Modified RNA agent with reduced off-target effect

Abstract

To provide effective nucleotide or chemical motifs for dsRNA molecules, which are advantageous for inhibition of target gene expression, while having reduced off-target gene silencing effects; and RNAi compositions suitable for therapeutic use.SOLUTION: A double-stranded RNA (dsRNA) molecule capable of inhibiting expression of a target gene comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, where the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, where the antisense strand comprises at least one thermally destabilizing modification of a duplex within first 9 nucleotide positions of the 5' region or a precursor thereof, where the sense strand comprises an ASGPR ligand, and where the destabilizing modification is selected from a modified unlocked nucleic acid (mUNA) and a glycol nucleic acid (GNA).SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 672,405, filed May 16, 2018, and U.S. Provisional Patent Application No. 62 / 719,291, filed Aug. 17, 2018, under 35 U.S.C. § 119(e), the contents of both of which are hereby incorporated by reference in their entirety.

[0002] The present invention relates to RNAi double-stranded agents having specific motifs that are advantageous for inhibiting target gene expression by reducing unwanted off-target effects and RNAi compositions suitable for therapeutic use. In addition, the present invention provides methods for inhibiting the expression of target genes by administering these RNAi double-stranded agents, for example, for the treatment of various diseases.

Background Art

[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 post-transcriptional silencing in many organisms, including vertebrates, and provide a new tool for testing gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multi-component nuclease that destroys messenger RNA homologous to its silencing trigger. RISC is known to have short RNAs (about 22 nucleotides) derived from its double-stranded RNA trigger, but the active protein component remained unknown.

[0004] One of the off-target effects of siRNA is the miRNA-like effect, where the argonaute protein, which is a core effector in RNA interference, processes the artificially introduced siRNA for inducing RNA interference as miRNA (microRNA) (Non-Patent Document 3). miRNAs recognize many target genes for gene silencing through base pairing between the seed region (positions 2 to 9 from the 5' end) and the target mRNA. The off-targets caused by siRNA are due to the base complementarity between the seed region of the RISC-loading antisense strand of siRNA and one or more mRNAs. The miRNA-like off-target effect in siRNA has been reported in several studies, affects the expression of numerous genes depending on the sequence of the seed region, and is significant enough to cause up to 30% of the positive hits in siRNA-based phenotypic screening. In addition, in the case of miRNAs, it has also been reported that when the interaction between the seed region and the target weakens, they silence the target gene through complementary pairing (3'-complementary pairing) within their 3' terminal region, which means that the miRNA-like off-target effect is likely mediated by such a mechanism.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, efforts are being continued to remove or reduce the miRNA-like off-target effects of siRNA by regulating the design of siRNA through the rational application of chemical modifications without impairing the effectiveness of gene silencing by siRNA gene therapy. The present invention is directed to that effect.

Means for Solving the Problems

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

[0008] The inventors have found that, in particular, when the dsRNA molecule contains at least one thermolabile modification of the duplex within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the 5' end of the antisense strand), and the dsRNA molecule has a melting temperature in the range of about 40°C to about 80°C, it can be more effective for mediating RNA interference than the parental dsRNA molecule lacking the destabilizing modification. In some embodiments, the destabilizing modification is selected from the modified unlocked nucleic acid (mUNA) and glycol nucleic acid (GNA) components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, 2'-mUNA, and their structures are as follows:

Chemical

[0009] In some embodiments of various aspects disclosed herein, the destabilizing modification is 2'-5' RNA, i.e., Mod8. In some preferred embodiments, when the destabilizing modification is 2'-5' RNA, the destabilizing modification is present at the 7th position (counting from the 5' end) of the antisense strand.

[0010] In some embodiments of various aspects disclosed herein, the 5'-mUNA is 5'-(S)-Me-UNA(Y95) or 5'-(R)-Me-UNA(Y97), and its structure is shown in FIG. 32. In some embodiments of various aspects disclosed herein, the 2'-mUNA is 2'-(S)-Me-UNA(Y96) or 2'-(R)-Me-UNA(Y98), and its structure is shown in FIG. 32. In some embodiments of various aspects disclosed herein, the 3'-mUNA is 3'-(S)-Me-UNA(Y99), 3'-(R)-Me-UNA(Y100) or 3'-(R)-Me-4'-(S)-hydroxymethyl-UNA(Y102), and its structure is shown in FIG. 32. In some embodiments of various aspects disclosed herein, it is 4'-(β)-OMe-UNA(Y101), and its structure is shown in FIG. 32.

[0011] In some embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA(ModA-ModK), and its structure is as follows:

Chemical formula

[0012] Exemplary Hyp-spacer nucleosides are as follows:

Chemical formula

[0013] Examples of TNA nucleosides are as follows:

Chemical formula

[0014] Also provided herein are destabilized modified monomers and phosphoramidites. For example, the monomers and phosphoramidites can be selected from the modified unlocked nucleic acids (mUNA) and glycol nucleic acids (GNA) components described in Examples 1-3. In some embodiments, the monomer or its phosphoramidite is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. Also provided herein are nucleic acids comprising one or more of the mUNA and / or GNA monomers described herein. Without limitation, the nucleic acids comprising one or more of the mUNA and / or GNA monomers described herein can be single-stranded, double-stranded, partially double-stranded, hairpin, or circular nucleic acids.

[0015] Accordingly, in one aspect, the invention is a dsRNA molecule capable of inhibiting the 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, the antisense strand comprises at least one heat destabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 counting from the 5' end of the 5' end of the antisense strand), and the dsRNA has the following characteristics: (i) a melting temperature (T m); (ii) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications; (iii) the antisense contains 1, 2, 3 or 4 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 or 4 phosphorothioate internucleotide linkages; (vii) the dsRNA contains at least 4 2'-fluoro modifications; (viii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (ix) further has at least one blunt end (e.g., all 1, 2, 3, 4, 5, 6, 7, 8 or 9) at the 5'-end of the antisense strand, to provide a dsRNA molecule. In some embodiments, the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA.

[0016] In some embodiments, the invention is a dsRNA molecule capable of inhibiting the 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, the antisense strand contains at least one thermally destabilizing modification of a double strand within the seed region (i.e., positions 2 to 9, preferably 3 to 8, counting from the 5'-end at the 5'-end of the antisense strand), and the dsRNA molecule has the following characteristics: (i) a melting temperature (T m); (ii) the antisense contains 6, 7, 8, 9, 10, 11 or 12 2'-OMe modifications; (iii) the antisense contains 1, 2, 3 or 4 phosphorothioate nucleotide 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 nucleotide 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) further has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8 or 9) blunt ends at the 5'-end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein, to provide a dsRNA molecule. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0017] In some embodiments, the dsRNA has a melting temperature ranging from the lower end of the range from about 40°C, 45°C, 50°C, 55°C, 60°C or 65°C and 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 some particular 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.

[0018] 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.

[0019] The inventors have also discovered that for dsRNA molecules to be more effective in vivo, after administration, for example in the mouse liver, at least 40-50% of the antisense strand needs to be present on day 7 in vivo.

[0020] In another aspect, the present invention further provides a method for delivering the 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.

[0021] This patent or application file has at least one drawing created in color. Copies of the patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figures 19 - 20

Figure 21

Figure 22

Figure 23

Figure 24A

Figure 24B

Figure 25A

Figure 25B

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30A

Figure 30B

Figure 31A

Figure 31B

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Mode for Carrying Out the Invention

[0023] The inventors have in particular discovered 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 thermolabilizing modifications at specific positions in the antisense strand, the dsRNA molecules were able to retain gene silencing activity similar to that of the parental dsRNA while reducing off-target gene silencing. Furthermore, the number of off-target genes that are downregulated or upregulated by these dsRNA molecules containing thermolabilizing modifications is also reduced compared to the parental dsRNA.

[0024] From such, in one aspect, the present invention provides a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. Generally, the dsRNA molecules of the present invention exhibit high on-target gene silencing while reducing or minimizing off-target gene silencing and / or toxicity. Without limitation, the dsRNA molecules of the present invention can be used in RNA interference-based gene silencing techniques, including, but not limited to, in vitro or in vivo applications, and can be substituted with such dsRNA molecules.

[0025] Generally, the dsRNA molecule includes a sense strand (also referred to as the passenger strand) and an antisense strand (also referred to as the guide strand). Each strand of the dsRNA molecule can range in length from 12 to 40 nucleotides. 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 length or of unequal length.

[0026] 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 some particular embodiments, the antisense strand is 23 nucleotides in length. Similar to the antisense strand, the sense strand, in some embodiments, is 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 some particular embodiments, the antisense strand is 21 nucleotides in length.

[0027] The inventors also discovered that for dsRNA molecules that should be more effective in vivo, the antisense strand should have some metabolic stability. In other words, for dsRNA molecules that should be more effective in vivo, some amount of the antisense strand may need to be present in vivo for some time after administration. Thus, in some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 5th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 6th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 7th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 8th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 9th day after in vivo administration.In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 10th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 11th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 12th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 13th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 14th day after in vivo administration. In some embodiments, at least 40%, such as 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 the dsRNA is present in vivo, for example in the mouse liver, on the 15th day after in vivo administration.

[0028] 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 thermally destabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 counted from the 5' end of the 5' end of the antisense strand), and the dsRNA has a melting temperature (T m ) of from 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 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 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 in total) blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the T m from about 40°C to about 80°C is optional.

