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the antisense strand to reduce expression of a target gene, and wherein Dicer cleavage of the dsRNA preferentially yields siRNA comprising the 3' end of the antisense strand, thereby reducing expression of the target gene in the mammal, and wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile The stabilizing nucleotides are present in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense strand contains 1, 2, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand contains 1, 2, 4, or 5 phosphorothioate internucleotide linkages; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA has a double-stranded region 12 to 29 nucleotide pairs in length; and (viii) the sense strand comprises one, two, three, four, five, seven, eight, nine, or ten LNA modifications.In some embodiments, a melting temperature of about 40° C. to about 80° C. is optional.

[0131] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense strand comprises three or four phosphodiesterase inhibitors; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (viii) the dsRNA has a blunt end at the 5' end of the antisense strand; and (ix) the sense strand further contains one, two, three, four, five, six, seven, eight, or all nine of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0132] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); ... (i) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications; (iv) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (viii) the dsRNA has a blunt end at the 5' end of the antisense strand; and (ix) the sense strand further contains one, two, three, four, five, six, seven, eight, or all nine of the following: (i) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, five, six, seven, eight, or nine LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0133] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phospho- (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (viii) the dsRNA has a blunt end at the 5' end of the antisense strand; and (ix) the sense strand further contains one, two, three, four, five, six, seven, eight, or all nine of the following: LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0134] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the dsRNA optionally has any of the following characteristics: (i) the antisense strand has 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications, where no 2'-fluoro modifications are present at positions 3 to 9 (counting from the 5' end of the antisense strand); (iv) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (vii) the dsRNA has a blunt end at the 5' end of the antisense strand; and (viii) the sense strand further contains at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, or 8) of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0135] In one aspect, the present invention provides a dsRNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, at positions 2 to 9, counting from the 5' end), and the dsRNA molecule has the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3 to 9 (counting from the 5' end); (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phospho- (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (viii) the antisense strand has a blunt end at the 5' end; and (ix) the sense strand contains one, two, three, four, five, six, seven, eight, nine, or ten LNA modifications. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0136] In certain embodiments, the thermally destabilizing modification of the duplex is present in the antisense strand at position 5, 6, or 7, counting from the 5' end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is present in the antisense strand at position 2, 3, 4, 8, or 9, counting from the 5' end of the antisense strand.

[0137] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at the 5' end of the antisense strand, from positions 2 to 9, counting from the 5' end), and the antisense strand has any of the following characteristics: (i) 2, 3, 4, 5, or 6 2'-fluoro modifications ((of the antisense strand) and (ii) no 2'-modifications at positions 3-9 (counting from the 5' end); and (ii) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and the sense strand further comprises one, two, three, or four of the following features: (i) a ligand conjugated to the sense strand; (ii) 2, 3, 4, or 5 2'-fluoro modifications; (iii) 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (iv) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications.

[0138] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, a ligand is conjugated to the sense strand, and the dsRNA comprises at least four 2'-fluoro modifications and no 2'-modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand).

[0139] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and wherein the sense strand comprises a ligand and there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand). In some further embodiments thereof, the ligand is an ASGPR ligand.

[0140] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4 to 8, counting from the 5' end, wherein the sense strand comprises a ligand, and wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, and there are no 2'-modifications at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand). In some further embodiments thereof, the ligand is an ASGPR ligand.

[0141] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one double-stranded thermodestabilizing modification within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and wherein the antisense strand further comprises at least two of the following features: (i) the double-stranded thermodestabilizing modifications are located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro modifications (wherein no 2'-modification is present at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand)); (iii) a phosphorothioate internucleotide linkage between nucleotides 1 and 2 (counting from the 5' end); and the antisense strand has a length of 18 to 35 nucleotides. In some further embodiments, the ligand is an ASGPR ligand.

[0142] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and wherein the sense strand has any of the following characteristics: (i) the ligand is a nucleotide of one of the sense strands; (ii) the sense strand contains at least two 2'-fluoro modifications; (iii) the sense strand and antisense strand exhibit sufficient complementarity to form a double-stranded region spanning at least 19 nucleotide positions; (iv) the sense strand contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and the thermally destabilizing modifications of the duplex are located within the double-stranded region, and no 2'-modifications are present at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand).

[0143] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoros, wherein there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the antisense strand comprises at least one double-stranded thermodestabilizing modification within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and wherein the double-stranded thermodestabilizing modification is [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0144] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 4-8, counting from the 5' end, wherein the sense strand comprises a ligand, and each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), and wherein the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0145] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at positions 5, 6, or 7, counting from the 5' end of the antisense strand, and wherein the sense strand comprises a ligand.

[0146] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex located at position 5, 6, or 7, counting from the 5' end, wherein the sense strand comprises a ligand, and each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), and wherein the thermodestabilizing modifications of the duplex are [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0147] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoro, wherein there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), wherein said antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and wherein said sense strand comprises a ligand, wherein the ligand comprises one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

[0148] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the dsRNA comprises at least four 2'-fluoros, wherein there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), wherein said antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and wherein said sense strand comprises a ligand, wherein the ligand has the structure: [ka] It is an ASGPR ligand.

