Modified oligonucleotide

Abstract

To provide effective nucleotide or chemical motifs of dsRNA molecules that are advantageous for inhibiting target gene expression, and to provide RNAi compositions suitable for therapeutic applications.SOLUTION: One aspect of the invention pertains to double-stranded RNA (dsRNA) agents capable of inhibiting the expression of a target gene. Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA molecules that are suitable for therapeutic use, and to methods of inhibiting the expression of a target gene by administering these dsRNA molecules, e.g., to treat various disease states.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 914,010, filed October 11, 2019, under 35 U.S.C. § 119(e), the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to modified oligonucleotides that are advantageous for inhibiting target gene expression and compositions suitable for therapeutic use. In addition, the present invention provides methods for inhibiting target gene expression by administering these modified oligonucleotides, for example, for the treatment of various diseases. [Background technology]

[0003] RNA interference, or "RNAi," is a term originally coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al., 2003; Fire et al., 2003). Short dsRNAs induce gene-specific posttranscriptional silencing in many organisms, including vertebrates, providing a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to possess short RNAs (approximately 22 nucleotides) derived from its double-stranded RNA trigger, but the protein components of this activity remained unknown.

[0004] The discovery of RNA-mediated post-transcriptional gene silencing in 1998 was a crucial breakthrough in biological research. This process, known as RNA interference (RNAi), enabled the specific knockdown of any gene, making it a widely used technique in all biological research. From a therapeutic perspective, RNAi has the advantage of being able to target any disease-related RNA-based factor, many of which are considered "undruggable" by small molecules. Furthermore, the sequence-specific nature of target recognition reduces concerns about off-target toxicity. Several RNAi-based strategies use different classes of RNA for efficient knockdown. One such example is small interfering RNA (siRNA), which is perfectly complementary to the target sequence on a transcript and is introduced into target cells as a duplex. After entering the cell, the siRNA is loaded into the RNA-induced silencing complex (RISC). During this loading process, the passenger strand (sense strand) is removed, leaving the guide strand (antisense strand) within the RISC, where it binds to its complementary site on the target mRNA. The bound mRNA is then cleaved by the nuclease activity of RISC and can then be further degraded by cellular nucleases.

[0005] Altritol-modified nucleic acids (ANAs) support an RNA-like A-form structure when incorporated into double-stranded nucleic acid structures, improving nuclease stability. Placing ANA modifications at the 3' end of the sense strand of siRNA can improve efficacy, whereas placing ANA modifications at the 5' end of the antisense strand completely abolishes activity. See (Non-Patent Document 3); (Non-Patent Document 4); (Non-Patent Document 5); and (Non-Patent Document 6). However, efforts continue to be made to adjust siRNA design by rational application of chemical modifications without compromising the gene silencing efficacy of siRNA gene therapy drugs. The present invention relates to this endeavor. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Fire et al.(1998)Nature 391,806-811 [Non-patent document 2] Elbashir et al.(2001)Genes Dev.15,188-200 [Non-patent document 3] Chem.Eur.J.1999,5,2424-2431 [Non-patent document 4] Nucleic Acids Res.2007,35,1064-1074 [Non-Patent Document 5] Eur.J.Pharm.2009,606,38-44 [Non-patent document 6] Nucleic Acids Res.2012,40,7573-7583 Summary of the Invention [Means for solving the problem]

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

[0008] The present inventors have unexpectedly discovered that the dsRNA molecule that comprises one or more altritol nucleotides in particular positions, for example, in the seed region of antisense strand and / or sense strand, is highly effective in mediating RNA interference and / or is not harmful.Therefore, in one aspect, the present invention provides a dsRNA molecule that can inhibit the expression of target gene.Generally, this dsRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand has sufficient complementarity with target sequence to mediate RNA interference, and this dsRNA molecule comprises at least one hexopyranose nucleotide or cyclohexene nucleotide.

[0009] In some embodiments of the various aspects disclosed herein, the hexopyranose nucleoside has the following structure: [ka] (In the formula, B is a modified or unmodified nucleobase; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, OH, protected OH, NH2, NHMe, NMe2, OR 9 , C1-C6 alkyl (e.g., methyl, ethyl, propyl, etc.), branched alkyl, or aminoalkyl; R 7 and R 8 are independently H, C1-C6 alkyl (e.g., methyl, ethyl, propyl, etc.), or OR 9 and R 9 is C1~C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar) It is of the type.

[0010] Exemplary hexopyranose nucleotides include, but are not limited to, allopyranose nucleotides, altropyranose nucleotides (altritol nucleotides), glucopyranose nucleotides, mannopyranose nucleotides, gulopyranose nucleotides, idopyranose nucleotides, galactopyranose nucleotides, and talopyranose nucleotides. In some embodiments, the hexopyranose nucleotide is a deoxyhexopyranose nucleotide, such as a 6-deoxyhexopyranose nucleotide. Exemplary deoxyhexopyranose nucleotides include, but are not limited to, fucopyranose nucleotides, rhamnopyranose nucleotides, quinovopyranose nucleotides, and pneumopyranose nucleotides.

[0011] In some embodiments, the hexopyranose nucleotide or cyclohexene nucleoside is selected from those shown in Figures 12 and 13.

[0012] In some embodiments, the hexopyranose nucleotides are altritol nucleotides.

[0013] In some embodiments, the hexopyranose nucleotide is an.

[0014] In some embodiments, altritol nucleotides are present in the antisense strand. Generally, altritol nucleotides can be present anywhere in the antisense strand. For example, altritol nucleotides can be present within the seed region (i.e., at the 5'-end of the antisense strand, at positions 3 to 8 counting from the 5'-end).

[0015] In some embodiments, altritol nucleotides are present in the sense strand. As with the antisense strand, altritol nucleotides can be present anywhere within the sense strand. For example, altritol nucleotides can be present within the 5' region (i.e., at the 5' end of the sense strand, positions 2 to 8 counting from the 5' end). In some embodiments, the sense strand includes altritol nucleotides in the central region of the sense strand.

[0016] In some embodiments, the dsRNA molecule comprises at least one 2'-fluoro nucleotide and / or at least one 2'-OMe nucleotide.

[0017] In some embodiments, the dsRNA molecule comprises at least 1, 2, 3, 4 or more phosphorothioate internucleotide linkages.

[0018] In some embodiments, the dsRNA molecule further comprises a ligand conjugated to the sense strand or antisense strand.For example, the dsRNA molecule further comprises an ASGPR ligand conjugated to the sense strand or antisense strand.Preferably, the ligand is conjugated to the 3' end of the sense strand.

[0019] In another aspect, the present disclosure provides a method for inhibiting the expression of target gene sequence.Generally, this method comprises administering to cells an amount of dsRNA molecule as described herein that is sufficient to inhibit the expression of target gene.Cells can be in vitro or in vivo.

[0020] In yet another aspect, the present disclosure provides pharmaceutical compositions comprising the dsRNA molecules described herein.

[0021] In yet another aspect, the present disclosure provides the method for treating subject using dsRNA molecules as described herein.Generally, this method for treating comprises administering therapeutically effective amount of dsRNA molecules as described herein to the subject that needs it. [Brief explanation of the drawings]

[0022] [Figure 1A] Sense and antisense strand sequences of exemplary dsRNAs are shown. [Figure 1B] The structures of 2'-OMe RNA, 2'-F RNA, and altritol nucleic acid (ANA) nucleotides are shown. [Figure 2] 1 is a bar graph showing the in vitro RNAi activity of duplexes with ANA in the antisense strand, where the nucleotide at the indicated position in the antisense strand, also referred to as the guide strand, was replaced with the corresponding ANA nucleotide. [Figure 3] 1 is a bar graph showing the in vitro RNAi activity of duplexes with ANA in the sense strand, where the nucleotide at the indicated position in the sense strand, also referred to as the passenger strand, was replaced with the corresponding ANA nucleotide. [Figure 4]1 is a line graph showing IC50 values ​​of select exemplary siRNAs containing ANA modifications, with the indicated positions of the antisense or sense strand bearing the corresponding ANA nucleotide. [Figure 5] Figure 5A shows line graphs depicting the on-target (Figure 5A) and off-target (Figure 5B) inhibitory effects of exemplary siRNAs in luciferase reporter assays. Luciferase reporter plasmids were co-transfected with the indicated siRNAs into COS-7 cells. Forty-eight hours after transfection, cells were harvested and subjected to dual-luciferase assays. The % target remaining rate was calculated by dividing the Renilla / Firefly ratio at each siRNA concentration by the ratio in the absence of siRNA. [Figure 6] Figure 6 shows the in vivo activity of subcutaneously administered ANA-containing siRNA in C57BL / 6 mice. Figure 6A is a bar graph showing circulating Ttr protein levels determined by ELISA (mean ± SD). Figure 6B is a bar graph showing Ttr transcript levels in the liver at the end of the study (day 14; mean ± SD). [Figure 7] A model schematic of the siRNA guide strand with ANA-G at position S7 bound to Ago2, with ANA residues highlighted by cyan carbon atoms and selected intrastrand P-P distances provided in Å. [Figure 8] Figure 8A shows the nuclease stability of oligonucleotides bearing ANA modifications against degradation by snake venom phosphodiesterase (SVPD) for oligonucleotides bearing a single or two ANA nucleotides at the 3' end of polydT. Figure 8B shows the stability of oligonucleotides bearing a single or two ANA nucleotides at the 5' end of polydT against degradation by phosphodiesterase II (PDEII). "s" indicates a phosphorothioate bond. U is uridine, and Uan is an ANA nucleotide with uracil as the nucleobase. For direct comparison, identical sequences with terminal uridines were prepared. [Figure 9]Diagram of the active site of Xrn1 5'-exoribonuclease with 5'P-dTdTdT-3' bound (phosphorus and carbon atoms colored orange and yellow, respectively) as observed in the crystal structure of the complex [Reference Jinek et al. Mol. Cell 2011]. The 5'P-aUaU-3'P ANA dimer (phosphorus and carbon atoms colored black and cyan, respectively) is superimposed to reveal the displaced P-P spacing between ANA and DNA (black and orange arrows, respectively) and the orientation of the phosphate relative to the Mg2+ ion (green sphere). [Figure 10A] Figure 10A is a line graph showing ANA 3'-exonuclease stability. Calculated t values ​​are UU-3': << 1 hour, (Uan)(Uan)-3': 4.7 hours, UsU-3': 10.2, Us(Uan)-3': 8.0 hours, and (Uans)(Uan)-3': 16.3 hours. Overall, Uan has increased 3'-exonuclease stability compared to its phosphodiester (PO) parent, with double incorporation providing greater stability than single incorporation. [Figure 10B] 1 is a line graph showing ANA 5'-exonuclease stability. Calculated t values ​​are: 5'-UU: << 1.5 hours, 5'-(Uan)U: 11.5, 5'-UsU: 26.9, 5'-(Uans)U: 113 hours, and 5'-(Uans(Uan): 458.5. Overall, Uan has increased 5'-exonuclease stability compared to its phosphodiester (PO) and phosphorothioate (PS) parents, respectively, with double incorporation providing greater stability than single incorporation. [Figure 11] Schematic showing the crystal structure of an ANA:RNA duplex; PDB ID 3ok2. The phosphate spacing in ANA (5.5 Å) is smaller than in RNA (6 Å). Adapted from Ovaere et al. Nucleic Acid Res., 40:7573-7583 (2012). [Figure 12] Some exemplary hexopyranose nucleoside phosphoramidites are shown. [Figure 13]Some exemplary hexopyranose nucleoside phosphoramidites are shown. [Figure 14] Some exemplary hexopyranose nucleosides and nucleotides are shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] In one aspect, the present invention provides a double-stranded RNA (dsRNA) molecule, the dsRNA capable of inhibiting expression of a target gene, the dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand has sufficient complementarity with a target sequence to mediate RNA interference, and the dsRNA molecule comprises at least one hexopyranose nucleotide, cyclohexene nucleotide, e.g., altritol nucleotide.

[0024] Hexopyranose nucleotide or cyclohexene nucleotide can be present at any position in dsRNA.For example, hexopyranose nucleotide or cyclohexene nucleotide can be present in the sense strand of siRNA molecule.It is noted that hexopyranose nucleotide or cyclohexene nucleotide can be present in the sense strand.For example, hexopyranose nucleotide or cyclohexene nucleotide can be at the 5'-end of sense strand, the 3'-end of sense strand and / or the internal position of sense strand.

[0025] In some embodiments, the hexopyranose or cyclohexene nucleotide is present at the 5'-end of the sense strand. In some embodiments, the hexopyranose or cyclohexene nucleotide is present at the 3'-end of the sense strand.

[0026] In some embodiments, the hexopyranose nucleotide or cyclohexene nucleotide can be present at an internal position in the sense strand. For example, the hexopyranose nucleotide or cyclohexene nucleotide can be present at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the sense strand. In some embodiments, the hexopyranose nucleotide or cyclohexene nucleotide can be present within the 5' region (i.e., at the 5' end of the sense strand, positions 2-8 counting from the 5' end). In some embodiments, the hexopyranose nucleotide or cyclohexene nucleotide can be present in the central region of the sense strand.