[0029] In some embodiments, the dsRNA molecule has a double-stranded region that is 12 to 40 nucleotide pairs in length, where the antisense strand contains at least one thermally labile modification of the double strand within the seed region (i.e., positions 2 to 9 counted from the 5' end at the 5' end of the antisense strand), and the dsRNA has a T of about 40°C to about 80°C m 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 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 4 2'-fluoro modifications; (vii) further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) blunt ends at the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0030] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, where the antisense strand has at least one thermally destabilizing 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, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0031] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, where the antisense strand has at least one thermally destabilizing 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), where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK), 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). In some embodiments, the T of about 40°C to about 80°C mis optional.

[0032] In some specific embodiments, the double-stranded heat destabilizing modification is present at the 5th, 6th, 7th or 8th position counted from the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0033] In some specific embodiments, the double-stranded heat destabilizing modification is present at the 5th position counted from the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0034] In some specific embodiments, the double-stranded heat destabilizing modification is present at the 6th position counted from the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0035] In some specific embodiments, the double-stranded heat destabilizing modification is present at the 7th position counted from the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0036] In some specific embodiments, the double-stranded thermal destabilizing modification is present at the 8th position counted from the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this part, the dsRNA molecule further comprises at least one thermal destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0037] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, where the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the antisense strand comprises at least one double-stranded thermal destabilizing modification within the seed region (i.e., positions 2 to 9 counted from the 5' end of the 5' end of the antisense strand), where the dsRNA has a melting temperature of about 40 °C to about 80 °C, and 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 further comprises one or both of; 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 comprises one, two or three of, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA(ModA-ModK). In some of these 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 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counted from the 5' end, and the ligand is conjugated to the sense strand, the dsRNA has a melting temperature of about 40°C to about 80°C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA(ModA-ModK).

[0039] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 thermolabile modification of the duplex within the first 9 nucleotide positions counted from the 5' end, the ligand is conjugated to the sense strand, the dsRNA comprises at least 4 2'-fluoro modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). 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 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the dsRNA comprises at least 4 2'-fluoro, wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counted from the 5' end, the sense strand comprises a ligand, the dsRNA has a melting temperature of about 40°C to about 80°C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand. In some embodiments, the T 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 the target sequence to mediate RNA interference, the antisense strand comprising at least one heat destabilizing modification of the duplex located at positions 4 to 8 counted from the 5' end, the sense strand comprising a ligand, and each of the sense and antisense strands comprising at least two 2'-fluoro modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand. 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 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 heat destabilizing modification of the duplex within the first nine nucleotide positions counted from the 5' end, the sense strand comprises a ligand, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the antisense has the following characteristics: (i) the heat destabilizing modification of the duplex is located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro modifications; (iii) a phosphorothioate internucleotide linkage between positions 1 and 2 (counted from the 5' end) of the nucleotides; the antisense strand further comprises at least two of having a length of 18 to 35 nucleotides, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). 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.

[0043] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 said antisense strand comprises at least one heat destabilizing modification of the duplex within the first nine 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 the sense strand has 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 duplex region spanning at least 19 nucleotide positions, wherein the heat destabilizing modification of the duplex has at least one located within said duplex region, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0044] 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 heat destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, the sense strand comprises a ligand, the dsRNA has a melting temperature of about 40°C to about 80°C, and the heat destabilizing modification of the duplex is selected from the group consisting of GNA-isoC, GNA-isoG, 5'm-UNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T m is optional.

[0045] 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, the antisense strand comprising at least one thermolabile modification of the duplex located at positions 4 to 8 counting from the 5' end, the sense strand comprising a ligand, and each of the sense and antisense strands comprising at least two 2'-fluoro modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the Tm of about 40°C to about 80°C is optional.

[0046] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 heat destabilizing modification of the duplex located at the 7th position counting from the 5' end of the antisense strand, wherein the sense strand comprises a ligand, the dsRNA has a melting temperature of from about 40 °C to about 80 °C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the T of about 40 °C to about 80 °C m is optional.

[0047] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, said antisense strand comprising at least one thermolabile modification at the 7th position counted from the 5’ end of the duplex, said sense strand comprising a ligand, and each of the sense and antisense strands comprising at least two 2’-fluoro modifications, wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK). In some embodiments, the T m is optional.

[0048] 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 4 2'-fluoro, and the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and the sense strand comprises a ligand, wherein the dsRNA optionally has a melting temperature of about 40°C to about 80°C, wherein the ligand comprises one or more GalNAc derivatives linked through a divalent or trivalent branched linker, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0049] 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 4 2'-fluoro, and the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, and the sense strand comprises a ligand, wherein the dsRNA optionally has a melting temperature of about 40°C to about 80°C, wherein the ligand has the structure: [Chemical formula] is an ASGPR ligand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand); where 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; where the antisense strand comprises 3, 4, 5, or 6 2'-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region that is 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0051] 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 thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5’ end of the antisense strand); where the sense strand is conjugated to a ligand and comprises 2’-fluoro modifications at positions 7, 10, and 11 or positions 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; where the antisense strand comprises 3, 4, 5, or 6 2’-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3’ end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0052] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing 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); where 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; where the antisense strand comprises 2'-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0053] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand); where 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; where the antisense strand comprises 2'-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense 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; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA comprises a double-stranded region that is 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0054] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 from the 5’ end of the antisense strand); where the sense strand is conjugated to a ligand and comprises 2’-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5’ end of the sense strand), and optionally, phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand comprises 2’-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA comprises a double-stranded region that is 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3’ end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0055] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, where the antisense strand comprises at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, and where 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, 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 4 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; and (viii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 in total) of blunt ends at the 5' end of the sense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some specific 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 of about 40°C to about 80°C m is optional.

[0056] 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 thermally labile nucleotide, where at least one thermally labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2-9 from the 5’ end of the antisense strand); where the sense strand is conjugated to a ligand and comprises 2’-fluoro modifications at positions 7, 10, and 11 or positions 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; where the antisense strand comprises 2’-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense 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; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA comprises a double-stranded region that is 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3’ end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0057] In some embodiments, one end of the dsRNA is blunt-ended and the other end has an overhang, where the antisense strand contains at least one thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., at positions 2 to 9 from the 5' end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has 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 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 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of a double-stranded region 12 to 40 nucleotide pairs in length, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the overhang is present on the 3' end of the antisense strand and the blunt end is present at the 5' end of the antisense strand. In some particular embodiments, the overhang is 2, 3, or 4 nucleotides in length. In some embodiments, the T of about 40°C to about 80°C m is optional.

[0058] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, where one end of the dsRNA is blunt-ended and the other end has an overhang, where the antisense strand has at least one thermally destabilizing 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), where 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 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 further has at least one (e.g., 1, 2, 3, 5, or all 6) of at least 4 2'-fluoro modifications, optionally the 2-nucleotide overhang is present on the 3' end of the antisense strand and the blunt end is present on the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the overhang is present on the 3' end of the antisense strand and the blunt end is present on the 5' end of the antisense strand. In some embodiments, the T m is optional.

[0059] In some embodiments, the dsRNA molecule of the present invention may also have two blunt ends at both ends of the dsRNA duplex.

[0060] In some embodiments, the dsRNA has blunt ends at both ends of the duplex, wherein the antisense strand comprises at least one thermally labile nucleotide, wherein at least one thermally labile nucleotide is 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, 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 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6 or 7 in total) of the following: a double-stranded region 12-40 nucleotide pairs in length, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further embodiment, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0061] 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 double strand, where one end of the dsRNA is a blunt end and the other end has an overhang, where the antisense strand has at least one thermally destabilizing modification of the double strand located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), where 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 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 further has at least one (e.g., 1, 2, 3, 5, or all 6) of at least 4 2'-fluoro modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0062] In some embodiments, the dsRNA molecule of the present invention comprises a 21 nucleotide (nt) sense strand and a 23 nucleotide (nt) antisense strand, wherein the antisense strand comprises at least one thermally labile nucleotide, wherein at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand), wherein one end of the dsRNA is blunt-ended while the other end comprises a 2nt 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 comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioate nucleotide 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 nucleotide linkages; (vi) the dsRNA comprises at least 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6 or all 7) of including a blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). Preferably, the 2nt overhang is present at the 3' end of the antisense. In some embodiments, the T of about 40°C to about 80°C m is optional.

[0063] 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 starting from the 5'-terminal nucleotide (position 1) of the sense strand contain at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and at least 8 ribonucleotides within that position starting from the 3'-terminal nucleotide pair with positions 1 to 23 of the sense strand to form a double strand; wherein at least the 3'-terminal nucleotide of the antisense strand does not pair with the sense strand, and at most 6 consecutive 3'-terminal nucleotides do not pair with the sense strand, thereby forming a 3'-single-stranded overhang of 1 to 6 nucleotides; wherein the 5'-terminal of the antisense strand contains 10 to 30 consecutive nucleotides that do not pair with the sense strand, thereby forming a single-stranded 5'-overhang of 10 to 30 nucleotides; wherein at least the 5'-terminal and 3'-terminal nucleotides of the sense strand base pair with the 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; when the double-stranded nucleic acid is introduced into mammalian cells, 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; the antisense strand has at least one thermally destabilizing nucleotide, wherein at least one thermally destabilizing nucleotide is present within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-terminal of the antisense strand), the dsRNA has a melting temperature of about 40 °C to about 80 °C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA.In some further embodiments of this part, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). For example, the heat destabilizing nucleotides are present between positions that are opposite or complementary to positions 14 to 17 at the 5' end of the sense strand, where the dsRNA optionally has 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 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 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following: containing a double-stranded region 12 to 30 nucleotide pairs in length. In some embodiments, the T of about 40 °C to about 80 °C. m is optional.