[0149] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; wherein the antisense strand comprises 3, 4, 5, or 6 2'-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0150] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally Optionally, the dsRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; wherein the antisense strand comprises 3, 4, 5, or 6 2'-fluoro modifications, wherein no 2'-fluoro modifications are present at positions 3-9 of the antisense strand and 2, 3, or 4 phosphorothioate internucleotide linkages; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0151] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications, 0 or 2 phosphorothioate internucleotide linkages, and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; wherein the antisense strand The strand comprises a 2'-fluoro modification at positions 2, 14, or 16, or at positions 2, 14, and 16; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22 and between nucleotides 22 and 23; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0152] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 3 or 4 2'-fluoro modifications and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally, 0 or 2 phosphorothioate internucleotide linkages; wherein the antisense strand The strand comprises a 2'-fluoro modification at positions 2, 14, or 16, or at positions 2, 14, and 16; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22 and between nucleotides 22 and 23; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0153] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand , 2, 14, or 16, or 2, 14, and 16; and the antisense comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22, and between nucleotides 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand.

[0154] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications and 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; and wherein the antisense strand is 2 , 14, or 16; and the antisense comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand.

[0155] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and comprises 3 or 4 2'-fluoro modifications, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand has at least one heat-labile nucleotide in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand). , 14, or 16; and the antisense comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5'-end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3'-end of the antisense strand.

[0156] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); wherein the sense strand is conjugated to a ligand and contains 3 or 4 2'-fluoro modifications, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and 0, 1, 2, or 3 phosphorothioate internucleotide linkages; and wherein the antisense strand is 2, 14, 16, 18, 20, 21, 22, 23, 24, or 25 nucleotides in length. or a 2'-fluoro modification at position 16; and the antisense comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand.

[0157] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; wherein the antisense strand comprises 2'-fluoro modifications at positions 2, 14, and 16; and wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0158] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and wherein the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally 1, wherein the antisense strand comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; wherein the antisense strand comprises 2'-fluoro modifications at positions 2, 14, and 16; and wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0159] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, where at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally between nucleotides 1 and 2, and between nucleotides 2 and 3. and 3; wherein the antisense strand comprises a 2'-fluoro modification at positions 2, 14, and 16; and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22 and between nucleotides 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region of 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0160] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, where at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and where the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and between nucleotides 1 and 2, and between nucleotides 2 and 3. wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 2, 14, and 16; and wherein the antisense strand comprises a 2'-fluoro modification at positions 2, 14, and 16; and wherein the antisense strand also comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a 2-nucleotide overhang at the 3' end of the antisense strand.

[0161] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, where the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally comprising phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. , 7, 8, 9, or 10 LNA modifications; wherein the antisense strand comprises a 2'-fluoro modification at position 2, 14, or 16; and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand.

[0162] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, where at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. , 9, or 10 LNA modifications; wherein the antisense strand comprises a 2'-fluoro modification at position 2, 14, or 16; and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand.

[0163] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; the antisense strand has at least one heat-labile nucleotide, wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the sense strand is conjugated to a ligand and comprises 2'-fluoro modifications at positions 7, 10, and 11 or 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally between nucleotides 1 and 2 and between nucleotides 2 and 3. wherein the antisense strand comprises a 2'-fluoro modification at position 2, 14, or 16; and wherein the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; and wherein the dsRNA optionally further comprises at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region 12 to 25 nucleotide pairs in length; (ii) the dsRNA comprises a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA has at least a two-nucleotide overhang at the 3' end of the antisense strand.

[0164] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermodestabilizing modification of the duplex within the seed region (i.e., at positions 2 to 9 of the 5' end of the antisense strand, counting from the 5' end), and the dsRNA has a melting temperature (T m), and the dsRNA optionally has the following features: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages. (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; (viii) a blunt end at the 5' end of the antisense strand; (ix) with the proviso that no 2'-fluoro modifications are present at positions 3 to 9 (counting from the 5' end) of the antisense strand (e.g., positions 1, 2, 3, 4, 5, 6, 7, 8, or all 9). In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0165] In some embodiments, the dsRNA molecule has a double-stranded region 12 to 40 nucleotide pairs in length, wherein the antisense strand contains at least one thermodestabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 of the 5' end of the antisense strand, counting from the 5' end), and the dsRNA has a T of about 40°C to about 80°C. mand wherein the dsRNA optionally further has at least one of the following characteristics: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and vii) a blunt end at the 5' end of the antisense strand, with the proviso that no 2'-fluoro modifications are present at positions 3 to 9 (counting from the 5' end) of the antisense strand (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0166] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length, where the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA has a melting temperature of about 40°C to about 80°C, provided that there are no 2'-fluoro modifications at positions 3-9 (counting from the 5' end) of the antisense strand. In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0167] In certain embodiments, the dsRNA molecules of the invention comprise: (a)(i) 21 nucleotides long; (ii) a 3'-terminally attached ASGPR ligand comprising three GalNAc derivatives linked via a trivalent branched linker; and (iii) 2′-F modifications at positions 7, 10, and 11 (counting from the 5′ end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0168] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0169] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0170] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) LNA modification; and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0171] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) an LNA modification at at least one of positions 1, 2, and 3 (e.g., 1, 2, or 3) (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0172] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2′-F modifications at positions 7, 9, 10, and 11 (counting from the 5′ end); (iv) at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) LNA modification; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0173] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) an LNA modification at at least one of positions 1, 2, and 3 (e.g., 1, 2, or 3) (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0174] In another specific embodiment, the dsRNA molecule of the invention comprises: (a)(i) 21 nucleotides long; (ii) a 3′-terminally attached ASGPR ligand containing three GalNAc derivatives linked via a trivalent branched linker; (iii) 2′-F modifications at positions 7, 9, 10, and 11 (counting from the 5′ end); (iv) an LNA modification at at least one (e.g., 1, 2, or 3) of positions 1, 2, and 3 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and a sense strand having (b)(i) 23 nucleotides long; (ii) 2′-F modifications at positions 2, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); and (iv) a duplex heat-destabilizing modification at position 5, 6, or 7 (counting from the 5′ end); and an antisense strand having: wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0175] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, wherein the antisense strand has at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) sense strand is conjugated with ligand; (iv) sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) dsRNA comprises at least 4 2'-fluoro modifications; (vii) dsRNA comprises 18, 19, 20, 21, 22, 23, 24 or 24 nucleotide pair long double-stranded region; and (viii) dsRNA comprises blunt end at the 5' end of sense strand (for example, 1, 2, 3, 4, 5, 6, 7 or 8).In some embodiments, sense strand is 19, 20 or 21 or 22 nucleotide long, and antisense strand is 20, 21 or 22 nucleotide long.