[0027] In some embodiments, the hexopyranose nucleotide or cyclohexene nucleotide may be present in the sense region of the sense strand at a position opposite the seed region of the antisense strand (i.e., at the 5' end of the antisense strand, positions 3 to 8 counting from the 5' end).

[0028] In some embodiments, the sense strand comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hexopyranose and / or cyclohexene nucleotides. In some embodiments, the sense strand comprises a hexopyranose and / or cyclohexene nucleotide at at least one of positions 3 and 12.

[0029] In some embodiments, the sense strand comprises at least two or more hexopyranose nucleotides and / or cyclohexene nucleotides. When two or more hexopyranose nucleotides and / or cyclohexene nucleotides are present, they may be adjacent to each other. Thus, in some embodiments, the sense strand comprises at least two, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more hexopyranose nucleotides and / or cyclohexene nucleotides adjacent to each other. In other words, the sense strand comprises a stretch of two or more independently selected hexopyranose nucleotides and / or cyclohexene nucleotides.

[0030] In some embodiments, the sense strand comprises three hexopyranose and / or cyclohexene nucleotides adjacent to one another, i.e., the sense strand comprises a stretch of three independently selected hexopyranose and / or cyclohexene nucleotides.

[0031] In some embodiments, hexopyranose nucleotides or cyclohexene nucleotides are present in the antisense strand. Generally, hexopyranose nucleotides and / or cyclohexene nucleotides can be present anywhere within the antisense strand. For example, hexopyranose nucleotides or cyclohexene nucleotides can be at the 5' end of the antisense strand, the 3' end of the antisense strand, and / or at an internal position within the antisense strand.

[0032] In some embodiments, the hexopyranose or cyclohexene nucleotide is present at the 5'-end of the antisense strand. In some embodiments, the hexopyranose or cyclohexene nucleotide is present at the 3'-end of the antisense strand.

[0033] In some embodiments, hexopyranose nucleotides or cyclohexene nucleotides can be present at internal positions in the antisense strand. For example, hexopyranose nucleotides or cyclohexene nucleotides can be present at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the antisense strand. In some embodiments, hexopyranose nucleotides or cyclohexene nucleotides can be present within the seed region (i.e., at the 5' end of the antisense strand, positions 2-8 counting from the 5' end). For example, the antisense strand includes a hexopyranose nucleotide and / or cyclohexene nucleotide at at least one of positions 3-8 counting from the 5' end of the antisense strand. In some embodiments, hexopyranose nucleotides or cyclohexene nucleotides can be present in the central region of the antisense strand.

[0034] In some embodiments, the antisense strand comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more, hexopyranose and / or cyclohexene nucleotide. For example, the antisense strand comprises a hexopyranose and / or cyclohexene nucleotide at at least one of positions 6, 7, 9, 12, 16, 21, and 22, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a hexopyranose and / or cyclohexene nucleotide at at least one of positions 6, 7, and 16, counting from the 5' end of the antisense strand. For example, the antisense strand comprises a hexopyranose and / or cyclohexene nucleotide at at least one of positions 6 and 7, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a hexopyranose or cyclohexene nucleotide at position 7, counting from the 5' end of the antisense strand.

[0035] In some embodiments, the antisense strand comprises at least two or more hexopyranose nucleotides and / or cyclohexene nucleotides. When two or more hexopyranose nucleotides and / or cyclohexene nucleotides are present, they may be adjacent to each other. Thus, in some embodiments, the antisense strand comprises at least two, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more hexopyranose nucleotides and / or cyclohexene nucleotides adjacent to each other. In other words, the antisense strand comprises a stretch of two or more independently selected hexopyranose nucleotides and / or cyclohexene nucleotides.

[0036] In some embodiments, the antisense strand comprises three hexopyranose and / or cyclohexene nucleotides adjacent to one another, i.e., the antisense strand comprises a stretch of three independently selected hexopyranose and / or cyclohexene nucleotides.

[0037] In some embodiments, the hexopyranose nucleotides are altritol nucleotides.

[0038] In some embodiments, the hexopyranose nucleotide is a glucopyranose nucleotide.

[0039] In some embodiments, the hexopyranose nucleotides are mannopyranose nucleotides.

[0040] In some embodiments, the hexopyranose nucleotides are galactopyranose nucleotides.

[0041] In some embodiments, the hexopyranose nucleotide is a fucopyranose nucleotide.

[0042] In some embodiments, the sense strand and / or the antisense strand may comprise nucleotides containing 5'-vinylphosphonate (VP) moieties. For example, the sense strand comprises nucleotides containing 5'-vinylphosphonate (VP) moieties. In another example, the antisense strand comprises nucleotides containing 5'-vinylphosphonate (VP) moieties.

[0043] In some embodiments, dsRNA comprises at least one altritol nucleotide. dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more altritol nucleotides. Without limitation, all altritol nucleotides can be present in one strand. In some embodiments, dsRNA molecule comprises one or two altritol nucleotides. In some preferred embodiments, dsRNA molecule comprises one altritol nucleotide.

[0044] In some embodiments, the antisense strand comprises at least one, for example, 2, 3, 4, 5, or more, altritol nucleotides. Without limitation, the altritol nucleotides in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises at least one altritol nucleotide within the seed region (i.e., at the 5'-end of the antisense strand, from positions 3 to 8, counting from the 5'-end). In some embodiments, the antisense strand comprises an altritol nucleotide at at least one of positions 6, 7, 9, 12, 16, 21, and 22, counting from the 5'-end. In some embodiments, the antisense strand comprises an altritol nucleotide at at least one of positions 6, 7, and 16, counting from the 5'-end. For example, the antisense strand comprises an altritol nucleotide at at least position 6, counting from the 5'-end. In another example, the antisense strand comprises an altritol nucleotide at at least position 7, counting from the 5'-end. In yet another example, the antisense strand comprises an altritol nucleotide at at least position 16, counting from the 5'-end.

[0045] The present inventors have discovered that a dsRNA in which the 5'-end of the antisense strand (AS1) is modified with an altritol nucleotide is effective. Thus, in some embodiments, the antisense strand does not contain an altritol nucleotide at positions 1 and / or 2 from the 5'-end.

[0046] In some embodiments, the altritol nucleotides are present in the sense strand. The antisense strand contains at least one, for example, two, three, four, five, or more, altritol nucleotides. As with the antisense strand, the altritol nucleotides can be present anywhere in the sense strand. In some embodiments, the sense strand can contain at least one altritol nucleotide at a position opposite or complementary to the seed region (i.e., at the 5' end of the antisense strand, between positions 3 and 8, counting from the 5' end). For example, the sense strand can contain an altritol nucleotide at at least one of positions 3 and 21, counting from the 5' end of the sense strand. In some embodiments, the sense strand contains an altritol nucleotide at position 1, counting from the 5' end. In some embodiments, the sense strand does not contain any altritol nucleotides.

[0047] In some embodiments of the various aspects disclosed herein, the altritol nucleotide has the structure: [ka] (In the formula, B is a modified or unmodified nucleobase; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, OH, NH2, NHMe, NMe2, OR 9 or C1-C6 alkyl (e.g., methyl, ethyl, propyl, etc.); R 7 and R 8 are independently H, C1-C6 alkyl (e.g., methyl, ethyl, propyl, etc.), or OR9 and R 9 is C1-C6 alkyl (e.g., methyl, ethyl, propyl, etc.), cycloalkyl, aryl, aralkyl, heteroaryl, or sugar) is a modified altritol nucleotide having the formula:

[0048] dsRNA molecule Aspects of the present invention include double-stranded RNA molecules. Generally, the dsRNA molecule comprises 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 independently range from 12 to 40 nucleotides in length. For example, each strand can independently be 14 to 40 nucleotides in length, 17 to 37 nucleotides in length, 25 to 37 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. Without limitation, the sense and antisense strands can be of equal or unequal length. In some embodiments, the antisense strand is longer than the sense strand, e.g., by 1, 2, 3, 4, or 5 nucleotides.

[0049] In some embodiments, the antisense strand is 18-35 nucleotides in length. In some embodiments, the antisense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In certain embodiments, the antisense strand is 23 nucleotides in length.

[0050] Like the antisense strand, the sense strand, in some embodiments, is 18-35 nucleotides in length. In some embodiments, the sense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In certain embodiments, the antisense strand is 21 nucleotides in length.

[0051] In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0052] A double-stranded RNA molecule has a double-stranded or duplex region. Generally, the duplex region (double-stranded region) is 12 to 40 nucleotide base pairs in length. For example, a dsRNA has a duplex region of 12 to 25 nucleotide base pairs in length. In some embodiments, a dsRNA has a duplex region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs in length. In certain embodiments, a dsRNA has a duplex region of 19, 20, 21, or 22 nucleotide base pairs in length.

[0053] The dsRNA molecules described herein may contain one or more nucleic acid modifications. It is noted that in addition to one or more altritol nucleotides, one or more nucleic acid modifications are present in the dsRNA.

[0054] In some embodiments, dsRNA molecules can contain 2'-fluoro nucleotides, i.e., 2'-fluoro modifications. For example, dsRNA molecules can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more 2'-fluoro nucleotides. Without limitation, all 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least two 2'-fluoro nucleotides. 2'-fluoro modifications can appear on any nucleotide in the sense strand or antisense strand. For example, 2'-fluoro modifications can appear on every nucleotide in the sense strand and / or antisense strand; each 2'-fluoro modification can appear in an alternating pattern on the sense strand or antisense strand; or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0055] The antisense strand of a dsRNA molecule may contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-fluoro nucleotides. In some embodiments, the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification of the antisense strand can be present at any position. In some embodiments, the antisense strand contains at least three 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at least 2, 14, and 16 positions from the 5' end. In some other embodiments, the antisense strand contains at least four 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at least 2, 6, 14, and 16 positions from the 5' end. In some further embodiments, the antisense strand contains at least five 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at least 2, 6, 9, 14, and 16 positions from the 5' end. In still some further embodiments, the antisense strand comprises at least six 2'-fluoro nucleotides, for example, the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0056] The sense strand of a dsRNA molecule may contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-fluoro nucleotides. In some embodiments, the sense strand contains 2, 3, 4, or 5 2'-fluoro nucleotides. For example, the sense strand contains 3 or 4 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification of the sense strand can be present at any position. In some embodiments, the sense strand contains at least three 2'-fluoro nucleotides. For example, the sense strand contains 2'-fluoro nucleotides at least 7, 10, and 11 positions from the 5' end. In some other embodiments, the sense strand contains at least four 2'-fluoro nucleotides. For example, the sense strand contains 2'-fluoro nucleotides at least 7, 9, 10, and 11 positions from the 5' end.

[0057] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a blockage of 2, 3, or 4 2'-fluoro nucleotides.

[0058] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, and 11 from the 5'-end, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 14, and 16 from the 5'-end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, and 11 from the 5'-end, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 9, 14, and 16 from the 5'-end. In yet some other embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, and 11 from the 5'-end, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14, and 16 from the 5'-end.

[0059] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, 10, and 11 from the 5'-end, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 14, and 16 from the 5'-end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, 10, and 11 from the 5'-end, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 9, 14, and 16 from the 5'-end. In yet some other embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, 10, and 11 from the 5'-end, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14, and 16 from the 5'-end.

[0060] In some embodiments, the antisense strand does not contain 2'-fluoro nucleotides at positions 3-9, counting from the 5' end.

[0061] A dsRNA molecule may contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-OMe nucleotides. Without limitation, all 2'-OMe nucleotides may be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least one 2'-OMe nucleotide. The 2'-OMe modification may occur on any nucleotide in the sense strand or the antisense strand. For example, the 2'-OMe modification may occur on every nucleotide in the sense strand and / or the antisense strand; each thermostabilizing modification may occur in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand contain the 2'-OMe modification in an alternating pattern. The alternating pattern of thermostabilizing modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of thermostabilizing modifications on the sense strand can be shifted relative to the alternating pattern of 2'-OMe modifications on the antisense strand.

[0062] The antisense strand of dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more 2'-OMe modifications. Without limitation, the thermostabilizing modifications of the antisense strand can be located at any position. In some embodiments, the antisense strand comprises at least three thermostabilizing modifications.

[0063] For example, the antisense strand does not contain 2'-OMe modifications at positions 2, 14, and 16 from the 5' end. In some other embodiments, the antisense strand does not contain 2'-OMe modifications at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand does not contain 2'-OMe modifications at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some further embodiments, the antisense strand does not contain 2'-OMe modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0064] The sense strand of a dsRNA molecule may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more 2'-OMe modifications. Without limitation, the 2'-OMe modifications of the sense strand may be present at any position. In some embodiments, the sense strand does not contain 2'-OMe modifications at least at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand does not contain 2'-OMe modifications at least at positions 7, 9, 10 and 11 from the 5' end.