[0064] In some embodiments, the 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, together with the sense strand, contains a modified nucleotide that is susceptible to enzymatic cleavage at position 11 from the 5'-end, wherein the 3'-end of the sense strand and the 5'-end of the antisense strand form blunt ends, and the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3'-end, wherein in the double-stranded region that is at least 25 nucleotides long, when the dsRNA molecule is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target mRNA along at least 19 nt of the antisense strand length to reduce the expression of the target gene, wherein the dicing of the dsRNA preferentially yields an siRNA comprising the 3'-end of the antisense strand, thereby reducing the expression of the target gene in mammals, wherein the antisense strand contains at least one thermally labile nucleotide, wherein at least one thermally labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-end of the antisense strand), wherein the dsRNA has a melting temperature of about 40 °C to about 80 °C, wherein the dsRNA optionally has 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 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 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6 or all 7) of having a double-stranded region 12 to 29 nucleotide pairs in length, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein.In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA(ModA-ModK). In some embodiments, the T of about 40°C to about 80°C. m is optional.

[0065] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23, wherein the antisense strand has at least one thermally destabilizing 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 the dsRNA optionally has the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense comprises 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 4 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12-40 nucleotide pairs in length; and (viii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 in total) of having blunt ends at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0066] In some embodiments, the antisense strand contains phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23, where the antisense strand has at least one thermally destabilizing 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), where 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 sense strand is conjugated to 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 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length; (vii) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length; and (viii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 all) of having blunt ends at the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0067] In some embodiments, the sense strand contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, where the antisense strand has at least one thermally destabilizing 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), where 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 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 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length; and (viii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 in total) of having blunt ends at the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the T of about 40°C to about 80°C m is optional.

[0068] 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, where the antisense strand has at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), where 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 sense strand is conjugated to a ligand; (iii) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (iv) the sense strand comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least 4 2'-fluoro modifications; (vi) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of having a blunt end at the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the T m is optional.

[0069] In one aspect, the present invention is a dsRNA molecule capable of inhibiting the 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 contains at least one thermolabile modification of the double strand within the seed region (i.e., at positions 2 to 9 of the 5' end counted from the 5' end of the antisense strand), and the dsRNA has 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 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 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or all 8) of the blunt ends at the 5' end of the antisense strand is further provided, a dsRNA molecule.

[0070] In some specific embodiments, the double-stranded heat destabilizing modification is present at the 7th position counted from the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0071] In some embodiments, the double-stranded heat destabilizing modification is present at the 2nd, 3rd, 4th, 5th, 6th, 8th, or 9th position counted from the 5' end of the antisense strand of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0072] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, where the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, the antisense strand comprises at least one double-stranded heat destabilizing modification within the seed region (i.e., at the 2nd to 9th positions of the 5' end counted from the 5' end of the antisense strand), and the antisense strand has the following characteristics: (i) 2, 3, 4, 5, or 6 2'-fluoro modifications; and (ii) one or both of 1, 2, 3, 4 or 5 phosphorothioate nucleotide linkages further comprises one or both of, 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, 4 or 5 phosphorothioate nucleotide linkages comprises 1, 2 or 3 of. Here, the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0073] 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, the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counted from the 5' end, and the ligand is conjugated to the sense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0074] 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, the antisense strand comprising at least one thermolabile modification of the duplex within the first 9 nucleotide positions counted from the 5' end, the ligand being conjugated to the sense strand, the dsRNA comprising at least 4 2'-fluoro modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0075] 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, and the antisense strand comprises at least one heat destabilizing 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 of this, the ligand is an ASGPR ligand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[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, wherein the antisense strand comprises at least one thermolabile modification of the duplex located at positions 4 to 8 counted 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 destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand.

[0077] 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, and the antisense strand comprises at least one heat destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, the sense strand comprises a ligand, and the antisense has the following characteristics: (i) the heat destabilizing modification of the duplex is located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro modifications; (iii) at least two phosphorothioate internucleotide linkages between nucleotides 1 and 2 (counting from the 5' end), and the antisense strand has a length of 18 to 35 nucleotides, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments, the ligand is an ASGPR ligand.

[0078] 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 heat destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and the sense strand has 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 duplex region spanning at least 19 nucleotide positions, wherein the heat destabilizing modification of the duplex is located within the duplex region, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0079] 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, and the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, the sense strand comprises a ligand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0080] 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 heat destabilizing modification of the duplex located at positions 4 to 8 counting from the 5' end, the sense strand comprises a ligand, wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0081] 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, and the antisense strand comprises at least one thermally destabilizing modification of the duplex located at position 7 counted from the 5' end of the antisense strand, wherein the sense strand comprises a ligand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0082] 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 thermolabile modification at position 7 counted from the 5' end of the duplex, the sense strand comprises a ligand, wherein each of the sense and antisense strands comprises at least two 2'-fluoro modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0083] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 4 2'-fluoro, wherein the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, wherein the ligand comprises one or more GalNAc derivatives linked through a divalent or trivalent branched linker, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0084] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 4 2'-fluoro, wherein the antisense strand comprises at least one heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, wherein the ligand has the structure:

Chemical formula

[0085] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand); where the sense strand is conjugated to a ligand, contains 3 or 4 2'-fluoro modifications, and contains 0, 1, 2, or 3 phosphorothioate internucleotide linkages; where the antisense strand contains 3, 4, 5, or 6 2'-fluoro modifications and contains 2, 3, 4, or 5 phosphorothioate internucleotide linkages; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0086] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 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 contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 3, 4, 5 or 6 2'-fluoro modifications and contains 2, 3, 4 or 5 phosphorothioate internucleotide linkages; where the dsRNA optionally has the following characteristics: (i) the dsRNA contains a double-stranded region 12-25 nucleotides in length; (ii) the dsRNA contains a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA further has at least one (e.g., 1, 2 or all 3) of having at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0087] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where the sense strand is conjugated to a ligand, contains 3 or 4 2'-fluoro modifications, and contains 0, 1, 2, or 3 phosphorothioate internucleotide linkages; where the antisense strand contains 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; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following features: (i) the dsRNA contains a double-stranded region that is 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0088] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where 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; where the antisense strand contains 2'-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0089] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2 - 9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following features: (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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0090] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 6, 8, 9, 14, or 16, or positions 2, 6, 14, or 16, or positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of a at least 2 nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0091] In certain embodiments, the dsRNA molecule of the invention is (a) a sense strand, where (i) 21 nucleotides in length; (ii) An ASGPR ligand attached to the 3'-end, comprising three GalNAc derivatives linked via a trivalent linker; and (iii) 2'-F modifications at positions 7, 10, and 11 (counting from the 5'-end) of the sense strand, and (b) An antisense strand, comprising (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 6 - 8, 9, 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 double-stranded thermolabile modification at position 7 (counting from the 5'-end) of the antisense strand, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0092] In another specific embodiment, the dsRNA molecule of the present invention comprises (a) A sense strand, comprising (i) 21 nucleotides in length; (ii) An ASGPR ligand attached to the 3'-end, comprising three GalNAc derivatives linked via a trivalent linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5'-end); and (iv) Phosphorothioate nucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 6, 14, and 16 (counting from the 5' end); (iii) Phosphorothioate nucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end); and (iv) A double-stranded thermal destabilizing modification at position 7 (counting from the 5' end) comprising an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermal destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0093] In another specific embodiment, the dsRNA molecule of the present invention (a) is a sense strand, (i) 21 nucleotides in length; (ii) An ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) Phosphorothioate nucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5'-end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5'-end); (iii) Phosphorothioate nucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5'-end); and (iv) A double-stranded heat destabilizing modification at position 6 or 7 (counting from the 5'-end) comprising an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0094] In another specific embodiment, the dsRNA molecule of the present invention (a) is a sense strand, (i) 21 nucleotides in length; (ii) An ASGPR ligand attached to the 3'-end, comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5'-end); and (iv) Phosphorothioate nucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5'-end) and a sense strand having (b) an antisense strand, wherein (i) is 23 nucleotides in length; (ii) has 2'-F modifications at positions 2, 6, 8, 9, 14 and 16 (counting from the 5'-end); (iii) has phosphorothioate nucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22 and between nucleotides 22 and 23 (counting from the 5'-end); and (iv) has a duplex thermal destabilizing modification at position 7 (counting from the 5'-end) and an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermal destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0095] In another specific embodiment, the dsRNA molecule of the present invention (i) has 2'-F modifications at positions 2, 14 and 16 (counting from the 5'-end); and (ii) has a duplex thermal destabilizing modification at position 6 or 7 (counting from the 5'-end) comprising an antisense strand having, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0096] In another specific embodiment, the dsRNA molecule of the present invention (a) a sense strand, (i) an ASGPR ligand comprising three GalNAc derivatives linked via a trivalent linker; (ii) phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5'-end) having a sense strand, and (b) an antisense strand, (i) 2'-F modifications at positions 2, 14, and 16 (counting from the 5'-end); (ii) a double-stranded heat destabilizing modification at position 6 or 7 (counting from the 5'-end) having an antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0097] In another specific embodiment, the dsRNA molecule of the present invention is (a) a sense strand comprising (i) an ASGPR ligand attached to the 3'-end, comprising three GalNAc derivatives linked via a trivalent branched linker; (ii) phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5'-end); and a sense strand having (b) an antisense strand comprising (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5'-end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5'-end); and (iv) a double-stranded thermally destabilizing modification at position 6 or 7 (counting from the 5'-end) and an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0098] 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 divalent or trivalent branched linker, such as

Chemical formula

[0099] In one example, the ASGPR ligand is attached to the 3'-end of the sense strand.