[0176] The sense strand and the antisense strand typically form a double-stranded dsRNA. The double-stranded region of the dsRNA molecule may be 12 to 40 nucleotide pairs in length. For example, the double-stranded region may be 14 to 40 nucleotide pairs, 17 to 30 nucleotide pairs, 25 to 35 nucleotide pairs, 27 to 35 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.

[0177] In some embodiments, a dsRNA molecule of the invention has a double-stranded region 12-40 nucleotide pairs in length, wherein the antisense strand has at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has any of the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense contains 1, 2, 3, 4, or 5 2'-fluoro modifications; (iii) sense strand is conjugated with a ligand; (iv) sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide bonds; and (vi) dsRNA comprises at least four 2'-fluoro modifications; and (vii) dsRNA comprises a blunt end at the 5' end of antisense strand (for example, 1, 2, 3, 4, 5, 6 or 7).In some embodiments, double-stranded region is 18, 19, 20, 21, 22 or 23 nucleotide pairs in length.In certain embodiments, double-stranded region is 21 nucleotide pairs in length.

[0178] In some embodiments, the dsRNA molecules of the present invention comprise one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of the strand. The overhangs can be 1-10 nucleotides, 1-6 nucleotides, e.g., 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, be complementary to the targeted gene sequence, or be other sequences. The first and second strands can also be linked by additional bases, e.g., to form a hairpin, or by other non-basic linkers.

[0179] In some embodiments, the nucleotides in the overhang region of the dsRNA molecule of the present invention can each independently be modified or unmodified nucleotides, for example but not limited to 2'-sugar modified, such as 2'-fluoro 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyl adenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof.For example, TT can be the overhang sequence at one end of one strand.Overhang can form mismatch with target mRNA, or can be complementary to the gene sequence being targeted, or can be other sequences.

[0180] The 5' or 3' overhang of the sense strand, antisense strand or both strands of the dsRNA molecule of the present invention can be phosphorylated.In some embodiments, the overhang region comprises two nucleotides with phosphorothioate between them, and these two nucleotides can be the same or different.In some embodiments, the overhang is present at the 3' end of the sense strand, antisense strand or both strands.In some embodiments, this 3' overhang is present in the antisense strand.In some embodiments, this 3' overhang is present in the sense strand.

[0181] The dsRNA molecule of the present invention may contain only a single overhang, which can enhance the interference activity of dsRNA without affecting its overall stability.For example, the single-stranded overhang is located at the 3'-end of the sense strand, or alternatively at the 3'-end of the antisense strand.The dsRNA may also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa.Generally, the antisense strand of dsRNA has a nucleotide overhang at its 3'-end, and the 5'-end is blunt.Without being bound by theory, the asymmetric blunt ends at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand support the guide strand loading in the RISC process.For example, the single overhang comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.In some embodiments, the dsRNA has a 2-nucleotide overhang at the 3'-end of the antisense strand and the blunt end at the 5'-end of the antisense strand.

[0182] In some embodiments, one end of the dsRNA is blunt and the other end has an overhang, wherein the antisense strand has at least one heat-labile nucleotide, and wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, an overhang is present on the 3' end of the antisense strand, and a blunt end is present at the 5' end of the antisense strand. In certain embodiments, the overhang is 2, 3, or 4 nucleotides in length.

[0183] In some embodiments, the dsRNA molecule has a double-stranded region 19, 20, 21, 22, or 23 nucleotide base pairs in length, wherein one end of the dsRNA is blunt and the other end has an overhang, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense contains 1, 2, 3, 4, or 5 phosphodiesterases; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide bonds; and (vi) the dsRNA comprises at least four 2'-fluoro modifications, and optionally further comprises at least one (e.g., 1, 2, 3, 5 or 6) of the following: a 2-nucleotide overhang is present on the 3'-end of the antisense strand, and a blunt end is present on the 5'-end of the antisense strand. In some embodiments, an overhang is present on the 3'-end of the antisense strand, and a blunt end is present on the 5'-end of the antisense strand.

[0184] In some embodiments, the dsRNA molecules of the invention may also have two blunt ends at either end of the dsRNA duplex.