[0065] dsRNA molecules may contain locked nucleic acids (LNA). For example, dsRNA molecules may contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more LNA modifications. Without limitation, all LNA nucleotides may be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least LNA modifications. LNA modifications may occur on any nucleotide of the sense strand or the antisense strand. For example, LNA modifications may occur on all nucleotides of the sense strand and / or the antisense strand; each LNA modification may occur in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand contain LNA modifications in an alternating pattern. The alternating pattern of LNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of LNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0066] The antisense strand of the dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNA modifications. Without limitation, the LNA modifications of the antisense strand can be present in any position.

[0067] The sense strand of a dsRNA molecule may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNA modifications. Without limitation, the LNA modifications of the sense strand may be present at any position. In some embodiments, the sense strand contains at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNA modifications, and the antisense strand does not contain 2'-fluoro nucleotides at positions 3 to 9, counting from the 5' end.

[0068] dsRNA molecules may contain bridged nucleic acids (BNA). For example, dsRNA molecules may contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more BNA modifications. Without limitation, all BNA nucleotides may be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least a BNA modification. BNA modifications may appear on any nucleotide of the sense strand or the antisense strand. For example, BNA modifications may appear on every nucleotide of the sense strand and / or the antisense strand; each BNA modification may appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand contain BNA modifications in an alternating pattern. The alternating pattern of BNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of BNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0069] The antisense strand of the dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more BNA modifications. Without limitation, the BNA modifications of the antisense strand can be present in any position.

[0070] The sense strand of a dsRNA molecule may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more BNA modifications. Without limitation, the BNA modifications of the sense strand may be present at any position. In some embodiments, the sense strand contains at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more BNA modifications, and the antisense strand does not contain 2'-fluoro nucleotides at positions 3 to 9, counting from the 5' end.

[0071] The dsRNA molecule may contain cyclohexene nucleic acid (CeNA). For example, the dsRNA molecule may contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more CeNA modifications. Without limitation, all CeNA nucleotides may be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least CeNA modifications. The CeNA modification may appear on any nucleotide of the sense strand or the antisense strand. For example, the CeNA modification may appear on every nucleotide of the sense strand and / or the antisense strand; each CeNA modification may appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand contain the ceNA modification in an alternating pattern. The alternating pattern of CeNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of ceNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0072] The antisense strand of a dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ceNA modifications. Without limitation, the ceNA modifications of the antisense strand can be present in any position.

[0073] The sense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ceNA modifications. Without limitation, the ceNA modifications of the sense strand can be present at any position. In some embodiments, the sense strand contains at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ceNA modifications, and the antisense strand does not contain a 2'-fluoro nucleotide at positions 3 to 9, counting from the 5' end.

[0074] In some embodiments, the dsRNA molecule may comprise a thermostabilizing modification. For example, the dsRNA molecule may comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more thermostabilizing modifications.

[0075] Without limitation, all of the thermostabilizing modifications may be present on one strand. In some embodiments, both the sense strand and the antisense strand contain at least one, for example, two, three, four, or more, thermostabilizing modifications. The thermostabilizing modifications may appear on any nucleotide of the sense strand or the antisense strand. For example, the thermostabilizing modifications may appear on every nucleotide of the sense strand and / or the antisense strand; each thermostabilizing modification may appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand contain thermostabilizing modifications in an alternating pattern. The alternating pattern of the thermostabilizing modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of the thermostabilizing modifications on the sense strand may be shifted relative to the alternating pattern of the thermostabilizing modifications on the antisense strand.

[0076] The antisense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more thermostabilizing modifications. In some embodiments, the antisense strand contains 2, 3, 4, 5, or 6 thermostabilizing modifications. Without limitation, the thermostabilizing modifications of the antisense strand can be located at any position. In some embodiments, the antisense strand contains at least three thermostabilizing modifications. For example, the antisense strand contains a thermostabilizing modification at at least one, for example, two or three, of positions 2, 14, and 16 from the 5' end. In some other embodiments, the antisense strand contains at least four thermostabilizing modifications. For example, the antisense strand contains a thermostabilizing modification at at least positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand contains at least five thermostabilizing modifications. For example, the antisense strand contains a thermostabilizing modification at at least positions 2, 6, 9, 14, and 16 from the 5' end. In yet some further embodiments, the antisense strand contains at least six thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0077] The sense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more thermostabilizing modifications. In some embodiments, the sense strand contains 2, 3, 4, or 5 thermostabilizing modifications. For example, the sense strand contains 3 or 4 thermostabilizing modifications. Without limitation, the thermostabilizing modifications of the sense strand can be located at any position. In some embodiments, the sense strand contains at least three thermostabilizing modifications. For example, the sense strand contains thermostabilizing modifications at at least positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand contains at least four thermostabilizing modifications. For example, the sense strand contains thermostabilizing modifications at at least positions 7, 9, 10, and 11 from the 5' end.

[0078] In some embodiments, the sense strand comprises thermostabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises thermostabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four thermostabilizing modifications.

[0079] In some embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0080] In some embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0081] In some embodiments, the sense strand does not contain a thermostabilizing modification at the position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0082] Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications, locked nucleic acids (LNAs), abasic modifications; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs). 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.

[0083] In some embodiments, the dsRNA molecule comprises one or more overhang regions (i.e., single-stranded regions) and / or capping groups at the 3'-end, 5'-end, or both ends of the strand. Without limitation, the overhangs can be 1-10 nucleotides, 1-6 nucleotides, 1-5 nucleotides, 1-4 nucleotides, 1-3 nucleotides, 2-6 nucleotides, 2-5 nucleotides, 2-4 nucleotides, 2-3 nucleotides, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other or the result of two strands of the same length being offset. The overhangs can form mismatches with the target sequence, or they can be complementary to the target sequence or other sequences. The first and second strands can be linked, for example, by forming a hairpin with additional bases or by other non-basic linkers. Without limitation, the overhangs can be present at the 3'-end of the sense strand, the antisense strand, or both strands.

[0084] In some embodiments, the dsRNA molecule comprises a single overhang. For example, the dsRNA molecule has a single overhang, and this overhang is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In some embodiments, the overhang is located at the 3'-end of the antisense strand. In some specific embodiments, the dsRNA comprises a 2-nucleotide overhang at the 3'-end of the antisense strand.

[0085] dsRNA can also have blunt ends. For example, one end of dsRNA is blunt, and the other end has an overhang. Without limitation, the blunt end can be located at the 5'-end of antisense strand (or the 3'-end of sense strand), or vice versa. Generally, the antisense strand of dsRNA has a nucleotide overhang at its 3'-end, and the 5'-end is blunt. Without being bound by theory, the asymmetric blunt end at the 5'-end of antisense strand and the 3'-end overhang of antisense strand favors the process of loading guide strand into RISC. In some embodiments, dsRNA has a 2-nucleotide overhang at the 3'-end of antisense strand and a blunt end at the 5'-end of antisense strand.

[0086] In some other embodiments, the dsRNA molecule has two blunt ends, ie, at both ends of the dsRNA.

[0087] The nucleotides in the overhang region of a dsRNA molecule can each independently be modified or unmodified, including, but not limited to, 2'-sugar modified nucleotides such as 2'-fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyl adenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof. For example, TT (or UU) can be the overhang sequence for either end of either strand. The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of a dsRNA molecule can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate internucleotide bond between them, where the two nucleotides in the overhang region can be the same or different.

[0088] A dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate or methylphosphonate internucleotide linkages. The phosphorothioate or methylphosphonate internucleotide linkage modification can occur on any nucleotide at any position on the sense strand or antisense strand or both strands. For example, the internucleotide linkage modification can occur on all nucleotides on the sense strand and / or antisense strand; each internucleotide linkage modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modification on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modification on the sense strand can have a change relative to the alternating pattern of internucleotide linkage modification on the antisense strand.

[0089] In some embodiments, the dsRNA molecule comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region comprises two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be provided to link the overhang nucleotide to the terminal paired nucleotide within the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked through phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. Preferably, these terminal three nucleotides can be located at the 3' end of the antisense strand.

[0090] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein either the phosphorothioate or methylphosphonate internucleotide linkages are positioned at any position within the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0091] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0092] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0093] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0094] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0095] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0096] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0097] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, four, five, or six phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0098] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0099] In some embodiments, the dsRNA molecule comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions of the sense and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked through phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense and / or antisense strand.

[0100] In some embodiments, the dsRNA molecule comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 positions of the internal region of the double strand of each of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked through phosphorothioate methylphosphonate internucleotide linkages at positions 8-16, counting from the 5'-end of the sense strand of the double-stranded region; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the terminal positions 1-10.

[0101] In some embodiments, the dsRNA molecule comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0102] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0103] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end).

[0104] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0105] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0106] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end).

[0107] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0108] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0109] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0110] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0111] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end).

[0112] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end).

[0113] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0114] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0115] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0116] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0117] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0118] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0119] In some exemplary dsRNA molecules, the sense strand can contain 0, 1, 2, 3, or 4 phosphorothioate internucleotide linkages. For example, the sense strand contains phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3.

[0120] In some exemplary dsRNA molecules, antisense strand can comprise 1, 2, 3 or 4 phosphorothioate internucleotide bonds.For example, sense strand comprises phosphorothioate internucleotide bonds between nucleotide position 21 and 22 and between nucleotide position 22 and 23.In another example, antisense strand comprises phosphorothioate internucleotide bonds between nucleotide position 1 and 2, between nucleotide position 2 and 3, between nucleotide position 21 and 22 and between nucleotide position 22 and 23.

[0121] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23. For example, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23.

[0122] 5'-modified In some embodiments, the dsRNA molecules may be 5' phosphorylated or contain a phosphoryl analog at the 5' terminus. Exemplary 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (dithiophosphate; (HO)(HS)(S)PO-5'), 5 '-phosphorothiolate ((HO)2(O)PS-5'); any further combination of oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). Modifications may be placed within the antisense strand of the dsRNA molecule. For example, the antisense strand can include a 5'-vinylphosphonate nucleotide at the 5' end.

[0123] In some embodiments, the antisense strand comprises a 5'-E-vinyl phosphonate. In some embodiments, the antisense strand comprises a 5'-E-vinyl phosphonate and a nucleoside at the N-1 position that reduces or inhibits the activity of the siRNA compared to an siRNA having the same antisense strand sequence but an unmodified N-1 position, and a nucleoside at the N-1 position that reduces or inhibits the activity of the siRNA compared to an siRNA having the same antisense strand sequence but an unmodified N-1 position.

[0124] In some embodiments, the sense strand comprises a 5'-morpholino modification, a 5'-dimethylamino modification, a 5'-deoxy modification, an inverted abasic modification, or an inverted abasic locked nucleic acid modification at the 5' end.

[0125] Generally, dsRNAs have melting temperatures ranging from about 40°C to about 80°C. For example, dsRNAs have melting temperatures ranging from about 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C at the lower end of the range and from about 70°C, 75°C, or 80°C at the upper end of the range. In some embodiments, dsRNAs have melting temperatures ranging from about 55°C to about 70°C or from about 60°C to about 75°C. In some embodiments, dsRNAs have melting temperatures ranging from about 57°C to about 67°C. In certain embodiments, dsRNAs have melting temperatures ranging from about 60°C to about 67°C. In some further embodiments, dsRNAs have melting temperatures ranging from about 62°C to about 66°C.

[0126] Without wishing to be bound by theory, dsRNA molecules with 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.

[0127] Without being bound by theory, in order for dsRNA molecules to be more effective in vivo, antisense strand must have a certain metabolic stability.In other words, in order for dsRNA molecules to be more effective in vivo, after administration, a certain amount of antisense strand may need to exist in vivo after a certain period of time.Therefore, in some embodiments, after in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example, in the liver of mice, on the 5th day.In some embodiments, after in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example, in the liver of mice, on the 6th day. In some embodiments, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of the dsRNA is present in vivo, for example, in the liver of a mouse, on the 7th day after in vivo administration.In some embodiments, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of the dsRNA is present in vivo, for example, in the liver of a mouse, on the 8th day after in vivo administration.In some embodiments, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of the dsRNA is present in vivo, for example, in the liver of a mouse, on the 9th day after in vivo administration. In some embodiments, at least 40%, e.g., 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, e.g., in the liver of a mouse, 10 days after in vivo administration.In some embodiments, after in vivo administration, on day 11, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of the dsRNA is present in vivo, for example, in the liver of a mouse. In some embodiments, after in vivo administration, on day 12, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of the dsRNA is present in vivo, for example, in the liver of a mouse. In some embodiments, after in vivo administration, on day 13, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of the dsRNA is present in vivo, for example, in the liver of a mouse. 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 liver of a mouse, on day 14 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 liver of a mouse, on day 15 after in vivo administration.