[0100] In some cases, 2'-fluoro modifications in the seed region of the antisense strand, such as positions 2 to 9, particularly positions 3 to 9, may have a minimal effect on the in vitro potency of the dsRNA while adversely affecting the in vivo activity of the dsRNA. The inventors have found that, in particular, the in vivo activity of such dsRNA can be restored to a level comparable to that of the parental dsRNA by removing some or all of the 2'-fluoro modifications from the seed region of the antisense strand, i.e., positions 2 to 9, particularly positions 3 to 9, counted from the 5'-end.

[0101] Accordingly, in some embodiments, the present invention provides a dsRNA molecule capable of inhibiting the 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 heat destabilizing modification of the duplex within the seed region (i.e., positions 2 to 9, counted from the 5'-end of the 5'-end of the antisense strand), 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 nucleotide 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 nucleotide internucleotide linkages; (vii) the dsRNA contains at least 4 2'-fluoro modifications; (viii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (ix) blunt ends at the 5'-end of the antisense strand; (x) the sense strand further has at least one (e.g., all of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein, to provide a dsRNA molecule. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0102] In some embodiments, the present invention is a dsRNA molecule capable of inhibiting the 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 contains at least one heat destabilizing modification of the double strand within the seed region (i.e., positions 2 to 9 counted from the 5'-end at the 5'-end of the antisense strand), 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 4 2'-fluoro modifications; (viii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (ix) blunt ends at the 5'-end of the antisense strand; and (x) the sense strand further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 all) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and the 2'-fluoro modifications are not present at positions 3 to 9 (counting from the 5'-end) of the antisense strand, to provide a dsRNA molecule, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[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, and the antisense strand comprises at least one thermolabile modification of the duplex within the seed region (i.e., at positions 2 to 9 counting from the 5' end 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 antisense strand has the following characteristics: (i) 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro modifications (wherein the antisense strand does not have 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end)); and (ii) further comprises one or both of 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; the sense strand has the following characteristics: (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) comprises 1, 2 or 3 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the 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 from 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 thermally destabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 counting from the 5' end 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 antisense strand further comprises (i) 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro modifications; and (ii) 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages, and the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, wherein the sense strand optionally has 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 or 3 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the 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 from 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 thermally labile modification of the duplex within the seed region (i.e., positions 2 to 9 counting from the 5' end 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 antisense strand further comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages, wherein the antisense strand optionally comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro modifications; the sense strand comprises 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, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the 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 the target sequence to mediate RNA interference, and the antisense strand comprises at least one thermolabile modification of the duplex within the seed region (i.e., positions 2 to 9 counting from the 5' end 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 antisense strand further comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages, wherein the antisense strand optionally comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro modifications, provided that there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end); the sense strand comprises 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, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the 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, and the antisense strand comprises at least one thermolabile modification of the duplex within the first 9 nucleotide positions counted from the 5' end, the ligand is conjugated to the sense strand, the dsRNA comprises at least 4 2'-fluoro modifications and there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counted from the 5' end), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0108] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 said antisense strand comprises at least one heat destabilizing modification of the duplex within the first nine 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 there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand. In some embodiments, the 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 from 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 heat destabilizing modification of the duplex located at positions 4 to 8 counting 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 from about 40°C to about 80°C and there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand. In some embodiments, the melting temperature of from about 40°C to about 80°C is optional.

[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 heat destabilizing modification of the duplex within the first nine 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 antisense has the following characteristics: (i) the heat destabilizing modification of the duplex is located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro modifications; (iii) a phosphorothioate internucleotide bond between nucleotides 1 and 2 (counting from the 5' end); and (iv) the antisense strand further comprises at least two of having a length of 18 to 35 nucleotides and the absence of 2'-fluoro modifications at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments, the ligand is an ASGPR ligand. In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0111] 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, 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 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; (iii) the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications; and (iv) the sense strand and the antisense strand have at least one of sufficient complementarity to form a duplex region spanning at least 19 nucleotide positions, wherein the thermally destabilizing modification of the duplex is located within the duplex region and there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0112] 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 thermolabile modification of the duplex located at positions 4 to 8 counted 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 there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counted from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0113] 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 located at position 5, 6 or 7 counting from the 5' end of the antisense strand, wherein there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counting from the 5' end), wherein the sense strand comprises a ligand, optionally at least one LNA modification, wherein the dsRNA has a melting temperature of about 40 °C to about 80 °C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0114] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 thermolabile modification of the duplex located at positions 5, 6 or 7 counted 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 there is no 2'-fluoro modification at positions 3 to 9 of the antisense strand (counted from the 5' end), wherein the dsRNA has a melting temperature of from about 40°C to about 80°C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of from about 40°C to about 80°C is optional.

[0115] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 4 2'-fluoro, wherein the antisense strand comprises at least one heat destabilizing 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 there is no 2'-fluoro modification at positions 3-9 of the antisense strand (counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, wherein the ligand comprises one or more GalNAc derivatives linked through a divalent or trivalent branched linker, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0116] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 4 2'-fluoro, wherein the antisense strand comprises at least one heat destabilizing 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 there is no 2'-fluoro modification at positions 3-9 of the antisense strand (counting from the 5' end), wherein the dsRNA has a melting temperature of about 40°C to about 80°C, wherein the ligand has the structure:

Chem.

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and optionally includes at least one LNA modification, includes 3 or 4 2'-fluoro modifications, and includes 0, 1, 2, or 3 phosphorothioate internucleotide linkages; where the antisense strand includes 3, 4, 5, or 6 2'-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages, provided that there is no 2'-modification at positions 3-9 of the antisense strand (counting from the 5' end); where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA includes a double-stranded region that is 12-25 nucleotide pairs in length; (ii) the dsRNA includes a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA further has at least one (e.g., 1, 2, or all 3) of a at least 2 nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further includes at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally contains at least one LNA modification; where the antisense strand contains 3, 4, 5, or 6 2'-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages, provided that there are no 2'-modifications at positions 3-9 of the antisense strand (counting from the 5' end); where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the 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; the antisense strand has at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and includes 3 or 4 2'-fluoro modifications, 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and optionally at least one LNA modification; where the antisense strand includes a 2'-fluoro modification at positions 2, 14, or 16; the antisense includes phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA includes a double-stranded region 12-25 nucleotide pairs in length; (ii) the dsRNA includes a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further includes at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the 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; the antisense strand has at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5’ end of the antisense strand); where the sense strand is conjugated to a ligand and contains 3 or 4 2’-fluoro modifications, at least one LNA modification, and optionally 0, 1, 2, or 3 phosphorothioate internucleotide linkages; where the antisense strand contains a 2’-fluoro modification at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has one or more of the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 2-nucleotide overhang at the 3’ end of the antisense strand (e.g., 1, 2, or all 3), where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK). In some embodiments, the 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 thermally labile nucleotide, where at least one thermally 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); where the sense strand is conjugated to a ligand and includes 3 or 4 2'-fluoro modifications, 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and optionally, at least one LNA modification; where the antisense strand includes a 2'-fluoro modification at positions 2, 14, or 16; the antisense includes phosphorothioate internucleotide linkages between nucleotides 21 and 22, nucleotides 22 and 23, nucleotides 1 and 2, and nucleotides 2 and 3; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA includes a double-stranded region that is 12 - 25 nucleotide pairs in length; (ii) the dsRNA includes a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further includes at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2 - 9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and includes 3 or 4 2'-fluoro modifications, 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and at least one LNA modification; where the antisense strand includes a 2'-fluoro modification at positions 2, 14, or 16; the antisense includes phosphorothioate internucleotide linkages between nucleotides 21 and 22, nucleotides 22 and 23, nucleotides 1 and 2, and nucleotides 2 and 3; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA includes a double-stranded region 12 - 25 nucleotide pairs in length; (ii) the dsRNA includes a blunt end at the 5' end of the antisense strand; and (iii) the dsRNA further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further includes at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0123] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or at positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally containing phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally containing at least one LNA modification; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of at least 2 nucleotide overhangs at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0124] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2 - 9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), contains at least one LNA modification, and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0125] In some embodiments, the sense and antisense strands are independently 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, where the antisense strand has at least one thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5’ end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense 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 (counting from the 5’ end); (ii) the antisense 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 contains at least 4 2’-fluoro modifications; (vii) the dsRNA contains a double-stranded region 18, 19, 20, 21, 22, 23, 24, or 24 nucleotide pairs in length; (viii) the dsRNA contains a blunt end at the 5’ end of the sense strand; and (ix) the sense strand further has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK).In some specific 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.

[0126] In some embodiments, the sense and antisense strands are, independently, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, where the antisense strand has at least one thermally labile nucleotide and 1, 2, 3, or 4 phosphorothioate nucleotide linkages, where at least one thermally labile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2 - 9 from the 5' end of the antisense strand), the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense 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 (counting from the 5' end); (ii) the sense strand is conjugated to 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 nucleotide linkages; (v) the dsRNA contains at least 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region that is 18, 19, 20, 21, 22, 23, 24, or 24 nucleotide pairs in length; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of a blunt end at the 5' end of the sense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some particular 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 from about 40°C to about 80°C is optional.