[0185] In some embodiments, the dsRNA has blunt ends at both ends of the duplex, wherein the antisense strand has at least one heat-labile nucleotide, and wherein the at least one heat-labile nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate modifications. (iii) the sense strand is conjugated to a ligand; (iv) the sense strand includes two, three, four, or five 2'-fluoro modifications; (v) the sense strand includes one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA includes at least four 2'-fluoro modifications; and (vii) the dsRNA includes a double-stranded region 12 to 40 nucleotide pairs in length (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0186] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length and has blunt ends at both ends of the duplex, wherein one end of the dsRNA is blunt and the other end has an overhang, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has any of the following characteristics: (i) the antisense strand has 2, 3, 4, 5, or 6 nucleotide base pairs in length; (ii) the antisense strand contains one, two, three, four, or six 2'-fluoro modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications.

[0187] Thermal destabilization modification As described above, dsRNA molecules can be optimized for RNA interference by incorporating a thermodestabilizing modification within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. The inventors have discovered that dsRNAs having an antisense strand containing at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5, or more) thermodestabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the thermodestabilizing modification of the duplex is located between positions 2-9, or preferably between positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermodestabilizing modification of the duplex is located between positions 6, 7, or 8 from the 5' end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermal destabilizing modification" refers to a modification that results in a lower total melting temperature (T m ) (preferably, the T of the dsRNA when it does not have such modifications) m T 1, 2, 3, or 4°C lower than m In some embodiments, the thermodestabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0188] Thermally destabilizing modifications can include, but are not limited to, abasic modifications; mismatches with the opposing nucleotide on the opposite strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0189] Exemplary abasic modifications include, but are not limited to, the following: [ka] (Wherein R=H, Me, Et, or OMe; R'=H, Me, Et, or OMe; R''=H, Me, Et, or OMe) [ka] wherein B is a modified or unmodified nucleobase. Includes.

[0190] Exemplary sugar modifications include, but are not limited to, the following: [ka] wherein B is a modified or unmodified nucleobase. Includes.

[0191] In some embodiments, the thermally destabilizing modification of the duplex is [ka] (wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic). is selected from the group consisting of:

[0192] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, when any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) of the sugar has been removed (see Mikhailovet et al., Tetrahedron Letters, 26(17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009) (incorporated herein by reference in their entireties)). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0193] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA but differing in its "backbone" composition from repeating glycerol units linked by phosphodiester bonds. [ka]

[0194] The double-stranded thermolabile modification can be mismatched (i.e., non-complementary base pair) between the thermolabile nucleotide and the opposite nucleotide in the opposite strand within the dsRNA duplex.Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatched base pairings known in the art are also in accordance with the present invention.Mismatches can occur between either naturally occurring or modified nucleotides, that is, mismatched base pairing can occur between the nucleobases from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule has at least one nucleobase in mismatched pairing that is a 2'-deoxynucleobase; for example, the 2'-deoxynucleobase is present in the sense strand.

[0195] In some embodiments, the duplex thermodestabilizing modifications in the seed region of the antisense strand include nucleotides with impaired WCH bonds to complementary bases on the target mRNA, e.g., [ka] Examples include:

[0196] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0197] Thermodestabilizing modifications may also include universal bases that have reduced or eliminated ability to form hydrogen bonds with opposing bases, and phosphate modifications.

[0198] In some embodiments, the thermal destabilizing modification of double strand comprises but is not limited to the nucleotide with non-standard base, such as the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA double strand, as described in WO2010 / 0011895 (its entirety is incorporated herein by reference).Exemplary nucleobase modifications are as follows: [ka]

[0199] In some embodiments, the duplex thermodestabilizing modifications in the seed region of the antisense strand include one or more α-nucleotides complementary to bases on the target mRNA, e.g., [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes.

[0200] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages include: [ka] Examples include:

[0201] The alkyl in the R group is a C1-C6 alkyl. Specific alkyl in the R group includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0202] In some embodiments, exemplary destabilizing modifications are shown in FIG.

[0203] In addition to the antisense strand containing thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both sense and antisense strands contain at least two stabilizing modifications.Stabilizing modifications can be present on any nucleotide of the sense strand or antisense strand.For example, stabilizing modifications can be present on every nucleotide of the sense strand and / or antisense strand; each stabilizing modification can be present in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand contains both stabilizing modifications in an alternating pattern.The alternating pattern of stabilizing modifications on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand can have a change to the alternating pattern of stabilizing modifications on the antisense strand.

[0204] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0205] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be at the 5'-end or 3'-end of destabilizing modification, that is, the nucleotide at -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at each of the 5'-end and 3'-end of destabilizing modification, that is, the nucleotide at -1 and +1 position from the position of destabilizing modification.

[0206] In some embodiments, the antisense strand comprises at least two stabilizing modifications 3'-terminal to the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0207] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications.

[0208] In some embodiments, the sense strand does not contain a stabilizing modification at the position opposite or complementary to the thermodestabilizing modification of the duplex in the antisense strand.

[0209] Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermostabilizing modifications include, but are not limited to, LNA.

[0210] In some embodiments, the dsRNA of the present invention comprises at least four (for example, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, all of the 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense and antisense strands comprise at least two 2'-fluoro nucleotides. The 2'-fluoro modification can be present on any nucleotide of the sense strand or antisense strand. For example, the 2'-fluoro modification can be present on every nucleotide of the sense strand and / or antisense strand; each 2'-fluoro modification can be present in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand contains both 2'-fluoro modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can have a change to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0211] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14 and 16 from the 5' end.