[0128] Without wishing to be bound by theory, thermodestabilizing modifications in the 5' region of the antisense strand (i.e., at positions 2-9 from the 5' end of the antisense strand) can reduce or inhibit off-target gene silencing. Thus, in some embodiments, the antisense strand comprises at least one (e.g., 1, 2, 3, 4, 5, or more) thermodestabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. The term "one or more thermodestabilizing modifications" includes one or more modifications that will result in a dsRNA with an overall lower melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower) than the Tm of a dsRNA lacking such one or more modifications.

[0129] In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8, or 9 from the 5' end of the antisense strand, or preferably at position 4, 5, 6, 7, or 8. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand. In some other embodiments, the thermally destabilizing modification is located at position 6, 7, or 8 from the 5' end of the antisense strand. In some particular embodiments, the thermally destabilizing modification is located at position 7 from the 5' end of the antisense strand.

[0130] Thermally destabilizing modifications can include, but are not limited to, abasic modifications; mismatches with the opposing nucleotide on the opposite strand; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, e.g., unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0131] Exemplary abasic modifications include, but are not limited to, the following: [ka] wherein R is H, Me, Et, or OMe; R' is H, Me, Et, or OMe; R'' is H, Me, Et, or OMe; and * represents either R, S or racemic).

[0132] Exemplary destabilizing sugar modifications include, but are not limited to, the following: [ka] where B is a modified or unmodified nucleobase.

[0133] Further sugar modifications include, but are not limited to, the following: [ka] where B is a modified or unmodified nucleobase.

[0134] In some embodiments, the thermodestabilizing modification is [ka] (wherein B is a modified or unmodified nucleobase, and the asterisk on each structure represents R, S, or a racemic mixture) is selected from the group consisting of:

[0135] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, where any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar linkages has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are hereby incorporated by reference in their entireties). Acyclic derivatives allow for backbone flexibility without affecting Watson-Crick pairing. The acyclic nucleotides can be linked by a 2'-5' or a 3'-5' linkage.

[0136] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA but differs in the composition of its "backbone" in that it is made up of repeating units of glycerol linked by phosphodiester bonds. [ka]

[0137] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide in the dsRNA duplex and the opposite nucleotide in the opposite strand. Exemplary mismatch 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 mismatch base pairings known in the art are also suitable for the present invention. Mismatches can occur between any nucleotide, whether naturally occurring or modified, i.e., mismatch base pairing can occur between nucleobases from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide. In certain embodiments, the dsRNA molecule comprises at least one nucleobase of mismatch pairing, which is 2'-deoxynucleobase; for example, the 2'-deoxynucleobase is in the sense strand.

[0138] In some embodiments, the thermodestabilizing modifications in the seed region of the antisense strand include nucleotides that exhibit impaired WCH bonding with complementary bases on the target mRNA. Exemplary nucleotides that exhibit impaired WCH bonding with complementary bases on the target mRNA include, but are not limited to, nucleotides containing nucleobases independently selected from the following: [ka]

[0139] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0140] Thermodestabilizing modifications can also include universal nucleobase and phosphate modifications that reduce or eliminate the ability to form hydrogen bonds with opposing bases.

[0141] In some embodiments, thermal destabilizing modifications include but are not limited to the nucleotides with non-standard bases, such as the nucleobase modifications that impair or completely eliminate the ability to form hydrogen bonds with the base of the opposite strand.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in International Publication No. 2010 / 0011895 (which is incorporated herein by reference in its entirety).Exemplary such nucleobase modifications are: [ka]

[0142] In some embodiments, the thermodestabilizing modifications include: [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. and so forth, which contain one or more □-nucleotides complementary to bases on the target mRNA.

[0143] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include, but are not limited to, the following: [ka]

[0144] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0145] In some embodiments, the destabilizing modification is selected from the following: [ka]

[0146] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 or +1 nucleotide position from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0147] In some embodiments, the antisense strand comprises at least two stabilizing modifications 3'-terminal to the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0148] In some embodiments, the sense strand does not contain a thermostabilizing modification at the position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0149] In some embodiments, the antisense strand comprises at least one 2'-fluoro nucleotide adjacent to the destabilizing modification.For example, the 2'-fluoro nucleotide can be the nucleotide at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 position or +1 position from the position of the destabilizing modification.In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 position and +1 position from the position of the destabilizing modification.

[0150] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0151] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at the position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0152] In some embodiments, all nucleotides of the sense strand and / or antisense strand of a dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more linking phosphate oxygen; changes to the components of the ribose sugar, such as the 2' hydroxyl on the ribose sugar; large-scale substitutions that make the phosphate moiety a "dephospho" linker; modifications or substitutions of naturally occurring bases; and substitutions or modifications of the ribose-phosphate backbone.

[0153] Because nucleic acids are polymers of monomers, many modifications occur at positions that are repeated within the nucleic acid, such as modifications of bases or phosphate moieties or non-linked Os at phosphate moieties. In some cases, modifications will occur at all of the target positions within the nucleic acid, but in many cases, this will not be the case. For example, modifications can occur only at the 3' or 5' terminal positions, or only at positions in the terminal region, e.g., on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only at positions in the terminal region, e.g., on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the chain, or can occur in double-stranded and single-stranded regions, particularly at the ends. One or more 5' ends can be phosphorylated.

[0154] For example, to enhance stability, it may be possible to include specific bases in the overhang or to include modified nucleotides or nucleotide substitutes in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang can be modified, e.g., with the modifications described herein. Modifications can include, for example, the use of art-known modifications at the 2' position of the ribose sugar, e.g., deoxyribonucleotides, modified 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl in place of the ribosugar of the nucleobase, and modifications at the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0155] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro. A strand can contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications may be present in dsRNA in addition to one or more altritol nucleotides.

[0156] In some embodiments, in addition to one or more altritol nucleosides, at least two different modifications may be present on the sense strand and / or antisense strand. These two modifications may be 2'-deoxy, 2'-O-methyl, 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two different modified nucleotides selected from 2'-O-methyl, 2'-fluoro, and / or 2'-deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it should be understood that these modifications may be present in addition to one or more altritol nucleotides in the dsRNA.

[0157] In some embodiments, the dsRNA molecule comprises an alternating pattern of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The term "alternating motif" or "alternating pattern," as used herein, refers to a motif having one or more modifications, with each modification occurring at alternating nucleotides on one strand. Alternating nucleotides can refer to every other nucleotide, every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0158] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modification of every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0159] In some embodiments, the dsRNA molecule comprises an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. This shift can be such that a group of modified nucleotides on the sense strand corresponds to a group of differently modified nucleotides on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA duplex, the alternating motif on the sense strand can begin at the 5'-3' end of the strand, and the alternating motif on the antisense strand can begin at the 3'-5' end of the strand, with "ABABAB" within the duplex region. As another example, the alternating motif on the sense strand can begin at the 5'-3' end of the strand, and the alternating motif on the antisense strand can begin at the 3'-5' end of the strand, with "BABABA" within the duplex region, with "AABBAABB" within the 5'-3' end of the strand, and the alternating motif on the antisense strand can begin at the 3'-5' end of the strand, thus resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0160] In some embodiments, the dsRNA molecule contains one or more mismatches with the target, either within the duplex or in combination. Mismatches can appear in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on each base pair, but adjacent base analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, such as non-standard or non-standard pairings (as described elsewhere herein), are preferred over standard (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over standard pairings.

[0161] In some embodiments, the dsRNA molecule comprises at least one of the first 1, 2, 3, 4, or 5 base pairs (which may be independently selected from the group A:U, G:U, I:C) within the duplex region from the 5' end of the antisense strand and a mismatch pair, e.g., a non-canonical or non-standard pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0162] In some embodiments, the nucleotide at one position in the duplex region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

[0163] Without wishing to be bound by theory, the introduction of 4'- and / or 5'-modified nucleotides at the 3' end of the phosphodiester (PO), phosphorothioate (PS) and / or phosphorodithioate (PS2) linkage of a dinucleotide at any position in a single- or double-stranded oligonucleotide may exert a steric effect on the internucleotide bond, thus protecting or stabilizing it from nucleases.

[0164] In some embodiments, 5'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in a dsRNA molecule. For example, 5'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in a dsRNA molecule. The alkyl group at the 5' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl can be racemic or chirally pure R or S isomer.

[0165] In some embodiments, a 4'-modified nucleoside is introduced at the 3'-end of a dinucleotide at any position in a dsRNA. For example, a 4'-alkylated nucleoside can be introduced at the 3'-end of a dinucleotide at any position in a dsRNA. The alkyl group at the 4'-position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced at the 3'-end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. The 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0166] In some embodiments, 5'-alkylated nucleosides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the efficacy of the dsRNA. The 5'-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl can be either racemic or chirally pure R or S isomer.

[0167] In some embodiments, 4'-alkylated nucleosides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the efficacy of the dsRNA. The 4'-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl can be either racemic or chirally pure R or S isomer.

[0168] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the efficacy of the dsRNA. The 5'-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. The 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0169] In some embodiments, dsRNA molecules can contain 2'-5' linkages (including 2'-H, 2'-OH, and 2'-OMe, and P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance, or inhibit the sense strand from binding to the antisense strand, or can be used at the 5' end of the sense strand to prevent the sense strand from being activated by RISC. In some embodiments, the sense strand contains a 2'-5' linkage between the N-1 position and the N-2 position counting from the 5' end.

[0170] In some embodiments, dsRNA molecules can contain L-sugars (e.g., L-ribose, L-arabinose, containing 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to promote nuclease resistance, or to inhibit the sense strand from binding to the antisense strand, or can be used at the 5' end of the sense strand to prevent the sense strand from being activated by RISC. In some embodiments, the sense strand contains an L-sugar nucleotide at the 5' end.

[0171] Thus, at least one of the sense strand and the antisense strand may contain at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven or more 2'-deoxy modifications at positions 5 to 17, e.g., positions 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 10 to 16, 10 to 15, 10 to 14, 10 to 13, or 10 to 12, counting from the 5' end of the sense strand or antisense strand.

[0172] In some embodiments, the dsRNA comprises at least three 2'-deoxy modifications, where the 2'-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at position 11 of the sense strand, counting from the 5' end of the sense strand.

[0173] In some embodiments, the dsRNA comprises at least five 2'-deoxy modifications, where the 2'-deoxy modifications are at positions 2, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0174] In some embodiments, the dsRNA comprises at least seven 2'-deoxy modifications, where the 2'-deoxy modifications are at positions 2, 5, 7, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0175] In some embodiments, the antisense strand comprises at least five 2'-deoxy modifications at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand. In some further embodiments thereof, the antisense strand is 18-25 nucleotides in length, preferably 18-23 nucleotides in length.

[0176] In some embodiments, a dsRNA agent may contain one or more non-natural nucleotides. For example, a dsRNA agent may contain less than 20%, for example, less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the dsRNA does not contain any non-natural nucleotides. For example, a dsRNA agent contains all natural nucleotides. Some exemplary non-natural nucleotides include, but are not limited to, acyclic nucleotides, locked nucleic acids (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0177] Ligand A variety of entities can be coupled to the oligonucleotides of the invention. Preferred moieties are ligands, preferably covalently coupled, either directly or indirectly via an intervening tether.

[0178] In preferred embodiments, the ligand alters the distribution, targeting, or lifetime of the molecule into which it is incorporated. In preferred embodiments, the ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, a receptor, e.g., a cell or organ compartment, a tissue, an organ, or a body region, compared to, e.g., a species not possessing such a ligand. A ligand that provides enhanced affinity for a selected target is also referred to as a targeting ligand.

[0179] Some ligands may have endosomolytic properties. Endosomolytic ligands promote endosome lysis and / or transport of the compositions of the present invention or components thereof from endosomes to the cytoplasm of cells. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenicity. In some embodiments, the endosomolytic ligand adopts its active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes endosome lysis and / or transport of the compositions of the present invention or components thereof from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972, incorporated by reference in its entirety), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586, incorporated by reference in its entirety), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, incorporated by reference in its entirety). In some embodiments, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0180] The ligands may improve the transport, hybridization and specificity properties, and may improve the nuclease resistance of the resulting natural or modified oligoribonucleotides or polymeric molecules comprising any combination of monomers and / or natural or modified ribonucleotides described herein.

[0181] Ligands may generally include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0182] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids, oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylate), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0183] Ligands can also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, such as antibodies that bind to specific cell types, such as kidney cells. The targeting group can be thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD peptidomimetics, or aptamers. Table 2 shows some examples of targeting ligands and their associated receptors.

[0184] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid. , O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ conjugates of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0185] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for a co-ligand; or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharide, p38 MAP kinase activator, or NF-κB activator.

[0186] The ligand can be, for example, a substance such as a drug that can increase uptake of an iRNA agent into a cell by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0187] Ligands can enhance cellular uptake of oligonucleotides, for example, by activating an inflammatory response. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF-α), interleukin-1 beta, or gamma interferon.

[0188] In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the distribution of the conjugate to target tissues, such as non-renal target tissues in the body. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the degradation resistance of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to regulate the binding of serum proteins, such as HSA.