[0127] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10 and 11 or positions 7, 9, 10 and 11 (counting from the 5' end of the sense strand), optionally contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 9, 14 or 16 or positions 2, 14 or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2 or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0128] 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 thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LAN modifications, and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 9, 14, or 16 or positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA has a melting temperature of about 40°C to about 80°C; where the dsRNA optionally has the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0129] In some embodiments, one end of the dsRNA is blunt-ended and the other end has an overhang, where the antisense strand has at least one thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., at positions 2 to 9 from the 5'-end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5'-end); (ii) the antisense 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 contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; and (vii) the sense strand further has at least one (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of LNA modifications, where the destabilizing modifications are selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modifications are selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the overhang is present on the 3'-end of the antisense strand and the blunt-end is present at the 5'-end of the antisense strand. In some particular embodiments, the overhang is 2, 3 or 4 nucleotides in length. In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0130] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, where one end of the dsRNA is blunt-ended and the other end has an overhang, where the antisense strand has at least one thermally destabilizing 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), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense 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, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least 4 2'-fluoro modifications; and (vii) the sense strand further has at least one (e.g., 1, 2, 3, 5, 6, or 7 in total) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, optionally present on the 3' end side of the 2-nucleotide overhang antisense strand and the blunt end is present at the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the overhang is present on the 3' end of the antisense strand and the blunt end is present at the 5' end of the antisense strand.In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0131] In some embodiments, the dsRNA molecule of the present invention may also have two blunt ends at both ends of the dsRNA duplex.

[0132] In some embodiments, the dsRNA has blunt ends at both ends of the double strand, where the antisense strand has at least one thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, where there is no 2'-fluoro modification at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense 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 contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length; and (viii) the sense strand further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 in total) of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0133] 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 double strand, where one end of the dsRNA is a blunt end and the other end has an overhang, where the antisense strand has at least one thermally destabilizing modification of the double strand located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense 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 contains at least 4 2'-fluoro modifications; and (vii) the sense strand further has at least one (e.g., 1, 2, 3, 5, 6, or 7 in total) of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0134] In some embodiments, the dsRNA molecule of the present invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand has at least one thermally labile nucleotide, and wherein at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2 to 9 from the 5' end of the antisense strand), and wherein one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and 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 comprises 2, 3, 4, 5 or 6 2'-fluoro modifications, provided that there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end of the antisense strand); (ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioate nucleotide 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 nucleotide linkages; (vi) the dsRNA comprises at least 4 2'-fluoro modifications; (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand, preferably the 2-nt overhang is present at the 3' end of the antisense; and (viii) the sense strand further has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) LNA modifications, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further embodiment, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0135] In some embodiments, in the dsRNA molecule of the invention comprising a sense and an antisense strand, the sense strand is 25 to 30 nucleotide residues in length, wherein positions 1 to 23 starting from the 5'-terminal nucleotide (position 1) of the sense strand contain at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and at least 8 ribonucleotides within its position starting from the 3'-terminal nucleotide pair with positions 1 to 23 of the sense strand to form a double strand; wherein at least the 3'-terminal nucleotide of the antisense strand does not pair with the sense strand, and at most 6 consecutive 3'-terminal nucleotides do not pair with the sense strand, thereby forming a 3'-single-stranded overhang of 1 to 6 nucleotides; wherein the 5'-terminal of the antisense strand contains 10 to 30 consecutive nucleotides that do not pair with the sense strand, thereby forming a single-stranded 5'-overhang of 10 to 30 nucleotides; wherein at least the 5'-terminal and 3'-terminal nucleotides of the sense strand base pair with the 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; when the double-stranded nucleic acid is introduced into mammalian cells, 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; the antisense strand has at least one thermally destabilizing nucleotide, wherein at least one thermally destabilizing nucleotide is present within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-terminal of the antisense strand), and the dsRNA has a melting temperature of about 40°C to about 80°C, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA.In some further embodiments of this part, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0136] For example, the heat destabilizing nucleotides are present between positions that are opposite or complementary to positions 14 to 17 at the 5' end of the sense strand, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end of the antisense strand); (ii) the antisense 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 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 30 nucleotide pairs in length; and the sense strand further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or all 1, 2, 3, 4, 5, 6, 7, or 8) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this part, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0137] In some embodiments, the 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 that is susceptible to enzymatic cleavage at the 11th position from the 5'-end, wherein the 3'-end of the sense strand and the 5'-end of the antisense strand form blunt ends, and the antisense strand is 1 to 4 nucleotides longer than the sense strand at its 3'-end, wherein in the double-stranded region that is at least 25 nucleotides in length, when the dsRNA molecule is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target mRNA along at least 19 nt of the antisense strand length, thereby reducing the expression of the target gene, wherein the dicing of the dsRNA preferentially yields an siRNA comprising the 3'-end of the antisense strand, thereby reducing the expression of the target gene in mammals, wherein the antisense strand has at least one thermolabile nucleotide, wherein at least one thermolabile nucleotide is present within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-end of the antisense strand), wherein the dsRNA has a melting temperature of about 40 °C to about 80 °C, wherein the dsRNA optionally has the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications, wherein there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5'-end of the antisense strand); (ii) the antisense comprises 1, 2, 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; and (vi) the dsRNA comprises at least 4 2'-fluoro modifications; (vii) the dsRNA has a double-stranded region with a length of 12 to 29 nucleotide pairs;and (viii) the sense strand further has at least one of including 1, 2, 3, 4, 5, 7, 8, 9 or 10 LNA modifications (for example, all of 1, 2, 3, 4, 5, 6, 7 or 8), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.;

[0138] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, where the antisense strand has at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5' end of the antisense strand), where the dsRNA has a melting temperature of from about 40 °C to about 80 °C, where the dsRNA optionally has the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end of the antisense strand); (ii) the antisense comprises 3 or 4 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 4 2'-fluoro modifications; (vii) the dsRNA comprises 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 has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10) of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of from about 40 °C to about 80 °C is optional.

[0139] In some embodiments, the antisense strand contains phosphorothioate internucleotide linkages between nucleotides 1 and 2, nucleotides 2 and 3, nucleotides 21 and 22, and nucleotides 22 and 23, where the antisense strand has at least one thermally destabilizing 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), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the sense strand is conjugated to 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 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length; (vii) the dsRNA contains a double-stranded region 12-40 nucleotide pairs in length; (viii) the dsRNA has blunt ends at the 5' end of the antisense strand; and (ix) the sense strand further has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0140] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, wherein the antisense strand has at least one thermolabile 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, wherein the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, wherein there are no 2'-fluoro modifications at positions 3-9 (counting from the 5' end of the antisense strand); (ii) the antisense 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 contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12-40 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 has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) LNA modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0141] In some embodiments, the sense strand contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and the antisense strand contains phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23, where the antisense strand has at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., positions 2 to 9 from the 5' end of the antisense strand), where the dsRNA has a melting temperature of about 40°C to about 80°C, where the dsRNA optionally has the following characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end of the antisense strand); (iv) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA contains at least 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (vii) the dsRNA has blunt ends at the 5' end of the antisense strand; and (viii) the sense strand further has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) LNA modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further embodiment, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0142] In one aspect, the present invention is a dsRNA molecule capable of inhibiting the 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 contains at least one heat destabilizing modification of the duplex within the seed region (i.e., at positions 2 to 9 counting from the 5' end of the 5' end of the antisense strand), and the dsRNA molecule has the following characteristics: (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end); (ii) the antisense 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 contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs in length; (viii) blunt ends at the 5' end of the antisense strand; and (ix) the sense strand further has at least one (e.g., all 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) LNA modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some embodiments, a melting temperature of about 40°C to about 80°C is optional.

[0143] In some particular embodiments, the double-stranded heat destabilizing modification is present at the 5th, 6th or 7th position counted from the 5'-end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA(ModA-ModK).

[0144] In some embodiments, the double-stranded heat destabilizing modification is present at the 2nd, 3rd, 4th, 8th or 9th position counted from the 5'-end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA(ModA-ModK).

[0145] 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 thermolabile modification of the duplex within the seed region (i.e., at positions 2 to 9 counting from the 5' end of the antisense strand), and the antisense strand has the following characteristics: (i) 2, 3, 4, 5 or 6 2'-fluoro modifications (no 2'-modification at positions 3 to 9 counting from the 5' end of the antisense strand); and (ii) further comprises one or both of 1, 2, 3, 4 or 5 phosphorothioate nucleotide linkages; the sense strand has the following characteristics: (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 nucleotide linkages; and (iv) comprises 1, 2, 3 or 4 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0146] 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, the antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counted from the 5' end, the ligand being conjugated to the sense strand, the dsRNA comprising at least 4 2'-fluoro modifications and having no 2'-modifications at positions 3 to 9 of the antisense strand (counted from the 5' end of the antisense strand), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0147] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 heat destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand and there is no 2'-modification at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand.

[0148] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 thermolabile modification of the duplex located at positions 4 to 8 counted 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 there is no 2'-modification at positions 3 to 9 of the antisense strand (counted from the 5' end of the antisense strand), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments of this, the ligand is an ASGPR ligand.

[0149] 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 said antisense strand comprises at least one heat destabilizing modification of the duplex within the first nine nucleotide positions counted from the 5' end, wherein said sense strand comprises a ligand, wherein the antisense has the following characteristics: (i) the heat destabilizing modification of the duplex is located at positions 4 to 8 of the antisense strand; (ii) at least two 2'-fluoro modifications (wherein there are no 2'-modifications at positions 3 to 9 of the antisense strand (counted from the 5' end of the antisense strand)); (iii) further comprising at least two phosphorothioate internucleotide linkages between nucleotides 1 and 2 counted from the 5' end; and the antisense strand has a length of 18 to 35 nucleotides, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some further embodiments, the ligand is an ASGPR ligand.