[0212] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5' end or 3' end of destabilizing modification, that is, the nucleotide at -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, the nucleotide at -1 and +1 position from the position of destabilizing modification.

[0213] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0214] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises 2, 3 or 4 2'-fluoro nucleotide blocks.

[0215] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at the position opposite or complementary to the thermally destabilizing modification of the duplex in the antisense strand.

[0216] In some embodiments, a dsRNA molecule of the invention comprises a 21 nucleotide (nt) sense strand and a 23 nucleotide (nt) antisense strand, wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and the dsRNA optionally comprises any of the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-full nucleotides; (ii) the antisense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand (for example, 1, 2, 3, 4, 5, 6 or 7). Preferably, a 2nt overhang is present at the 3' end of the antisense strand.

[0217] In some embodiments, in the dsRNA molecule of the present invention comprising a sense and an antisense strand, the sense strand is 25 to 30 nucleotide residues in length, wherein positions 1 to 23 of the sense strand, starting from the 5'-terminal nucleotide (position 1), contain at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, wherein, starting from the 3'-terminal nucleotide, at least 8 ribonucleotides within its positions are paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5' end of the antisense strand contains 10 to 30 consecutive nucleotides that are not paired with the sense strand. wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; and wherein, when the double-stranded nucleic acid is introduced into a mammalian cell, the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression; and wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 of the 5'-end of the antisense strand).For example, the heat-destabilizing nucleotide is present between positions 14 and 17 opposite or complementary to positions 14 and 17 at the 5' end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following characteristics: (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a double-stranded region 12 to 30 nucleotide pairs in length.

[0218] In some embodiments, a dsRNA molecule of the present invention comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides, and the antisense strand having a length of at most 30 nucleotides comprises, together with the sense strand, a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, and the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3' end, wherein in the double-stranded region having a length of at least 25 nucleotides, when the dsRNA molecule is introduced into a mammalian cell, the antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the antisense strand to reduce expression of a target gene, and wherein Dicer cleavage of the dsRNA preferentially yields an siRNA comprising the 3' end of the antisense strand, thereby reducing expression of the target gene in a mammal. and wherein the expression of the antisense strand is reduced, wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and wherein the dsRNA optionally comprises any of the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages. (iii) the sense strand is conjugated to a ligand; (iv) the sense strand includes two, three, four, or five 2'-fluoro modifications; (v) the sense strand includes one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA includes at least four 2'-fluoro modifications; and (vii) the dsRNA has a double-stranded region 12 to 29 nucleotide pairs in length (e.g., all 1, 2, 3, 4, 5, 6, or 7).

[0219] In some embodiments, all the nucleotides in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and this modification can include one or more of the non-bonded phosphate oxygen and / or one or more bonded phosphate oxygens, one or both of the modification;Modification of ribose sugar component, for example, the 2' hydroxyl of ribose sugar;Modification or substitution of phosphate moiety with " dephosphorylation " linker;Modification or substitution of natural base;And substitution or modification of ribose-phosphate backbone.

[0220] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at positions that are repeated within the nucleic acid. In some cases, modifications will occur at every target position in the nucleic acid, but in many cases, this is not the case. For example, modifications may occur only at the 3' or 5' terminal position, or only within the terminal region, such as a position on the terminal nucleotide or within the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only within the double-stranded region of the RNA, or only within the single-stranded region of the RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both ends, or only within the terminal region, such as a position on the terminal nucleotide or within the last 2, 3, 4, 5, or 10 nucleotides of the chain, or within double-stranded and single-stranded regions, especially at the ends. The 5' end may be phosphorylated.

[0221] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications known in the art at the 2' position of the ribose sugar, such as deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications, in place of the ribosugar of the nucleobase, and modifications at the phosphate group, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0222] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro.These strands can contain two or more modifications.In some embodiments, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.It should be understood that these modifications are present in addition to at least one thermostabilizing modification of the double strand that exists in antisense strand.

[0223] At least two different modifications are typically present on the sense strand and the antisense strand. These two modifications may be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, or other. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it should be understood that these modifications are present in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0224] In some embodiments, the dsRNA molecules of the present invention contain alternating patterns of modifications, particularly within the B1, B2, B3, B1', B2', B3', and B4' regions. The term "alternating motif" or "alternating pattern," as used herein, refers to a motif having one or more modifications, with each modification occurring on alternating nucleotides in a single strand. The alternating nucleotides may refer to every other nucleotide, every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one modification type for a nucleotide, the alternating motif may be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0225] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one modification type to a nucleotide, the alternation pattern, i.e., the modifications to every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0226] In some embodiments, the dsRNA molecules of the present invention comprise a modification pattern in an alternating motif on the sense strand that is different from the modification pattern in an alternating motif on the antisense strand. The modification groups of the nucleotides in the sense strand may correspond to different modification groups of the nucleotides in the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, within the double-stranded region, the alternating motif in the sense strand may begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from the 3'-5' end of the strand. As another example, within the double-stranded region, the alternating motif in the sense strand may begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from the 3'-5' end of the strand, thereby resulting in a complete or partial change in the modification pattern between the sense and antisense strands.