[0189] For example, lipid-based ligands can be used to inhibit, such as by regulating the binding of the complex to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the complex to the kidney.

[0190] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

[0191] In other preferred embodiments, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can also be used instead of or in addition to the lipid-based ligand.

[0192] In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, and other vitamins or nutrients that are taken up by cancer cells. Also included are HAS and low-density lipoproteins (LDL) and high-density lipoproteins (HDL).

[0193] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents are peptides such as tat or antenopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidylmimetic, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having a lipophilic and lipophobic phase.

[0194] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. The peptide or peptidomimetic moiety can be, for example, about 5 to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can contain a hydrophobic membrane translocating sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide capable of transporting numerous polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) have been shown to function as delivery peptides. Peptides or peptidomimetics, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-94, 1991, incorporated by reference in its entirety), can be encoded by random sequences of DNA. Preferably, the peptide or peptidomimetic tethered to the iRNA agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide moiety can range from about 5 amino acids in length to about 40 amino acids in length. The peptide moiety may have structural modifications, for example to enhance stability or to direct conformational properties. Any of the following structural modifications may be used:RGD peptide moieties can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002, incorporated by reference in its entirety). RGD peptides can facilitate targeting of iRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001, incorporated by reference in its entirety). Preferably, RGD peptides will facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, such as glycosylated or methylated, to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing αVβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, incorporated by reference in its entirety). Peptides that target markers abundant in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells that display integrins. Thus, RGD peptides, cyclic peptides with RGD, RGD peptides containing D-amino acids, and synthetic RGD mimics can be used. In addition to RGD, other moieties that target integrin ligands can be used. Generally, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or other oncogenes, such as those described herein.

[0195] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or cecropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003, incorporated by reference in its entirety).

[0196] In some embodiments, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. Clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, escalentin-1, and caerin. To maintain the integrity of helical stability, several factors are preferably considered. For example, a maximum number of helix-stabilizing residues will be used (e.g., leu, ala, or lys) and a minimum number of helix-destabilizing residues will be used (e.g., proline or cyclic monomer units). Capping residues will be considered (e.g., Gly is an exemplary N-capping residue), and / or C-terminal amidation can be used to provide additional H-bonds and stabilize the helix. The formation of salt bridges between oppositely charged residues separated by the i±3 or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homoarginine, ornithine, or histidine can form salt bridges with the anionic residues glutamic acid or aspartic acid.

[0197] Peptide and peptidomimetic ligands include natural or modified peptides, such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds, i.e., peptides in which a bond is replaced with one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides.

[0198] The targeting ligand can be any ligand capable of targeting a specific receptor. Examples include folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters, such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. The ligand can also be based on nucleic acids, such as aptamers. The aptamer can be unmodified or have any combination of the modifications disclosed herein.

[0199] Endosomal release agents include imidazoles, poly- or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charge, dendrimers with masked or unmasked cationic or anionic charge.

[0200] PK modifiers refer to pharmacokinetic modifiers. PK modifiers include lipophilic agents, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modifiers include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins, i.e., short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, and the inclusion of multiple phosphorothioate linkages in the backbone further makes them suitable as ligands (e.g., as PK-modulating ligands) for the present invention.

[0201] Furthermore, aptamers that bind to serum components (eg, serum proteins) are also suitable as PK-modulating ligands in the present invention.

[0202] Other ligand conjugates suitable for the present invention are described in U.S. patent applications U.S. Patent Application Publication Nos. 10 / 916,185, filed August 10, 2004; 10 / 946,873, filed September 21, 2004; 10 / 833,934, filed August 3, 2007; 11 / 115,989, filed April 27, 2005; and 11 / 944,227, filed November 21, 2007, which are incorporated by reference in their entireties for all purposes.

[0203] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while others have different properties. For example, the ligands may have targeting properties, endosomolytic activity, or PK modulating properties. In a preferred embodiment, all ligands have different properties.

[0204] The ligand can be coupled to the oligonucleotide at various locations, e.g., the 3' end, the 5' end, and / or an internal position. In preferred embodiments, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier as described herein. The ligand or tethered ligand can be present on a monomer when the monomer is incorporated into a growing chain. In some embodiments, the ligand can be incorporated via coupling to a "precursor" monomer after the "precursor" monomer is incorporated into a growing chain. For example, TAP-(CH2) nMonomers bearing an amino-terminated tether such as NH (i.e., without an associated ligand) can be incorporated into a growing oligonucleotide chain. In a subsequent operation, i.e., after incorporation into the precursor monomer chain, a ligand bearing an electrophilic group, such as a pentafluorophenyl ester or aldehyde group, can then be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the tether of the precursor monomer.

[0205] In another example, a monomer can be incorporated bearing a chemical group suitable for participation in a click chemistry reaction, such as an azide or alkyne terminated tether / linker. In a subsequent operation, i.e., after incorporation into the precursor monomer chain, a ligand bearing a complementary chemical group, such as an alkyne or azide, can be attached to the precursor monomer by coupling the alkyne and azide together.

[0206] In a double-stranded oligonucleotide, the ligand can be attached to one or both strands. In some embodiments, a double-stranded iRNA agent has a ligand conjugated to the sense strand. In other embodiments, a double-stranded iRNA agent has a ligand conjugated to the antisense strand.

[0207] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or a derivative thereof can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is bound to a conjugate moiety. Conjugation to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be bound to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be bound to a conjugate moiety, for example, at an abasic residue. The internucleoside linkage can also have a conjugate moiety. In phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, dithiophosphate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0208] In some embodiments, the ligand is conjugated to the sense strand. As described herein, the ligand can be conjugated to the 3'-end, 5'-end, or internal position of the sense strand. In some embodiments, the ligand is conjugated to the 3'-end of the sense strand. Furthermore, the ligand can be conjugated to a nucleobase, sugar moiety, or internucleotide linkage of the sense strand.

[0209] While any suitable ligand in the field of RNA interference can be used, the ligand is typically a carbohydrate, such as a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, or polysaccharide.

[0210] Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylgalactosamine) derivatives attached through a monovalent, divalent, or trivalent branched linker.

[0211] In some embodiments, the dsRNAs of the invention are conjugated to bivalent and trivalent branched linkers and have formulas (IV)-(VII): [ka] (In the formula, q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5C independently represents 0 to 20 occurrences, where the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C each independently represents the absence, presence of CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C independently represent each occurrence of absence, alkylene, and substituted alkylene, where one or more methylenes are O, S, S(O), SO, N(RN ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or each occurrence of heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each independently represents each occurrence of a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R a is H or an amino acid side chain) The structure includes any of the structures shown in

[0212] The trivalent conjugated GalNAc derivative can be used to transfect a target gene, for example, a gene of formula (VII): [ka] (In the formula, L 5A , L 5B and L 5C represents a monosaccharide, e.g., a GalNAc derivative) These are particularly useful in combination with RNAi agents to inhibit the expression of

[0213] Examples of suitable bivalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the following compounds: [ka] [ka] [ka] Examples include:

[0214] In some embodiments, the dsRNA described herein comprises Ligand 1, a ligand having the following structure: [ka]

[0215] In some embodiments, the dsRNA described herein comprises a ligand described in U.S. Pat. No. 5,994,517 or U.S. Pat. No. 6,906,182, the contents of each of which are incorporated by reference in their entirety.

[0216] In some embodiments, the ligand may be a triantennary ligand as described in Figure 3 of U.S. Patent No. 6,906,182. For example, the dsRNA described herein may include a ligand selected from the following triantennary ligands: [ka]

[0217] In some embodiments, the ligand is a multivalent ligand, e.g., a ligand of formula (VII). In some further embodiments, the ligand is a GalNAc derivative, e.g., a ligand selected from ligands 1-8 disclosed herein.

[0218] The ligand can be attached to the dsRNA via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally refers to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, such as a phosphate backbone or a modified phosphate backbone, such as a sulfur-containing backbone. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier by an intervening tether. Thus, the cyclic carriers will often contain functional groups, such as amino groups, or generally provide suitable bonds for incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring.

[0219] In one embodiment, the dsRNA molecule of the present invention is conjugated to the ligand via a carrier, wherein the carrier can be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0220] The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the ligand can be conjugated to the sense strand, particularly the 3' end of the sense strand.

[0221] In some embodiments, the dsRNA molecule comprises at least one ASGPR ligand. For example, the ASGPR ligand is [ka] and one or more GalNAc derivatives attached via a bivalent or trivalent branched linker, such as:

[0222] In one example, the ASGPR ligand is attached to the 3' end of the sense strand.

[0223] Linker Embodiments of the conjugates disclosed herein include linkers, which may contain one or more linker components. The term "linker" refers to an organic moiety that connects two parts of a compound, e.g., a DVD immunoglobulin to a dsRNA. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR 1 , C(O), C(O)O, C(O)NR 1, SO, SO2, SO2NH, and the like, a unit or series of atoms, such as, for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocyclyl alkyl, heterocyclyl alkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylaryl alkyl, alkenylaryl alkenyl, alkenylaryl alkynyl, alkynylaryl alkyl, alkynylaryl alkenyl, alkynylaryl alkynyl, alkylheteroaryl alkyl, alkylheteroaryl alkenyl, alkynylaryl alkynyl, alkyl alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (wherein one or more methylenes are selected from the group consisting of O, S, S(O), SO, N(R 1 )2, which may be interrupted or terminal by C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic group; 1 is hydrogen, acyl, aliphatic or substituted aliphatic), and the like.

[0224] Without limitation, the subject dsRNAs may include a variety of linker functionalities, including, but not limited to, cleavable and non-cleavable linkers and reversible and irreversible linkers.

[0225] In some embodiments, the linker is a cleavable linker. A cleavable linker relies on a process within the target cell, such as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by a specific enzyme (e.g., a protease) within the cell, to release the two parts held together by the linker. Thus, the cleavable linker allows the two parts to be released in their original form after internalization and processing within the target cell. Cleavable linkers include, but are not limited to, those whose bond can be cleaved by enzymes (e.g., peptide linkers); reducing conditions (e.g., disulfide linkers); or acidic conditions (e.g., hydrazones and carbonates).

[0226] Generally, a cleavable linker comprises at least one cleavable linking group that is sufficiently stable outside a cell but that, upon entry into a target cell, is cleaved, releasing the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least 10 times faster, preferably at least 100 times faster, in the target cell or under a first reference condition (e.g., which may be selected to mimic or represent intracellular conditions) or under a second reference condition (e.g., which may be selected to mimic or represent conditions found in blood or serum) than in the subject's blood or serum.

[0227] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate or do not have substrate specificity, such as intracellular oxidizing or reducing enzymes or reducing agents that can degrade redox-cleavable linking groups by reduction, such as mercaptans; esterases; endosomes or agents that can create an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0228] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or to a desired compartment of the cell.

[0229] The linker can contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the cell to be targeted. For example, a ligand targeting the liver can be linked to a cationic lipid through a linker containing an ester group. Liver cells are rich in esterase, and therefore the linker is cleaved more efficiently in liver cells than in cell types that are not rich in esterase. Other cell types that are rich in esterase include lung, renal cortex, and testicular cells. Linkers containing peptide bonds can be used to target cell types that are rich in peptidase, such as hepatocytes and synovial cells.

[0230] In general, the suitability of candidate cleavable linkers can be evaluated by testing the ability of degradable agents (conditions) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, with the first condition selected to be indicative of cleavage in target cells and the second condition selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0231] One class of cleavable linkers is redox-cleavable linkers, which can be used in dsRNA molecules according to the present invention and are cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be relied upon. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, a candidate compound is cleaved at a maximum of 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0232] Phosphate-based cleavable linkers can be used in dsRNA molecules according to the present invention and are cleaved by agents that decompose or hydrolyze phosphate groups. An example of an agent that cleaves phosphate groups in cells is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linkers are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0233] Acid-cleavable linkers can be used in dsRNA molecules according to the present invention and are cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.5, 5.0 or below) or by an agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when a carbon atom is attached to the oxygen of the ester (alkoxy group), it is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0234] Ester-based cleavable linking groups can be used in dsRNA molecules according to the present invention, and are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0235] Peptide-based cleavable linkers can be used in dsRNA molecules according to the present invention and are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. Peptide bonds are a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0236] Use of dsRNA The dsRNA described herein can be used to inhibit the expression of target gene.Therefore, in another aspect, the present invention provides a method for inhibiting the expression of target gene.This method comprises administering the dsRNA described herein to cell in an amount sufficient to inhibit the expression of target gene.In a preferred embodiment, the present invention further relates to the use of the dsRNA described herein for inhibiting the expression of target gene in target cell in vitro.

[0237] Exemplary target genes include, but are not limited to, β-catenin (CTNNB1), IRF4, Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, activated protein C, cyclin D gene, These include mutations in the VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, p73 gene, p21 (WAF1 / CIP1) gene mutation, p27 (KIP1) gene mutation, PPM1D gene mutation, RAS gene mutation, caveolin I gene mutation, MIB I gene mutation, MTAI gene mutation, M68 gene mutation, tumor suppressor gene mutation, and p53 tumor suppressor gene mutation.