[0150] 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 thermolabile modification of the duplex within the first nine nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, and the sense strand has 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; (iii) the sense strand and the antisense strand exhibit sufficient complementarity to form a duplex region spanning at least 19 nucleotide positions; (iv) the sense strand has at least one of comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, the thermolabile modification of the duplex is located within the duplex region and there is no 2'-modification at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0151] 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 there is no 2'-modification at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the antisense strand comprises at least one heat destabilizing modification of the duplex within the first nine nucleotide positions counting from the 5' end, wherein the sense strand comprises a ligand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0152] 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 thermolabile modification of the duplex located at positions 4 to 8 counted 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 there is no 2'-modification at positions 3 to 9 of the antisense strand (counted from the 5' end of the antisense strand), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0153] 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 there is no 2'-modification at positions 3 to 9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the antisense strand comprises at least one heat destabilizing modification of the duplex located at positions 5, 6 or 7 counting from the 5' end of the antisense strand, wherein the sense strand comprises a ligand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0154] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 thermolabile 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, there is no 2'-modification at positions 3-9 of the antisense strand (counting from the 5' end of the antisense strand), wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermolabile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0155] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 4 2'-fluoro, wherein there is no 2'-modification 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 heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5'-end, wherein the sense strand comprises a ligand, wherein the ligand comprises one or more GalNAc derivatives linked through a divalent or trivalent branched linker, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0156] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand, each strand having from 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 4 2'-fluoro, wherein there is no 2'-modification 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 heat destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5'-end, wherein the sense strand comprises a ligand, wherein the ligand has the structure:

Chemical formula

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where 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; where the antisense strand contains 3, 4, 5, or 6 2'-fluoro modifications and 2, 3, 4, or 5 phosphorothioate internucleotide linkages; where the dsRNA optionally has the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally containing phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; where the antisense strand contains 3, 4, 5, or 6 2'-fluoro modifications, where there are no 2'-fluoro modifications at positions 3-9 of the antisense strand, and contains 2, 3, or 4 phosphorothioate internucleotide linkages; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of having at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where 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; where the antisense strand contains 2'-fluoro modifications at positions 2, 14 or 16 or at positions 2, 14 and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2 or all 3) 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[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 thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand); where 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 optionally 0 or 2 phosphorothioate internucleotide linkages; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, or 16 or at positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where 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; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, or 16 or at positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following features: (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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5’ end of the antisense strand); where the sense strand is conjugated to a ligand and contains 3 or 4 2’-fluoro modifications, 0, 1, 2, or 3 phosphorothioate internucleotide linkages, and optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; where the antisense strand contains 2’-fluoro modifications at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 2-nucleotide overhang at the 3’ end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK).

[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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand); where 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 optionally, 0, 1, 2, or 3 phosphorothioate internucleotide linkages; where the antisense strand contains a 2'-fluoro modification at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, nucleotides 22 and 23, nucleotides 1 and 2, and nucleotides 2 and 3; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of a at least 2 nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0164] 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 thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and includes 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; where the antisense strand includes a 2'-fluoro modification at position 2, 14, or 16; the antisense includes phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) of the following features: (i) the dsRNA includes a double-stranded region that is 12-25 nucleotide pairs in length; (ii) the dsRNA includes 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, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further includes at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0165] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally containing phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of at least 2 nucleotide overhangs at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0166] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2 - 9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 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 contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications; where the antisense strand contains 2'-fluoro modifications at positions 2, 14 and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2 or all 3) of having at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 - 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0167] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2, or all 3) 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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0168] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10 and 11 or 7, 9, 10 and 11 (counting from the 5' end of the sense strand), contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA modifications, and contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2'-fluoro modifications at positions 2, 14 and 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23; where the dsRNA optionally has at least one (e.g., 1, 2 or all 3) of the following features: (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 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further subset of embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0169] 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 thermally labile nucleotide, where at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), optionally containing phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0170] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2'-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5' end of the sense strand), contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and optionally contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications; where the antisense strand contains 2'-fluoro modifications at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 further has at least one (e.g., 1, 2, or all 3) of at least a 2-nucleotide overhang at the 3' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0171] 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 thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is within the seed region of the antisense strand (i.e., positions 2-9 from the 5’ end of the antisense strand); where the sense strand is conjugated to a ligand and contains 2’-fluoro modifications at positions 7, 10, and 11 or positions 7, 9, 10, and 11 (counting from the 5’ end of the sense strand), optionally contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA modifications, and optionally contains phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3; where the antisense strand contains 2’-fluoro modifications at positions 2, 14, or 16; the antisense contains phosphorothioate internucleotide linkages between nucleotides 21 and 22, between nucleotides 22 and 23, between nucleotides 1 and 2, and between nucleotides 2 and 3; where the dsRNA optionally has the following characteristics: (i) the dsRNA contains a double-stranded region that is 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 further has at least one (e.g., 1, 2, or all 3) of a at least 2 nucleotide overhang at the 3’ end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In this further subset of embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK).

[0172] 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 thermally destabilizing modification of the duplex within the seed region (i.e., positions 2 to 9 counting from the 5’ end of the 5’ end of the antisense strand), the dsRNA has a melting temperature (T m ) 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, 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 4 2’-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; (viii) blunt ends at the 5’ end of the antisense strand; (ix) provided that there is at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or all 9) absence of 2’-fluoro modifications at positions 3 to 9 (counting from the 5’ end) of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA and 2’-mUNA. In this further some embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h’GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0173] In some embodiments, the dsRNA molecule has a double-stranded region that is 12 to 40 nucleotide pairs in length, where the antisense strand contains at least one thermally destabilizing modification within the seed region (i.e., at positions 2 to 9 from 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 m and where the dsRNA optionally has 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 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 4 2'-fluoro modifications; and (vii) a blunt end at the 5' end of the antisense strand, provided that there are no 2'-fluoro modifications at positions 3 to 9 (counting from the 5' end) of the antisense strand, having at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of these, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further subset of embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of about 40°C to about 80°C is optional.

[0174] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, where the antisense strand has at least one thermally destabilizing modification of the double strand located within the seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), and the dsRNA has a melting temperature of from about 40°C to about 80°C, provided that there is no 2'-fluoro modification at positions 3 to 9 (counting from the 5' end) of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the melting temperature of from about 40°C to about 80°C is optional.

[0175] In certain embodiments, the dsRNA molecule of the invention (a) a sense strand comprising (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end comprising three GalNAc derivatives attached 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) an antisense strand comprising (i) 23 nucleotides in length; (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 thermally destabilizing modification of the double strand at positions 5, 6, or 7 (counting from the 5' end) comprising an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0176] In another specific embodiment, the dsRNA molecule of the present invention is (a) a sense strand comprising (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) having a sense strand and (b) an antisense strand comprising (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5' end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end); and (iv) a double-stranded heat destabilizing modification at positions 5, 6, or 7 (counting from the 5' end) comprising an antisense strand, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0177] In another specific embodiment, the dsRNA molecule of the present invention (a) a sense strand, wherein (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives linked via a trivalent linker; (iii) 2'-F modifications at positions 7, 9, 10, and 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) having a sense strand, and (b) an antisense strand, wherein (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5' end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end); and (iv) a double-stranded heat destabilizing modification at positions 5, 6, or 7 (counting from the 5' end) comprising an antisense strand, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0178] In another specific embodiment, the dsRNA molecule of the present invention is (a) a sense strand, wherein (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives attached 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 having a sense strand, and (b) an antisense strand, wherein (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5' end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, nucleotides 2 and 3, nucleotides 21 and 22, and nucleotides 22 and 23 (counting from the 5' end); and (iv) a double-stranded thermal destabilizing modification at positions 5, 6, or 7 (counting from the 5' end) comprising an antisense strand, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0179] In another specific embodiment, the dsRNA molecule of the present invention is (a) a sense strand comprising (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives attached 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, or 3) LNA modification at positions 1, 2, and 3 (counting from the 5' end) having a sense strand, and (b) an antisense strand comprising (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5' end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, nucleotides 2 and 3, nucleotides 21 and 22, and nucleotides 22 and 23 (counting from the 5' end); and (iv) a duplex thermal destabilizing modification at positions 5, 6, or 7 (counting from the 5' end) comprising an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0180] In another specific embodiment, the dsRNA molecule of the present invention is (a) a sense strand comprising (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3'-end, comprising three GalNAc derivatives attached 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 nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5'-end) having a sense strand and (b) an antisense strand comprising (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5'-end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5'-end); and (iv) a double-stranded heat destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end) comprising an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0181] In another specific embodiment, the dsRNA molecule of the present invention is (a) a sense strand comprising (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3'-end, comprising three GalNAc derivatives attached 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, or 3) LNA modification at positions 1, 2, and 3 (counting from the 5'-end) having a sense strand, and (b) an antisense strand comprising (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5'-end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, nucleotides 2 and 3, nucleotides 21 and 22, and nucleotides 22 and 23 (counting from the 5'-end); and (iv) a duplex heat destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end) comprising an antisense strand having, wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0182] In another specific embodiment, the dsRNA molecule of the present invention (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives attached 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, or 3) LNA modification at positions 1, 2, and 3 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 having a sense strand, (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-F modifications at positions 2, 14, and 16 (counting from the 5' end); (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23; and (iv) a double-stranded heat destabilizing modification at positions 5, 6, or 7 (counting from the 5' end) comprising an antisense strand having , wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand, and wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0183] In some embodiments, the sense and antisense strands are, independently, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, where the antisense strand has at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is 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 characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense 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 contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region that is 18, 19, 20, 21, 22, 23, 24, or 24 nucleotide pairs in length; and (viii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 in total) of a blunt end at the 5' end of the sense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some particular 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.