[0227] The dsRNA molecule of the present invention can further comprise at least one phosphorothioate or methylphosphonate internucleotide bond.Phosphorothioate or methylphosphonate internucleotide bond modification can occur on any nucleotide at any position of sense strand or antisense strand or both strands.For example, internucleotide bond modification can occur on all nucleotides on sense strand and / or antisense strand; each internucleotide bond modification can occur in an alternating pattern on sense strand or antisense strand; or sense strand or antisense strand contains both internucleotide bond modifications in an alternating pattern.The alternating pattern of internucleotide bond modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide bond modification on sense strand can have changes with respect to the alternating pattern of internucleotide bond modification on antisense strand.

[0228] In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region.For example, the overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides.Internucleotide bond modification can also be provided to link the overhang nucleotide with the terminal paired nucleotide within the double-stranded region.For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked through phosphorothioate or methylphosphonate internucleotide bond, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond that links the overhang nucleotide with the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides, and the third is the paired nucleotide adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be located at the 3' end of the antisense strand.

[0229] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein either the phosphorothioate or methylphosphonate internucleotide linkages are positioned at any position within the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0230] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0231] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0232] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0233] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0234] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0235] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0236] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, or six phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0237] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0238] In some embodiments, the dsRNA molecules of the invention further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense and / or antisense strands.

[0239] In some embodiments, the dsRNA molecules of the invention further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 positions of the internal region of the double strand of each of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked via phosphorothioate methylphosphonate internucleotide linkages at positions 8-16, counting from the 5'-end of the sense strand of the double-stranded region; the dsRNA molecule may optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the terminal positions 1-10.

[0240] In some embodiments, dsRNA molecules of the invention further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0241] In some embodiments, dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0242] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0243] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0244] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0245] In some embodiments, the dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0246] In some embodiments, the dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0247] In some embodiments, the dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0248] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0249] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0250] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end).

[0251] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0252] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0253] In some embodiments, the dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0254] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0255] In some embodiments, the dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0256] In some embodiments, the dsRNA molecules of the invention further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0257] In some embodiments, the dsRNA molecules of the invention further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0258] In some embodiments, the compounds of the present invention comprise a pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises 8 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises 7 or fewer internucleotide linkages in the Rp configuration.In some embodiments, the common pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (by way of non-limiting example, phosphodiester). In some embodiments, the common pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises 4 or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises 3 or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises 2 or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises 1 or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and 8 or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and 7 or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and 6 or fewer non-chiral internucleotide linkages.In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration are optionally adjacent or non-adjacent. In some embodiments, the internucleotide linkages in the Rp configuration are optionally adjacent or non-adjacent. In some embodiments, the non-chiral internucleotide linkages are optionally adjacent or non-adjacent.

[0259] In some embodiments, compounds of the invention comprise blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, such that each internucleotide bond of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, such that each internucleotide bond of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks, such that each internucleotide bond is a natural phosphate bond.

[0260] In some embodiments, the compounds of the present invention include a 5'-block Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety includes a 2'-F modification. In some embodiments, the 3'-block includes 4 or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block includes 6 or more nucleoside units. In some embodiments, the 3'-block includes 7 or more nucleoside units.

[0261] In some embodiments, compounds of the invention include nucleosides of a type within a region, or oligonucleotides are followed by a particular type of internucleotide linkage, e.g., a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate bond (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0262] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 3, 4, or 5 phosphorothioate internucleotide linkages. (iii) the sense strand is conjugated to a ligand; (iv) the sense strand includes two, three, four, or five 2'-fluoro modifications; (v) the sense strand includes one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA includes at least four 2'-fluoro modifications; (vii) the dsRNA includes a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand (e.g., all 1, 2, 3, 4, 5, 6, 7, or 8).

[0263] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the sense strand comprises a ligand and a covalent bond; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a double-stranded region between 12 and 40 nucleotide pairs in length; (vii) the dsRNA comprises a double-stranded region between 12 and 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0264] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand includes two, three, four, or five 2'-fluoro modifications; (v) the sense strand includes three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA includes at least four 2'-fluoro modifications; (vii) the dsRNA includes a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8).

[0265] In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand has at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally comprises the following features: (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications; (iv) the sense strand contains three or four phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand (e.g., 1, 2, 3, 4, 5, 6, or all 7).

[0266] In some embodiments, the dsRNA molecule of the present invention comprises mismatches with the target or a combination thereof within the double strand. Mismatches can be present in the overhang region or in the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the simplest method is to consider the association or dissociation free energy of specific pairings based on each pairing, but similar or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C; G:U is more preferable than G:C; and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (as described elsewhere herein), are more preferable than canonical (A:T, A:U, G:C) pairings; and pairings containing universal bases are more preferable than canonical pairings.

[0267] In some embodiments, the dsRNA molecules of the invention comprise at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0268] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of antisense strand is an AU base pair.

[0269] The present inventors have found that the introduction of 4'- and / or 5'-modified nucleotides at the 3'-end of phosphodiester (PO), phosphorothioate (PS), and / or dithiophosphate (PS2) linkages of dinucleotides at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide bond, thus protecting or stabilizing it against nucleases.

[0270] In some embodiments, 5'-modified nucleoside is introduced at the 3'-end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3'-end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5'-position of ribose sugar can be racemic or chiral pure R or S isomer.An exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be racemic or chiral pure R or S isomer.

[0271] In some embodiments, 4'-modified nucleosides are introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 4'-position of the ribose sugar can be racemic or chiral pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl can be racemic or chiral pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or chiral pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. The 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0272] In some embodiments, 5'-alkylated nucleoside can be introduced at any position on the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chiral pure R or S isomer.An exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be either racemic or chiral pure R or S isomer.