[0238] In some aspects, the dsRNAs described herein can be used to treat a subject or mammal.

[0239] Pharmaceutical Composition For use in treatment, dsNRA as described herein can be formulated into pharmaceutical compositions.Therefore, in another aspect, the present invention provides pharmaceutical compositions comprising dsRNA as defined herein.Pharmaceutically acceptable compositions comprise one or more of the dsRNA as described herein in therapeutically effective amounts, which can be taken alone or be combined with one or more pharmaceutically acceptable carriers (additives), excipients and / or diluents.

[0240] Pharmaceutical compositions can be specifically formulated for administration in solid or liquid form, such as (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained-release formulation; (3) topical application, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin; (4) vaginally or rectally, e.g., as a pessary, cream, or foam; (5) sublingually; (6) intraocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.

[0241] The phrase "therapeutically effective amount," as used herein, means an amount of a compound, material, or composition, including a conjugate described herein, that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable risk-benefit ratio applicable to any medical treatment.

[0242] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with the tissues of human beings and animals, without excessive toxicity, irritation, allergic response, or other problems or complications, consistent with a reasonable risk-benefit ratio.

[0243] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (24) other non-toxic affinity substances used in pharmaceutical formulations.

[0244] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except as long as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0245] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of compound which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0246] In certain embodiments, a formulation of the invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and a conjugate described herein. In certain embodiments, the formulation renders the conjugate described herein orally bioavailable.

[0247] The dsRNA formulation may be formulated in combination with another agent, such as another therapeutic agent or an agent that stabilizes the dsRNA. Additional agents include chelating agents, such as EDTA (e.g., Mg 2+ (for removing divalent cations such as ribonuclease inhibitors), salts, ribonuclease inhibitors (for example, broad-specificity ribonuclease inhibitors such as RNAsin), and the like.

[0248] Methods of preparing these formulations or compositions include the step of bringing into association a dsRNA of the invention with the carrier(s), and, optionally, one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing into association a dsRNA of the invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0249] In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a suspension of crystalline or amorphous material with poor water solubility. Here, the absorption rate of the drug depends on its dissolution rate, which in turn depends on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0250] The dsRNA according to the invention, similar to other pharmaceutical agents, may be formulated for administration in any convenient manner used in human or veterinary medicine.

[0251] The compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and / or dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0252] The composition must be sterile and fluid to the extent that delivery of the composition by syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution.

[0253] Administration route The dsRNA described herein or pharmaceutical compositions comprising it can be administered by various methods known in the art.As those skilled in the art will understand, the route and / or mode of administration will vary depending on the target disease or condition and the desired results.In order to administer the dsRNA described herein by a specific administration route, it may be necessary to coat the dsRNA with a material that prevents its inactivation, or to co-administer the dsRNA with the material.For example, the dsRNA can be administered to a subject in a suitable carrier, such as liposomes or a diluent.Pharmaceutically acceptable diluents include physiological saline and aqueous buffer solution.

[0254] Exemplary routes of administration include, but are not limited to, intravenous, subcutaneous, intratumoral, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

[0255] The compositions of the present invention can be administered in a number of ways, depending on whether local or systemic treatment is desired and to the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

[0256] The route and site of administration can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the target muscle is a logical choice. Lung cells can be targeted by administering dsRNA in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with dsRNA and mechanically introducing the dsRNA.

[0257] dose The actual dosage level of active ingredient in pharmaceutical compositions of the present invention, for example, dsRNA as described herein, can be modified to achieve the amount of active ingredient that is effective for achieving the desired therapeutic response for specific patient, composition and administration mode, without toxicity to patient.The dosage level selected will depend on various pharmacokinetic factors, including the activity of the specific composition of the present invention used, route of administration, administration time, excretion rate of the specific compound used, duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health and past medical history of the patient under treatment and similar factors well known in the medical field.

[0258] In some embodiments, the unit dose is less than 10 mg / kg body weight or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg / kg body weight and less than 200 nmoles (e.g., about 4.4 x 10 copies) / kg body weight of dsRNA molecule or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmoles / kg body weight of dsRNA molecule.

[0259] The specified amount can be an amount effective for treating or preventing a disease or disorder, such as a disease or disorder associated with the target gene. For example, the unit dose can be administered by injection (e.g., intravenous, subcutaneous, or intramuscular), inhalation, or topical application. In some embodiments, the dose can be less than 10, 5, 2, 1, or 0.1 mg / kg body weight.

[0260] In some embodiments, the unit dose is administered less than once a day, for example, less than every 2, 4, 8, or 30 days. In other embodiments, the unit dose is not administered with any frequency (e.g., a regular frequency). For example, the unit dose may be administered once.

[0261] In some embodiments, the effective dose is administered with other conventional therapeutic modalities.

[0262] In some embodiments, a subject is administered an initial dose and one or more maintenance doses. The one or more maintenance doses can be the same as or lower than the initial dose, e.g., half a dose less than the initial dose. A maintenance dosing regimen can involve treating a subject with one or more doses ranging from 0.01 μg to 15 mg / kg body weight / day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg / kg body weight / day. Maintenance doses can be administered, for example, no more than once every 2, 5, 10, or 30 days. Furthermore, a treatment regimen can last for a period of time, the duration of which will vary depending on the nature of the patient's particular disease, its severity, and general condition. In certain embodiments, the dose can be delivered no more than once a day, e.g., no more than once every 24, 36, 48, or longer, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in their condition and for relief of symptoms of the condition. The dose of the compound may be increased if the patient does not respond significantly to the current dose level, or the dose may be decreased if a reduction in symptoms of the condition is observed, if the condition disappears, or if unwanted side effects are observed.

[0263] An effective dose may be administered in a single dose or in two or more doses as desired or as deemed appropriate under the particular circumstances. Where it is desired to facilitate repeated or frequent infusions, implantation of a delivery device, such as a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intra-articular), or reservoir may be advisable.

[0264] In some embodiments, the composition comprises multiple dsRNA species. In another embodiment, the dsRNA species have sequences that do not overlap with or are not adjacent to other species of naturally occurring target sequences. In another embodiment, the multiple dsRNA species are specific to different naturally occurring target genes. In another embodiment, the dsRNA molecules are allele-specific.

[0265] The dsRNA described herein can be administered to mammals, particularly large mammals such as non-human primates or humans, in several ways.

[0266] In some embodiments, administration of dsRNA is parenteral, for example intravenous (for example, as bolus or diffusion infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or intraocular administration.Administration can be provided by subject or another person, for example, health care provider.Dosage can be provided in measured dose or by dispensing device that delivers metered dose.

[0267] Liposomes and lipid formulations The dsRNA described herein can be formulated for delivery in membranous molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, for example, one or more bilayers. Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an internal aqueous solution. The aqueous solution portion contains the siRNA composition. The lipophilic material separates the internal aqueous solution from the external aqueous solution, which typically does not contain the dsRNA composition, but in some cases may. Liposomes are useful for transporting and delivering active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when the liposome is applied to a tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As the liposome and cell merge, the contents of the internal aqueous solution, including the dsRNA described herein, are delivered into the cell, where the dsRNA can specifically bind to the target RNA and mediate RNAi. In some cases, the liposomes are also specifically targeted, for example, to direct conjugation to a particular cell type.

[0268] The liposomes containing dsRNA described herein can be prepared by various methods. In one example, the lipid components of liposomes are dissolved in detergent, thus forming micelles with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. Next, the dsRNA formulation is added to the micelles containing the lipid components. The cationic groups on the lipids interact with the dsRNA and condense around the dsRNA to form liposomes. After condensation, the detergent is removed, for example, by dialysis, resulting in a liposome formulation of dsRNA.

[0269] During the condensation reaction, if necessary, a carrier compound that promotes condensation can be added, for example, by controlled addition. For example, this carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0270] Further description of methods for making stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle is provided, for example, in WO 96 / 37194. For liposome formation, see Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga,et al. Endocrinol. 115:757, 1984 (which are incorporated by reference in their entirety). Commonly used techniques for preparing lipid aggregates of an appropriate size for use as a delivery vehicle include sonication and freeze-thaw extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986 (which are incorporated by reference in their entirety)). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidic technology can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984 (which are incorporated by reference in their entirety). These methods are readily adapted to encapsulate siRNA formulations in liposomes.

[0271] pH-sensitive or negatively charged liposomes entrap nucleic acid molecules rather than complexing with them. Because both nucleic acid molecules and lipids have similar charges, repulsion occurs rather than complexation. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of such liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cultured cell monolayers. Expression of this exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated by reference in its entirety).

[0272] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Generally, anionic liposome compositions are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0273] Other examples of methods for introducing liposomes into cells in vitro include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0274] In some embodiments, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with plasma membrane effectively, but it can be taken up by macrophage in vivo, and can be used to deliver siRNA to macrophage.

[0275] Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect the encapsulated siRNA in their internal compartment from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important points to consider in the preparation of liposome formulations are the lipid surface charge of the liposome, the vesicle size and the aqueous solution volume.

[0276] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that have the ability to fuse with negatively charged lipids in the plasma membrane of tissue culture cells, resulting in delivery of siRNA (for a description of DOTMA and its use with DNA, see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355, which is incorporated by reference in its entirety).

[0277] The DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to live tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficient positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are linked by ester bonds rather than ether bonds.

[0278] Other reported cationic lipid compounds include those conjugated to various moieties, including carboxyspermine, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678), conjugated to one of two types of lipids.

[0279] Another cationic lipid conjugate includes cholesterol-derivatized lipid ("DC-Chol"), which is combined with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim.Biophys.Res.Commun.179:280,1991).It has been reported that lipopolylysine, which is made by conjugating polylysine with DOPE, is effective for transfection in the presence of serum (Zhou, X. et al., Biochim.Biophys.Acta1065:8,1991, which is incorporated by reference in its entirety).It is said that for certain cell lines, these liposomes containing conjugated cationic lipids exhibit lower toxicity and result in higher transfection efficiency than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0280] Liposome preparations are particularly suitable for topical administration.Liposomes have several advantages over other preparations.These advantages include reducing the side effects associated with high systemic absorption of administered drugs, increasing the accumulation of administered drugs in desired targets, and being able to administer siRNA to skin.In some implementations, liposomes are used to deliver siRNA to epidermal cells and enhance the penetration of siRNA into skin tissues, such as skin.For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987, which are incorporated by reference in their entireties.

[0281] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin.Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) nonionic liposome formulations have been used to deliver drugs to the dermis of mouse skin.Such formulations containing siRNA are useful for treating dermatological disorders.

[0282] Liposomes containing the conjugates described herein can be highly deformable. Such deformability can allow liposomes to pass through pores smaller than the average radius of the liposomes. For example, transfersomes are a type of deformable liposome. Transfersomes can be created by adding a surface edge activator, usually a surfactant, to a standard liposome composition. To deliver siRNA to keratinocytes in the skin, transfersomes containing siRNA can be delivered subcutaneously, for example, by infection. To cross intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to their lipid properties, these transfersomes are self-optimizing (e.g., they can adapt to the shape of pores in the skin), self-repairing, can frequently reach their target without fragmentation, and can often be self-loading.

[0283] Other formulations suitable for the present invention are described in U.S. Provisional Patent Applications Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT application PCT / US2007 / 080331, filed October 3, 2007, also describes formulations suitable for the present invention.

[0284] Surfactants. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). Conjugate formulations can include surfactants. In some embodiments, the conjugates described herein are formulated as emulsions containing surfactants. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means of distinguishing the various surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms," ​​Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0285] If the surfactant molecule does not ionize, it is classified as a nonionic surfactant. Nonionic surfactants have wide application in pharmaceutical products and can be used over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most well-known members of the nonionic surfactant class.

[0286] If surfactant molecule has negative charge when dissolved or dispersed in water, the surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactates, acyl amides of amino acids, sulfuric acid esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates, sulfosuccinates and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0287] If the surfactant molecule has a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0288] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0289] The use of surfactants in drug products, formulations, and emulsions has been reviewed (Rieger, "Pharmaceutical Dosage Forms," ​​Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0290] Micelle and other membranous formulations. Formulations containing the conjugates described herein can be provided as micellar formulations. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that the hydrophobic portions of the molecules all face inward and the hydrophilic portions are in contact with the surrounding aqueous phase. The opposite arrangement exists when the environment is hydrophobic.

[0291] Mixed micelle formulations suitable for delivery through transdermal membranes contain an aqueous solution of the siRNA composition, alkali metal C8-C 22 They can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable hyaluronates, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compounds can be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles can be formed by virtually any mixture of these components, provided that vigorously mixing is performed to produce small micelles.