[0184] The sense strand and the antisense strand typically form double-stranded dsRNA. The double-stranded region of the dsRNA molecule can be 12 to 40 nucleotide pairs in length. For example, the double-stranded region can be 14 to 40 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 25 to 35 nucleotides in length, 27 to 35 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 nucleotide pairs in length.

[0185] In some embodiments, the dsRNA molecule of the invention has a double-stranded region that is 12 to 40 nucleotide pairs in length, wherein the antisense strand has at least one thermally labile nucleotide, wherein at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., positions 2 to 9 from the 5' end of the antisense strand), 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 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 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6 or all 7) of a blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). In some particular embodiments, the double-stranded region is 18, 19, 20, 21, 22 or 23 nucleotide pairs in length. In a particular embodiment, the double-stranded region is 21 nucleotide pairs in length.

[0186] In some embodiments, the dsRNA molecule of the present invention comprises one or more overhang regions and / or capping groups at the 3'-end or 5'-end or both ends of the strand. The overhang can be 1 to 10 nucleotides in length, 1 to 6 nucleotides in length, such as 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length or 1 to 2 nucleotides in length. The overhang can result from one strand being longer than the other or from two strands of the same length being in a twisted form. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence. The first strand and the second strand can also be linked, for example, by additional bases or other non-base linkers for forming a hairpin.

[0187] In some embodiments, the nucleotides within the overhang region of the dsRNA molecule of the present invention can each independently be a modified or unmodified nucleotide, such as, but not limited to, a 2'-sugar modification, such as 2'-fluoro 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA and any combination thereof. For example, TT can be an overhang sequence at one end on one strand. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0188] The 5' or 3' overhangs 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 having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3' end of the sense strand, antisense strand or both strands. In some embodiments, this 3' overhang is present within the antisense strand. In some embodiments, this 3' overhang is present within the sense strand.

[0189] The dsRNA molecule of the present invention may contain only a single overhang such that it can enhance the interfering activity of the dsRNA without affecting its overall stability. For example, a 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 blunt ends located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3' end and a blunt end at the 5' end. 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 that loads the RISC process. For example, a 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 blunt ends at the 3' end of the antisense strand and the 5' end of the antisense strand.

[0190] In some embodiments, one end of the dsRNA is blunt-ended and the other end has an overhang, where the antisense strand has at least one thermally destabilizing nucleotide, and at least one thermally destabilizing nucleotide is 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 contains 2, 3, 4, 5, or 6 2’-fluoro modifications; (ii) the antisense 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 contains at least 4 2’-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following: a double-stranded region 12-40 nucleotide pairs in length, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In this further subset of embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h’GNA (ModA-ModK). In some embodiments, the overhang is present on the 3’ end of the antisense strand and the blunt end is present at the 5’ end of the antisense strand. In some particular embodiments, the overhang is 2, 3, or 4 nucleotides in length.

[0191] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, where one end of the dsRNA is blunt-ended and the other end has an overhang, where the antisense strand has at least one thermally destabilizing 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 characteristics: (i) the antisense contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense 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 4 2'-fluoro modifications and optionally further has at least one (e.g., 1, 2, 3, 5, or all 6) of the following: the 2-nucleotide overhang is present on the 3' end of the antisense strand and the blunt end is present on the 5' end of the antisense strand, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further contains at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK). In some embodiments, the overhang is present on the 3' end of the antisense strand and the blunt end is present on the 5' end of the antisense strand.

[0192] In some embodiments, the dsRNA molecule of the invention can also have two blunt ends at both ends of the dsRNA duplex.

[0193] In some embodiments, the dsRNA has blunt ends at both ends of the double strand, where the antisense strand has at least one thermally labile nucleotide, and at least one thermally labile nucleotide is within the seed region of the antisense strand (i.e., at positions 2 to 9 from the 5' end of the antisense strand), 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 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 contains at least 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of a double-stranded region 12 to 40 nucleotide pairs in length, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1 to 3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In this further some embodiments, the dsRNA molecule further contains at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0194] In some embodiments, the dsRNA molecule has a double-stranded region that is 19, 20, 21, 22, or 23 nucleotide base pairs in length, has blunt ends at both ends of the double strand, where one end of the dsRNA is a blunt end and the other end has an overhang, where the antisense strand has at least one thermally destabilizing modification of the double strand 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 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 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 further has at least one (e.g., 1, 2, 3, 5, or all 6) of at least 4 2'-fluoro modifications, where the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (ModA-ModK).

[0195] Thermally destabilizing modification As described above, dsRNA molecules can be optimized for RNA interference by incorporating a thermo-destabilizing modification within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. The inventors have discovered that dsRNAs having an antisense strand that includes at least one thermo-destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand includes at least one (e.g., 1, 2, 3, 4, 5 or more) thermo-destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the thermo-destabilizing modification of the duplex is located at positions 2-9 or preferably 4-8 from the 5' end of the antisense strand. In some further embodiments, the thermo-destabilizing modification of the duplex is located at positions 6, 7 or 8 from the 5' end of the antisense strand.

[0196] In yet some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification" refers to a modification that provides a lower complete melting temperature (T m ) (preferably, the T of dsRNA without such modifications) m T 1, 2, 3 or 4°C lower than m In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0197] Thermal destabilizing modifications can include, but are not limited to, depurination modifications; mismatches with opposing nucleotides in the reverse strand; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA). For example, as thermal destabilizing modifications, but not limited to, the mUNA and GNA components described in Examples 1-3 herein can be mentioned. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA.

[0198] In some embodiments, the destabilizing modification mUNA is

Chemical formula

[0199] In some embodiments, the destabilizing modification mUNA is [Chemistry] (wherein, R is H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O-alkyl; or O-alkylamino; R' is H or Me, B is A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-canonical monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; or non-canonical monocyclic, bicyclic and tricyclic heterocycles, and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers) is selected from the group consisting of.

[0200] In some embodiments, the destabilizing modification mUNA is [Chemistry] (wherein, R is H, OMe; F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; O-nPr; O-alkyl; or O-alkylamino; R' is H or Me, B is A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-canonical monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; or 7-deazapurine, and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers) selected from the group consisting of

[0201] In some embodiments, the destabilizing modification mUNA is

Chemical formula

[0202] In some embodiments, the destabilizing modification mUNA is

Chem.

[0203] In some embodiments, the modified mUNA is

Chem.

[0204] In some embodiments, the modified mUNA is

Chemical Structure

Chemical Structure

Chemical Structure

[0205] Exemplary abasic modifications include, but are not limited to, the following:

Chemical Structure

Chemical Structure

[0206] Exemplary sugar modifications include, but are not limited to, the following:

Chemical Structure

[0207] In some embodiments, the double-stranded thermal destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some of these further embodiments, the dsRNA molecule further comprises at least one thermal destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK).

[0208] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, when any of the bonds between 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') is absent from the nucleotide, either independently or in combination. In some embodiments, the acyclic nucleotide is

Chemical formula

[0209] The term "GNA" refers to glycol nucleic acid, a polymer that is similar to DNA or RNA but differs in that it is composed of repeating glycerol units linked by phosphodiester bonds in the composition of its "backbone". [Chemical formula]

[0210] Thermal destabilizing modifications in the double strand can be mismatches (i.e., non-complementary base pairs) between a thermally destabilizing nucleotide and the opposing nucleotide in the reverse strand within the dsRNA double strand. 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 pair formations known in the art are also in accordance with the present invention. The mismatches can occur between nucleotides of either naturally occurring nucleotides or modified nucleotides, i.e., mismatched base pair formation can occur between nucleobases from each nucleotide, independent of modifications on the ribose sugar of the nucleotide. In certain embodiments, the dsRNA molecule has at least one nucleobase in the mismatched pairing that is a 2'-deoxy nucleobase; for example, the 2'-deoxy nucleobase is present within the sense strand.

[0211] In some embodiments, the thermal destabilizing modification of the double strand within the seed region of the antisense strand comprises nucleotides having impaired Watson-Crick H-bonds to complementary bases on the target mRNA, for example,

Chemical formula

[0212] More examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is hereby incorporated by reference in its entirety.

[0213] The thermal destabilizing modifications can also include universal bases and phosphate modifications with reduced or abolished ability to form hydrogen bonds with opposing bases.

[0214] In some embodiments, the double-stranded heat destabilizing modification includes nucleotides having non-standard bases such as nucleobase modifications in which the ability to form hydrogen bonds with bases within the reverse strand is impaired or completely lost. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010 / 0011895 (which is hereby incorporated by reference in its entirety). Exemplary nucleobase modifications are as follows.

Chemical formula

[0215] In some embodiments, the double-stranded heat destabilizing modification within the seed region of the antisense strand includes one or more α-nucleotides complementary to bases on the target mRNA, such as

Chemical formula

[0216] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include

Chemical formula

[0217] The alkyl in the R group is C 1 ~C 6 alkyl. Specific alkyls in the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

[0218] In some embodiments, exemplary destabilizing modifications are shown in Figure 1.

[0219] In addition to the antisense strand containing heat destabilizing modifications, the dsRNA can also include one or more stabilizing modifications. For example, the dsRNA can include at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, all of the stabilizing modifications can be present within a single strand. In some embodiments, both the sense and antisense strands include at least two stabilizing modifications. The stabilizing modifications can be present on any nucleotide of the sense or antisense strand. For example, the stabilizing modifications can be present on all nucleotides on the sense strand and / or the antisense strand; each stabilizing modification can be present in an alternating pattern on the sense or antisense strand; or the sense or antisense strand can include both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand can be the same as or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand can have a variation with respect to the alternating pattern of stabilizing modifications on the antisense strand.