[0273] In some embodiments, 4'-alkylated nucleoside can be introduced at any position on the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chiral pure R or S isomer.An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be either racemic or chiral pure R or S isomer.

[0274] In some embodiments, 4'-O-alkylated nucleoside can be introduced at any position on the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-Alkyl can be either racemic or chiral pure R or S isomer.An exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside.4'-O-methyl can be either racemic or chiral pure R or S isomer.

[0275] In some embodiments, the dsRNA molecules of the present invention may contain 2'-5' linkages (having 2'-H, 2'-OH and 2'-OMe, and having P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0276] In another embodiment, the dsRNA molecule of the present invention can comprise L-sugar (for example, L-ribose, L-arabinose, with 2'-H, 2'-OH and 2'-OMe).For example, these L-sugar modifications can be used to promote nuclease resistance or inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to avoid the activation of sense strand by RISC.

[0277] Various publications describe the multimeric siRNA, which can all be used together with the dsRNA of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 (their entirety is incorporated herein by reference).

[0278] A dsRNA molecule comprising one or more carbohydrate moieties attached to the dsRNA molecule can optimize one or more properties of the dsRNA molecule. In many cases, the carbohydrate moiety will be attached to a modified subunit of the dsRNA molecule. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA molecule can be replaced with a non-carbohydrate (preferably cyclic) carrier to which another moiety, such as a carbohydrate ligand, is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0279] The ligand may be attached to the polynucleotide by a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally allow for a bond suitable for incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring.

[0280] In some embodiments, the dsRNA molecule of the present invention may be linked to a ligand via a carrier, wherein the carrier may be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0281] The double-stranded RNA (dsRNA) agent of the present invention can optionally be conjugated with one or more ligands.Ligand can be attached to sense strand, antisense strand or both strands at 3' end, 5' end or both ends.For example, ligand can be attached to sense strand, particularly 3' end of sense strand.

[0282] In some embodiments, the dsRNA molecules of the invention are 5' phosphorylated or contain a phosphoryl analog at the 5' prime end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (dithiophosphate; (HO)(HS)(S)PO-5'), 5 '-phosphorothiolate ((HO)2(O)PS-5'); any further combination of oxygen / sulfur substituted monophosphate, diphosphate and triphosphate (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidic acid ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonate (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). In one example, the modification can be placed in the antisense strand of the dsRNA molecule.

[0283] Ligand A wide variety of entities can be coupled to the oligonucleotides of the invention. Preferred moieties are ligands, preferably covalently coupled, either directly or indirectly via an intervening tether.

[0284] In a preferred embodiment, the ligand changes the distribution, targeting, or life span of the molecule into which it is incorporated. In a preferred embodiment, the ligand provides enhanced affinity to a selected target, such as a molecule, a cell or cell type, a compartment, a receptor, such as a cell or organ compartment, a tissue, an organ, or a body region, for example, compared to a species that does not have such a ligand. A ligand that provides enhanced affinity to a selected target is also referred to as a targeting ligand.

[0285] Some ligands may have endosomolytic properties. Endosomolytic ligands promote endosome lysis and / or transport of the compositions of the present invention or their components from the endosomes of cells to the cytoplasm. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and membrane fusogenicity. In some embodiments, endosomolytic ligands adopt their active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes endosome lysis and / or transport of the compositions of the present invention or their components from the endosomes of cells to the cytoplasm. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972, incorporated by reference in its entirety), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586, incorporated by reference in its entirety), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, incorporated by reference in its entirety). In some embodiments, the endosomolytic component may contain a chemical group (e.g., amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0286] The ligands may improve the transport, hybridization, and specificity properties, and may also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides or polymer molecules comprising any combination of the monomers and / or natural or modified ribonucleotides described herein.

[0287] Ligands may generally include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0288] Ligands can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids, oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylate), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0289] Ligands can also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells. The targeting group can be thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD peptidomimetics, or aptamers. Table 2 shows some examples of targeting ligands and their associated receptors.

[0290] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid. , O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ conjugates of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0291] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for co-ligands; or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0292] The ligand can be a substance, such as a drug, that can increase uptake of an iRNA agent into a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0293] Ligands can enhance cellular uptake of oligonucleotides, for example, by activating an inflammatory response. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF-α), interleukin-1β, or gamma interferon.

[0294] In one embodiment, the ligand is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue, for example, non-renal target tissue of the body.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of conjugate, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) be used to regulate the binding of serum protein, for example, HSA.

[0295] For example, lipid-based ligand can be used for inhibition, such as adjusting the binding of complex to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney, and therefore less likely to be removed from the body.The lipid or lipid-based ligand that binds weaker to HSA can be used to target complex to the kidney.

[0296] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

[0297] In other preferred embodiments, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells may also be used in place of or in addition to the lipid-based ligand.

[0298] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells. Also included are HAS and low-density lipoproteins (LDL) and high-density lipoproteins (HDL).

[0299] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent.Preferably, the cell-penetrating agent is amphipathic.An exemplary cell-penetrating agent is a peptide such as tat or antenopedia.When the cell-penetrating agent is a peptide, it can be modified, including peptidylmimetic, invertomer, non-peptide or pseudo-peptide bond, and D-amino acid use.The helical agent is preferably an α-helical agent with lipophilic and lipophobic phases.