[0292] In one method, a first micelle composition is prepared containing a conjugate described herein and at least an alkali metal alkyl sulfate. This first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing a conjugate described herein, an alkali metal alkyl sulfate, and at least one of the micelle-forming compounds, followed by adding the remaining micelle-forming compounds with vigorous mixing.

[0293] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and prevent bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0294] To deliver the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is charged with a propellant. The propellant, under pressure, is in liquid form inside the dispenser. The ratio of these components is adjusted so that the aqueous phase and the propellant phase are combined, i.e., there is one phase. If there are two phases, the dispenser must be shaken before dispensing a portion of the contents, for example, from a metered valve. The dispensed dose of the drug is ejected from the metered valve as a fine spray.

[0295] Propellants can include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) can be used.

[0296] The specific concentrations of the essential ingredients can be determined by relatively simple experimentation. For oral absorption, it is often desirable to increase the dose, e.g., at least double or triple, that given by injection or gastrointestinal administration.

[0297] Particles. In some embodiments, the conjugates described herein can be incorporated into particles, such as microparticles. Microparticles can be made by spray drying, but can also be made by other methods, including freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0298] kit The present invention also provides kits that include the dsRNAs described herein.

[0299] Any one exemplary embodiment of this aspect can be described as follows.

[0300] Embodiment 1: A double-stranded RNA (dsRNA) molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, the dsRNA molecule comprises a hexopyranose nucleoside, and the dsRNA comprises a ligand.

[0301] Embodiment 2: A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, and wherein the antisense strand comprises at least one hexopyranose nucleoside in a seed region of the antisense strand, or the sense strand comprises at least one altritol nucleotide in a central region of the sense strand.

[0302] Embodiment 3: A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, and the dsRNA comprises at least one hexopyranose nucleoside, at least one 2'-fluoronucleotide, and at least one 2'-OMe nucleotide.

[0303] Embodiment 4: A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14-40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, the dsRNA comprises at least one hexopyranose nucleoside, and the dsRNA comprises at least four phosphorothioate internucleotide linkages.

[0304] Embodiment 5: The double-stranded RNA molecule of any one of embodiments 1 to 4, wherein the antisense strand comprises a hexopyranose nucleoside in the seed region of the antisense strand.

[0305] Embodiment 6: The double-stranded RNA molecule of any one of embodiments 1 to 5, wherein the antisense strand comprises a hexopyranose nucleoside at least at one of positions 3 to 8, counting from the 5' end of the antisense strand.

[0306] Embodiment 7: The double-stranded RNA molecule of any one of embodiments 1 to 6, wherein the antisense strand comprises a hexopyranose nucleoside at least one of positions 6, 7, 9, 12, 16, 21, and 22, counting from the 5' end of the antisense strand.

[0307] Embodiment 8: The double-stranded RNA molecule of embodiment 7, wherein the antisense strand comprises a hexopyranose nucleoside at at least one of positions 6, 7, and 16.

[0308] Embodiment 9: The double-stranded RNA molecule of embodiment 8, wherein the antisense strand comprises a hexopyranose nucleoside at at least one of positions 6 and 7.

[0309] Embodiment 10: The double-stranded RNA molecule of embodiment 9, wherein the antisense strand comprises a hexopyranose nucleoside at position 7.

[0310] Embodiment 11: The double-stranded RNA molecule of any one of embodiments 1 to 10, wherein the sense strand comprises at least two or more consecutive independently selected hexopyranose nucleosides.

[0311] Embodiment 12: The double-stranded RNA molecule of any one of embodiments 1 to 11, wherein the sense strand comprises at least three or more consecutive independently selected hexopyranose nucleosides.

[0312] Embodiment 13: The double-stranded RNA molecule of any one of embodiments 1 to 12, wherein the sense strand comprises a hexopyranose nucleoside in the central region of the sense strand.

[0313] Embodiment 14: The double-stranded RNA molecule according to any one of embodiments 1 to 13, wherein the sense strand comprises a hexopyranose nucleoside at least one of positions 3 and 12.

[0314] Embodiment 15: The double-stranded RNA molecule of any one of embodiments 1 to 14, wherein the antisense strand comprises at least two or more consecutive independently selected hexopyranose nucleosides.

[0315] Embodiment 16: The double-stranded RNA molecule of any one of embodiments 1 to 15, wherein the antisense strand comprises at least three or more consecutive independently selected hexopyranose nucleosides.

[0316] Embodiment 17: The double-stranded RNA molecule of any one of embodiments 1 to 16, wherein the sense strand comprises a hexopyranose nucleoside at the 5' end of the sense strand.

[0317] Embodiment 18: The double-stranded RNA molecule of any one of embodiments 1 to 17, wherein the sense strand comprises a hexopyranose nucleoside at the 5' end of the sense strand.

[0318] Embodiment 19: The double-stranded RNA molecule according to any one of embodiments 1 to 18, wherein the sense strand comprises a 5'-vinylphosphonate (VP) group.

[0319] Embodiment 20: The double-stranded RNA molecule of any one of embodiments 1 to 19, wherein the hexopyranose nucleotide is selected from the group consisting of allopyranose nucleotides, altritol nucleotides, glucopyranose nucleotides, mannopyranose nucleotides, gulopyranose nucleotides, idopyranose nucleotides, galactopyranose nucleotides, talopyranose nucleotides, fucopyranose nucleotides, rhamnopyranose nucleotides, quinovopyranose nucleotides, pneumopyranose nucleotides, and any combination thereof.

[0320] Embodiment 21: The double-stranded RNA molecule of any one of embodiments 1 to 20, wherein the hexopyranose nucleotides are selected from the group consisting of altritol nucleotides, glucopyranose nucleotides, mannopyranose nucleotides, galactopyranose nucleotides, fucopyranose nucleotides, and any combination thereof.

[0321] Embodiment 22: A double-stranded RNA molecule according to any one of embodiments 2 to 21, comprising a ligand.

[0322] Embodiment 23: The double-stranded RNA molecule of embodiment 1 or 22, wherein the ligand is an ASGPR ligand.

[0323] Embodiment 24: The ASGPR ligand is [ka] 24. The double-stranded RNA molecule of embodiment 23, wherein

[0324] Embodiment 25: The double-stranded RNA molecule according to any one of embodiments 1 to 24, wherein the sense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages.

[0325] Embodiment 26: The double-stranded RNA molecule according to any one of embodiments 1 to 25, wherein the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end).

[0326] Embodiment 27: The double-stranded RNA molecule according to any one of embodiments 1 to 26, wherein the antisense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages.

[0327] Embodiment 28: The double-stranded RNA molecule of any one of embodiments 1 to 27, wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end).

[0328] Embodiment 29: The double-stranded RNA molecule of any one of embodiments 1 to 28, wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end).

[0329] Embodiment 30: The double-stranded RNA molecule of any one of embodiments 1 to 29, wherein the sense strand and the antisense strand are independently 19 to 25 nucleotides in length.

[0330] Embodiment 31: The double-stranded RNA molecule of any one of embodiments 1 to 30, wherein the sense strand is 21 nucleotides in length.

[0331] Embodiment 32: The double-stranded RNA molecule of any one of embodiments 1 to 31, wherein the antisense strand is 23 nucleotides in length.

[0332] Embodiment 33: The double-stranded RNA molecule according to any one of embodiments 1 to 32, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0333] Embodiment 34: The double-stranded RNA molecule of any one of embodiments 1 to 33, comprising a single-stranded overhang at the 3' end of the antisense strand.

[0334] Embodiment 35: The double-stranded RNA molecule of any one of embodiments 1 to 34, comprising a blunt end at the 5' end of the antisense strand.

[0335] Embodiment 36: A double-stranded RNA molecule according to any one of embodiments 1 to 35, comprising 2'-fluoro or 2'-OMe nucleotides at positions complementary to the hexopyranose nucleotides.

[0336] Embodiment 37: The double-stranded RNA molecule of any one of embodiments 1 to 36, wherein the double-stranded RNA molecule does not comprise any nucleotides other than hexopyranose, 2'-fluoro and 2-OMe nucleotides.

[0337] Embodiment 38: The double-stranded RNA molecule of any one of embodiments 1 to 36, comprising a nucleotide selected from the group consisting of acyclic nucleotides, locked nucleic acids (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, and 2'-deoxy.

[0338] Embodiment 39: The double-stranded RNA molecule of any one of embodiments 1 to 38, wherein the sense strand comprises a 5'-morpholino modification, a 5'-dimethylamino modification, a 5'-deoxy modification, an inverted abasic modification, or an inverted abasic locked nucleic acid modification at the 5' end.

[0339] Embodiment 40: The double-stranded RNA molecule according to any one of embodiments 1 to 39, having a melting temperature in the range of about 40°C to about 80°C.

[0340] Embodiment 41: A pharmaceutical composition comprising the dsRNA agent of any one of embodiments 1-40, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0341] Embodiment 42: A gene silencing kit comprising a dsRNA molecule according to any one of embodiments 1 to 40.

[0342] Embodiment 43: A method for silencing a target gene in a cell, comprising introducing into the cell a dsRNA molecule according to any one of embodiments 1 to 40.

[0343] Embodiment 44: The method of embodiment 43, wherein the dsRNA agent is administered via subcutaneous or intravenous administration.

[0344] Embodiment 45: A method for silencing a target gene in a cell, the method comprising the step of allowing the cell to express a dsRNA molecule according to any one of embodiments 1 to 40.

[0345] Some selected definitions For convenience, we now collect here certain terms used in the specification, examples, and appended claims. Unless otherwise specified or implicit from context, the following terms and phrases include the meanings provided below. Unless otherwise specified or apparent from context, the following terms and phrases do not exclude the meaning that the term or phrase has previously acquired in the relevant art. These definitions are provided to aid in the description of particular embodiments and are not intended to limit the invention as claimed, since the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0346] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials can be used in the practice or testing of the present invention, the methods, devices, and materials are now described in this connection.

[0347] Furthermore, the practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., eds., 1987 and regularly updated editions); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0348] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the scope of the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the scope of the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0349] Certain ranges are provided herein with the term "about" before the numerical values. The term "about" is used herein to provide literal support for the number it precedes as well as for numbers that are near or approximately the number it precedes. In determining whether a number is near or approximately a specifically stated number, the unstated near or approximately number may be a number that, given the context in which it is presented, provides a substantial equivalent to the specifically stated number.

[0350] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and one or more components thereof, respectively, which may include elements essential to the invention, but not specified, whether essential or not.

[0351] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this provision is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0352] As used herein, the terms "dsRNA", "siRNA" and "iRNA agent" are used interchangeably to refer to agents that can mediate the silencing of target RNA, such as mRNA, for example, the transcript of a gene encoding a protein.For convenience, this mRNA is also referred to herein as the mRNA to be silenced.This gene is also referred to as target gene.Generally, the RNA to be silenced is an endogenous gene, an exogenous gene or a pathogen gene.In addition, RNA other than mRNA, such as tRNA and viral RNA, can also be targeted.

[0353] As used herein, the phrase "mediating RNAi" refers to the ability to sequence-specifically silence a target gene, e.g., mRNA. Without wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., the antisense strand of a dsRNA, which is 21-23 nucleotides in length.

[0354] As used herein, "specifically hybridizable" and "complementary" are terms used to indicate that there is a sufficient degree of complementarity between the compound of the present invention and the target RNA molecule such that stable and specific binding occurs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatments, or under conditions where the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0355] In some embodiments, a dsRNA molecule is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the dsRNA molecule silences the production of a protein encoded by the target mRNA. In another embodiment, the dsRNA molecule is "exactly complementary" to the target RNA, e.g., the target RNA and the dsRNA duplex agent anneal to form a hybrid formed, e.g., by Watson-Crick base pairing only in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., at least 10 nucleotides) that is exactly complementary to the target RNA. Furthermore, in some embodiments, the dsRNA molecule specifically discriminates between single-nucleotide differences. In this case, the dsRNA molecule mediates RNAi only if exact complementarity is found in the region of the single-nucleotide difference (e.g., within 7 nucleotides of the difference).

[0356] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) that is, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0357] The term "BNA" refers to bridged nucleic acids, often referred to as constrained or inaccessible RNA. BNAs can include 5-, 6-, or even 7-membered bridge structures with a "locked" C3'-endo sugar pucker. This bridge is typically incorporated at the 2', 4' positions of the ribose, resulting in a 2',4'-BNA nucleotide (e.g., LNA or ENA). Examples of BNA nucleotides include the following nucleosides: [ka]

[0358] The term "LNA" refers to locked nucleic acid, often referred to as constrained or inaccessible RNA. LNA is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an additional bridge (e.g., a methylene or ethylene bridge) that connects the 2' hydroxyl to the 4' carbon of the same ribose sugar. For example, this bridge can "lock" the ribose in a 3'-endo (North) conformation. [ka]

[0359] The term "ENA" refers to ethylene-bridged nucleic acid, often referred to as constrained or inaccessible RNA.