[0220] In some embodiments, the antisense strand includes 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 present at any position. In some embodiments, the antisense includes stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense includes stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0221] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification can be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification. In some embodiments, the antisense strand comprises stabilizing modifications at each of the 5' and 3' ends of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification. In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at the +1 and +2 positions from the position of the destabilizing modification.

[0222] 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 present 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 that are opposite or complementary to positions 11, 12, and 15 of the antisense strand, counted from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions that are opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counted from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocking of 2, 3, or 4 stabilizing modifications.

[0223] In some embodiments, the sense strand does not comprise stabilizing modifications at positions that are opposite or complementary to the duplex thermal destabilizing modifications in the antisense strand.

[0224] Exemplary thermal stabilizing modifications include, without limitation, 2'-fluoro modifications. Other thermal stabilizing modifications include, without limitation, LNA.

[0225] In some embodiments, the dsRNA of the present invention comprises at least four (e.g., 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, all of the 2'-fluoronucleotides may be present in a single strand. In some embodiments, both the sense and antisense strands comprise at least two 2'-fluoronucleotides. The 2'-fluoro modification may be present on any nucleotide of the sense strand or the antisense strand. For example, the 2'-fluoro modification may be present on all nucleotides on the sense strand and / or the antisense strand; each 2'-fluoro modification may be present in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand may comprise both 2'-fluoro modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may have a variation relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0226] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, the 2'-fluoro modification in the antisense strand may be present at any position. In some embodiments, the antisense comprises 2'-fluoronucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense comprises 2'-fluoronucleotides at positions 2, 6, 14 and 16 from the 5' end. In yet some other embodiments, the antisense comprises 2'-fluoronucleotides at positions 2, 14 and 16 from the 5' end.

[0227] In some embodiments, the antisense strand comprises at least one 2'-fluoronucleotide adjacent to a destabilizing modification. For example, the 2'-fluoronucleotide can be at the 5' or 3' end of the destabilizing modification, i.e., the nucleotide at the -1 or +1 position from the position of the destabilizing modification. In some embodiments, the antisense strand comprises 2'-fluoronucleotides at each of the 5' and 3' ends of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0228] In some embodiments, the antisense strand comprises at least two 2'-fluoronucleotides at the 3' end of the destabilizing modification, i.e., at the +1 and +2 positions from the position of the destabilizing modification.

[0229] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, the 2'-fluoro modification in the sense strand can be present at any position. In some embodiments, the antisense comprises 2'-fluoronucleotides at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoronucleotides at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counted from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counted from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a block of 2, 3, or 4 2'-fluoronucleotides.

[0230] In some embodiments, the sense strand does not comprise 2'-fluoronucleotides at positions opposite or complementary to the duplex thermal destabilizing modification in the antisense strand.

[0231] In some embodiments, the dsRNA molecule of the present invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand comprises at least one thermally labile nucleotide, wherein at least one thermally labile nucleotide is present within the seed region of the antisense strand (i.e., positions 2-9 from 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 has the following features: (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioate nucleotide 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 nucleotide linkages; (vi) the dsRNA comprises at least 4 2'-fluoro modifications; and (vii) the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6 or all 7) of a blunt end at the 5' end of the antisense strand, wherein the destabilizing modification is selected from the mUNA and GNA components described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In this further some embodiments, the dsRNA molecule further comprises at least one thermally labile modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (ModA-ModK). Preferably, the 2-nt overhang is present at the 3' end of the antisense strand.

[0232] In some embodiments, in the dsRNA molecule of the present invention comprising sense and antisense strands, the sense strand is 25 - 30 nucleotide residues in length, wherein positions 1 - 23 starting from the 5'-terminal nucleotide (position 1) of the sense strand contain at least 8 ribonucleotides; the antisense strand is 36 - 66 nucleotide residues in length, and at least 8 ribonucleotides within its position starting from the 3'-terminal nucleotide pair with positions 1 - 23 of the sense strand to form a double strand; wherein at least the 3'-terminal nucleotide of the antisense strand does not pair with the sense strand, and up to 6 consecutive 3'-terminal nucleotides do not pair with the sense strand, thereby forming a 3'-single-s...

Claims

**Claim 1** A double-stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the dsRNA molecule has a double-stranded region 12 to 40 nucleotide pairs in length, the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, the antisense strand contains a thermolabile modification at the 7th position of the antisense strand, counting from the 5' end of the antisense strand, the destabilizing modification has the following formula: 【Chemical 1】 (wherein the base is a modified or unmodified nucleobase) and the dsRNA molecule is either conjugated or not conjugated to a ligand, dsRNA molecule. **Claim 2** The dsRNA molecule according to claim 1, wherein the double-stranded region is 19 to 23 base pairs in length. **Claim 3** The dsRNA molecule according to claim 1, comprising at least one ligand conjugated to the sense strand. **Claim 4** The dsRNA molecule according to claim 3, wherein the at least one ligand is a targeting ligand. **Claim 5** The dsRNA molecule according to claim 4, wherein the targeting ligand is an ASGPR ligand conjugated to the 3' end or 5' end of the sense strand. **Claim 6** The dsRNA molecule according to claim 5, wherein the ASGPR ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker. **Claim 7** The dsRNA molecule according to claim 6, wherein the ASGPR ligand is as follows: 【Chemical 2】 **Claim 8** The dsRNA molecule according to claim 4, wherein the targeting ligand is a lipid. **Claim 9** The dsRNA molecule according to claim 4, wherein the targeting ligand is a peptide that targets cells. **Claim 10** The dsRNA molecule according to claim 3, wherein the at least one ligand improves the nuclease resistance of the dsRNA molecule. **Claim 11** The dsRNA molecule according to claim 1, having a melting temperature of 55°C to 67°C. **Claim 12** The dsRNA molecule according to claim 1, having a blunt end at the 5' end of the antisense strand. **Claim 13** The dsRNA molecule according to claim 12, having a blunt end at the 3' end of the antisense strand. **Claim 14** The dsRNA molecule according to claim 12, having a single-stranded overhang of 1 to 10 nucleotides in length at the 3' end of the antisense strand.

15. The dsRNA molecule according to claim 1, wherein each strand is 19 to 25 nucleotides in length.

16. The dsRNA molecule according to claim 1, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

17. The dsRNA molecule according to claim 1, wherein the antisense strand contains 2, 3, 4, 5 or 6 2'-fluoro modifications.

18. The dsRNA molecule according to claim 17, wherein there is no 2'-fluoro modification at the 3rd to 9th nucleotide positions of the antisense strand, counted from the 5' end of the antisense strand.

19. The dsRNA molecule according to claim 17, wherein the antisense strand has 2'-F modifications at positions 2, 14 and 16, counted from the 5' end of the antisense strand.

20. The dsRNA molecule according to claim 17, wherein the antisense strand has 2'-F modifications at positions 2, 6, 14 and 16, counted from the 5' end of the antisense strand.

21. The dsRNA molecule according to claim 17, wherein the antisense strand has 2'-F modifications at positions 2, 6, 8, 9, 14 and 16, counted from the 5' end of the antisense strand.

22. The dsRNA molecule according to claim 1, wherein the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications.

23. The dsRNA molecule according to claim 22, wherein the sense strand has 2'-F modifications at positions complementary to positions 11, 12 and 15 of the antisense strand, counted from the 5' end of the antisense strand.

24. The dsRNA molecule according to claim 22, wherein the sense strand has 2'-F modifications at positions complementary to positions 11, 12, 13 and 15 of the antisense strand, counted from the 5' end of the antisense strand.

25. The dsRNA molecule according to claim 1, wherein the antisense strand contains 1, 2, 3 or 4 phosphorothioate internucleotide linkages.

26. The dsRNA molecule according to claim 1, wherein at one or both ends of the antisense strand, at least the terminal two nucleotides are linked via a phosphorothioate or methylphosphonate internucleotide linkage.

27. The dsRNA molecule according to claim 1, wherein at one or both ends of the antisense strand, the three nucleotides at the terminus are linked via phosphorothioate internucleotide linkages.

28. The dsRNA molecule according to claim 1, wherein the sense strand comprises one, two, three or four phosphorothioate internucleotide linkages.

29. The dsRNA molecule according to claim 1, wherein at one or both ends of the sense strand, at least the two nucleotides at the terminus are linked via internucleotide linkages of phosphorothioate or methylphosphonate.

30. The dsRNA molecule according to claim 1, wherein at one or both ends of the sense strand, the three nucleotides at the terminus are linked via phosphorothioate internucleotide linkages.

31. The dsRNA molecule according to claim 1, wherein the first base pair from the 5'-end of the antisense strand within the double-stranded region is an AU base pair.

32. The dsRNA molecule according to claim 1, wherein the antisense strand comprises 5'-(E)-vinylphosphonate (VP).

33. A pharmaceutical composition comprising the dsRNA molecule according to claim 1, alone or in combination with a pharmaceutically acceptable carrier or excipient.

34. A composition for silencing a target gene in a cell, comprising the dsRNA molecule according to claim 1.

35. The composition according to claim 34, which is administered via subcutaneous or intravenous administration.

36. A composition for suppressing an off-target effect caused by the antisense strand of a dsRNA molecule, comprising the dsRNA molecule according to claim 1.

37. A composition for delivering a polynucleotide to a specific target in a subject, comprising the dsRNA molecule according to claim 1.

38. The composition according to claim 37, which is administered by an administration means including intramuscular, intratracheal, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, intrathecal or a combination thereof.

Images