[0300] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. The peptide or peptidomimetic moiety can be, for example, about 5 to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. In another alternative, the peptide moiety can contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting numerous polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) have been shown to function as delivery peptides. Peptides or peptidomimetics, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-94, 1991, incorporated by reference in its entirety), can be encoded by random sequences of DNA. Preferably, the peptide or peptidomimetic tethered to the iRNA agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide moiety can range from about 5 amino acids in length to about 40 amino acids in length. The peptide moiety may have structural modifications, for example to enhance stability or to direct conformational properties.Any of the following structural modifications can be used: RGD peptide moieties can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002, incorporated by reference in its entirety). RGD peptides can facilitate targeting of iRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001, incorporated by reference in its entirety). Preferably, RGD peptides will facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified, such as glycosylated or methylated, to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing αVβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, incorporated by reference in its entirety). Peptides that target markers abundant in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells that display integrins. Thus, RGD peptides, cyclic peptides with RGD, RGD peptides containing D-amino acids, and synthetic RGD mimics can be used. In addition to RGD, other moieties that target integrin ligands can be used. Generally, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this...

Claims

1. 1. A double-stranded RNA (dsRNA) molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the antisense strand comprises at least one double-stranded thermodestabilizing modification or precursor thereof within the first 9 nucleotide positions of its 5' region, and wherein the sense strand comprises an ASGPR ligand.

2. The dsRNA molecule of claim 1, comprising at least four 2'-fluoro groups.

3. The dsRNA molecule of claim 2, wherein the antisense strand is free of 2'-fluoro modifications at nucleotide positions 3-9.

4. Features include: a) the thermodestabilizing modifications of the duplex are located at positions 4 to 8 of the 5' region of the antisense strand; b) each of the sense and antisense strands comprises at least two 2'-fluoro modifications; and c) an ASGPR ligand bound to one end of the sense strand; The dsRNA molecule of claim 1, having the following structure:

5. The dsRNA molecule of claim 4, wherein the antisense strand is free of 2'-fluoro modifications at nucleotide positions 3-9.

6. The antisense strand has the following characteristics: a) the thermodestabilizing modifications of the duplex modifications are located at positions 4-8 of the antisense strand; b) at least two 2'-fluoro modifications; c) a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2 (counting from the 5' end); d) has a length of 18 to 35 nucleotides; The dsRNA molecule of claim 1, comprising at least two of:

7. The dsRNA molecule of claim 6, wherein the antisense strand is free of 2'-fluoro modifications at nucleotide positions 3-9.

8. The sense strand has the following characteristics: a) the ASGPR ligand bound to one end of the sense strand; b) at least two 2'-fluoro modifications; c) the sense strand and the antisense strand exhibit sufficient complementarity to form a double-stranded region spanning at least 19 nucleotide positions, and the thermodestabilizing modifications of the duplex are located within the double-stranded region; The dsRNA molecule of claim 1, comprising at least one of:

9. 9. The dsRNA molecule of claim 8, wherein the antisense strand is free of 2'-fluoro modifications at nucleotide positions 3-9.

10. The thermodestabilizing modification of the duplex is 【Chemistry 1】 wherein B is a nucleobase. The dsRNA molecule of claim 1, selected from the group consisting of:

11. 2. The dsRNA molecule of claim 1, wherein the stabilizing modification is located at position 7 of the antisense strand.

12. 2. The dsRNA molecule of claim 1, wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

13. The ASGPR ligand is 【Chemistry 2】 The dsRNA molecule of claim 8, wherein

14. 1. A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein the antisense strand comprises at least one double-stranded thermodestabilizing modification within the first 9 nucleotide positions of the 5' region, and wherein the dsRNA has a melting temperature of about 40°C to about 80°C.

15. 15. The dsRNA molecule of claim 14, having a melting temperature of about 55°C to about 67°C.

16. 2. The dsRNA molecule of claim 1, wherein at least 50% of the antisense strand is present in the liver 7 days after administration.

17. 17. The dsRNA of claim 16, further comprising at least one of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises one, two, three, or four phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, or four phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; (viii) a blunt end at the 5' end of the antisense strand; and (ix) the sense strand comprises one or more LNA modifications.

18. 18. The dsRNA of claim 17, wherein there are no 2'-fluoro modifications at positions 3 to 9 of the antisense strand.

19. The dsRNA agent of any one of claims 1 to 18, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

20. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-19, alone or in combination with a pharmaceutically acceptable carrier or excipient.

21. A gene silencing kit comprising the dsRNA molecule of any one of claims 1 to 19.

22. 15. A method for silencing a target gene in a cell, the method comprising introducing into said cell a dsRNA molecule according to any one of claims 1 to 14.

23. 23. The method of claim 22, wherein the dsRNA agent is administered via subcutaneous or intravenous administration.

24. 15. A method for silencing a target gene in a cell, the method comprising the step of expressing in said cell a dsRNA molecule according to any one of claims 1 to 14.

25. A method for suppressing off-target effects caused by the antisense strand of a dsRNA molecule, the method comprising the step of introducing the dsRNA molecule of any one of claims 1 to 19 into a cell.

26. A method for delivering a polynucleotide to a specific target in a subject by administering the dsRNA agent of any one of claims 1-19.

27. 27. The method of claim 26, wherein the administering step is performed by an administration means comprising intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, intralymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.

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