[0360] As used herein, "cleavage site" refers to the backbone linkage group in the target gene or sense strand that is cleaved by the RISC machinery using an iRNA agent. The target cleavage site region also includes at least one or at least two nucleotides on either side of the cleavage site. For the sense strand, the cleavage site is the backbone linkage group in the sense strand that would undergo cleavage if the sense strand itself were the target to be cleaved by the RNAi machinery. The cleavage site can be determined using methods known in the art, such as a 5'-RACE assay as detailed in Soutschek et al., Nature (2004) 432, 173-178 (incorporated by reference in its entirety). As is well understood in the art, the cleavage site region of a standard double-stranded RNAi agent contains two 21-nucleotide long strands (wherein these strands form a 19-contiguous base pair double-stranded region with a two-nucleotide single-stranded overhang at the 3' end), the cleavage site region corresponds to positions 9-12 from the 5' end of the sense strand.

[0361] The terms "decreased," "reduced," "reduction," or "inhibition" are all used herein to refer to a statistically significant amount of reduction. In some embodiments, "reduce," "reduction," "reduce," or "inhibit" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease may preferably be down to a level generally accepted as being within the normal range for individuals without the given disorder.

[0362] As used herein, the "central region" of a strand refers to positions 8-16, e.g., positions 8-15, 8-14, 8-13, 8-12, 9-16, 9-15, 9-14, 9-13, 9-12, 10-16, 10-15, 10-14, 10-13, or 10-12, counting from the 5' end of the strand. For example, the central region of a strand refers to positions 8, 9, 10, 11, 12, 13, 14, 15, and 16 of the strand. Preferred central regions of the sense strand are positions 8, 9, 10, 11, 12, 13, and 14, counting from the 5' end of the sense strand. More preferred central regions of the sense strand are positions 10, 11, 12, and 13, counting from the 5' end of the sense strand. Preferred central regions of the antisense strand, counting from the 5' end of the antisense strand, are positions 9, 10, 11, 12, 13, 14, 15, and 16. More preferred central regions of the antisense strand, counting from the 5' end of the antisense strand, are positions 10, 11, 12, 13, 14, and 15.

[0363] The terms "specific binding," or "specifically binds to," or "specific for," refer to binding by a binding moiety to a binding target, such as binding by an immunoglobulin to a target antigen, e.g., an epitope, on a particular polypeptide, peptide, or other target (e.g., a glycoprotein target), and mean binding that is measurably different from nonspecific interactions (e.g., nonspecific interactions can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining binding by a binding moiety or immunoglobulin to a target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. The terms "specific binding," or "specifically binds to," or "specific for," as used herein, refer to a binding activity that is at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM or stronger. d In certain instances, the term "specific binding" refers to binding of a molecule to a specific target molecule without binding to substantially any other molecules.

[0364] "Binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an immunoglobulin) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an immunoglobulin and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K d ) can be expressed as follows. For example, K dThe affinity can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens quickly and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art.

[0365] As used herein, "K d " or "K d "Value" refers to the dissociation constant measured by a technique appropriate for the immunoglobulin-target pair, for example, using a surface plasmon resonance assay, for example, using a Biacore X100 or Biacore T200 (GE Healthcare, Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip.

[0366] "Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow (reduce) the targeted pathological condition or disorder. For example, a subject or mammal is successfully "treated" for cancer if, after receiving a therapeutic amount of a conjugate described herein, the subject experiences an observable and / or measurable reduction or lack of one or more of the following: a reduction in the number of cancer cells or the absence of cancer cells; a reduction in tumor size; inhibition (i.e., slowing to some extent, and preferably stopping) of cancer cell invasion into peripheral organs, including the spread of cancer to soft tissue and bone; inhibition (i.e., slowing to some extent, and preferably stopping) of tumor metastasis; inhibition (i.e., slowing to some extent, and preferably stopping) of tumor growth; and / or alleviation to some extent of one or more symptoms associated with the particular cancer; a reduction in morbidity and / or mortality rate and an improvement in quality of life. In some embodiments, "treating" or "treatment" refers to therapeutic treatment.

[0367] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cows, horses, pigs, deer, bison, water buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, e.g., a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0368] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can advantageously be used as subjects representing animal models of cancer. The subject may be male or female.

[0369] The subject may be a subject who has been previously diagnosed with, or who has been identified as suffering from, a condition (e.g., cancer) for which treatment is required, or one or more complications associated with such a condition, and optionally has already received treatment for cancer or one or more complications associated with cancer. Alternatively, the subject may be a subject who has not previously been diagnosed with cancer or one or more complications associated with cancer. For example, the subject may be a subject who exhibits one or more risk factors for cancer or one or more complications associated with cancer, or a subject who does not exhibit risk factors.

[0370] A subject "in need" of treatment for a particular condition can be a subject who has the condition, a subject who has been diagnosed with the condition, or a subject who is at risk of developing the condition.

[0371] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features which may be readily separated or combined with the features of any of the other aspects without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events described or in any other order which is logically possible.

[0372] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. [Example]

[0373] Example 1: Altritol-modified siRNA: in vitro and in vivo RNAi activity and structural studies Natural RNA nucleotides contain a ribose sugar that adopts the C3'-endo conformation to form an A-form duplex. Interestingly, when the ribose sugar is replaced with a six-membered altritol sugar, the backbone of the resulting oligonucleotide (termed altritol nucleic acid, or ANA) maintains the A-form geometry. ANA can therefore be considered an RNA analog and may find useful applications when the RNA geometry must be preserved. In this study, we investigated ANA as a modification of small interfering RNA (siRNA) duplexes, which are originally fully modified by 2'-F RNA and 2'-OMe RNA chemical modifications. This siRNA was designed to silence the expression of the transthyretin (Ttr) gene and conjugated to N-acetylgalactosamine (GalNAc) for targeted delivery to hepatocytes. Single-nucleotide position walks of the ANA modification along the sense and antisense strands of the parent duplex were performed, and the resulting modified duplexes were screened for their potential ability to target Ttr mRNA in vitro. This analysis unexpectedly and surprisingly revealed that ANA was well-adapted to the seed region, particularly positions 6 and 7 of the antisense strand. The corresponding duplexes also showed good on- and off-target activity in luciferase reporter assays. Finally, duplexes with ANA at position 7 of the antisense strand in the seed region maintained RNAi activity in vivo, and incorporation of ANA at the 5' end of the oligonucleotide was shown to improve metabolic stability due to its stability against 5'-exonucleases.

[0374] The enormous potential of small interfering RNA (siRNA) in the development of novel medicines is evident from the success of Onpattro®, a first-in-class drug for the treatment of polyneuropathy in hereditary transthyretin-mediated amyloidosis 1 Many other siRNAs are being evaluated in early- to late-stage clinical trials. 2 siRNAs utilize a natural gene regulatory mechanism called RNA interference (RNAi) to silence the expression of a given gene of interest. 3、4siRNAs, which are composed of natural RNA nucleotides, are prone to rapid enzymatic degradation and do not easily reach target organs when administered systemically. Therefore, chemically modified siRNAs must be used for therapeutic applications. 5 2'-deoxy-2'-fluoro- (2'-F RNA, Figure 1b) 6、7 2'-F and 2'-O-methyl- (2'-OMe RNA, Figure 1b) RNA modifications are compatible with the natural RNA-induced silencing complex (RISC) and are widely used to stabilize siRNAs. 2'-F and 2'-OMe RNAs with strategically placed phosphorothioate linkages and conjugated to N-acetylgalactosamine (GalNAc) ligands are under clinical investigation. 8 The present inventors have investigated several additional chemically modified nucleotides that can be used synergistically with 2'-F RNA and 2'-OMe RNA to generate potent and safe siRNAs. 9~12 For example, rational use of glycol nucleic acids (GNAs) in the seed region of siRNAs, which are originally fully modified with 2'-F RNA and 2'-OMe RNA, may reduce seed-mediated off-target toxicity. 10、12 Similarly, placing 5'-morpholino-2'-OMe RNA nucleotides in the sense strand can enhance RNAi activity. 9 On the other hand, 4'-C-methoxy-2'-deoxy-2'-fluorouridine can improve metabolic stability while maintaining gene silencing. 13 Thus, the strategy of using 2'-F RNA and 2'-OMe RNA modifications and modified nucleotides in synergy is a validated approach to generate siRNAs with improved potency and specificity.

[0375] Altritol nucleic acids (ANAs) have a six-membered ring sugar with a nucleobase at the 2'-(S)-position and a hydroxyl group at the 3'-position (Figure 1). 14、15ANAs have a pre-organized structure that mimics N-type furanose sugars, while the presence of a hydroxyl group at the 3'-position contributes to duplex stabilization through hydration. ANAs have an RNA-like (A-type) geometric structure, making them suitable candidates for incorporation into siRNA, where maintaining an RNA-like structure is advantageous. ANA-modified siRNAs have been evaluated and have shown better RNAi activity compared to unmodified siRNAs. 16~19 However, it remains unclear how ANA behaves when used synergistically with other modified nucleotides / ligands. In this context, we present for the first time the in vitro and in vivo gene silencing activity of siRNA duplexes modified with ANA, 2'-F RNA, and 2'-OMe RNA nucleotides. We also evaluate the impact of ANA on protection from nucleolytic degradation when present at the 3' or 5' end of the oligonucleotide. We determined the crystal structure of an RNA octamer duplex incorporating ANA-A residues at 2 Å resolution. To help rationalize the advantageous activity and metabolic stability of RNAs incorporating ANA nucleotides position-specifically, we also constructed models of an siRNA guide strand bearing an ANA modification at position 7 bound to Argonaute 2 (Ago2) and an RNA with two 5'-terminal ANA-U residues bound to Xrn1 RNA 5'-exonuclease.

[0376] Materials and Methods Oligonucleotide Synthesis: Published Protocols 12 ANA-modified oligonucleotides were obtained according to the method described above, except that detritylation was performed using 3% trichloroacetic acid in CHCl. ​​Briefly, all oligonucleotides were prepared on an ABI 394 or MerMade 192 synthesizer at the 1 μmol scale using standard or custom supports. For ABI, all phosphoramidites were dissolved in 100% acetonitrile or 9:1 acetonitrile:DMF (2'-OMe-C, 2'-OMe-U) at a concentration of 0.15 M. For MerMade 192, a concentration of 0.10 M was used. ETT was used as the activating agent and detritylation was performed using 3% trichloroacetic acid in dichloromethane.

[0377] MerMade 192 Workup: After trityl removal synthesis using MerMade 192, the column was incubated with 150 μL of 40% aqueous methylamine for 30 minutes at room temperature, and the solution was drained into a 96-well plate by vacuum. After repeating the incubation and draining with a fresh aliquot of aqueous methylamine, the plate containing the crude oligonucleotides was sealed and shaken for an additional 60 minutes at room temperature to ensure complete removal of all protecting groups. Precipitation of the crude oligonucleotides was achieved by adding 1.2 mL of 9:1 acetonitrile:EtOH to each well, followed by overnight incubation at -20°C. The plate was then centrifuged at 3000 rpm for 45 minutes at 4°C, the supernatant removed from each well, and the pellet resuspended in 950 μL of 20 mM aqueous NaOAc. Finally, the final oligonucleotide products were eluted by desalting each crude solution with water on a GE Hi-Trap desalting column (Sephadex G25 Superfine). The identity and purity of all oligonucleotides were confirmed using ESI-MS and IEX HPLC, respectively.

[0378] ABI 394 workup: Standard protocols were used for cleavage and deprotection. Crude oligonucleotides were purified using strong anion exchange and phosphate buffer (pH = 8.5) containing sodium bromide. The identity and purity of all oligonucleotides were confirmed using ESI-LC / MS and IEX HPLC, respectively.

[0379] In vitro mRNA knockdown: 5 μL of siRNA was placed in a 384-well collagen-coated plate, followed by the addition of 4.90 μL of Opti-MEM and 0.1 μL of Lipofectamine RNAiMax (Invitrogen) to each well. The final siRNA concentration was 0.1 or 10 nM. The plate was incubated at room temperature for 15 minutes. Primary mouse hepatocyte cells were suspended in Invitrogro CP Rodent Medium (#Z990028 BioIVT), and 40 μL of this suspension (containing approximately 5,000 cells) was added to each well. Four replicates were evaluated for each siRNA. After incubating the cells for 24 hours, RNA was isolated using DynaBeads (ThermoFisher). The RNA was then reverse transcribed into cDNA according to the manufacturer's protocol (Applied Biosystems). Multiplex qPCR reactions were performed using gene-specific TaqMan assays for Ttr (ThermoFisher Scientific, #Mm00443267_m1) and mouse Gapdh (#4352339E) as an endogenous control. Real-time PCR was performed on a Roche LightCycler 480 using LightCycler 480 Probe Master Mix (Roche).

[0380] Off-target assay: COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were co-transfected in 96-well plates (15,000 cells / well) with 10 ng of luciferase reporter plasmid and 5-fold dilutions of 0.64 pM to 50 nM siRNA using 2 μg / mL Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were harvested 48 hours post-transfection and subjected to a dual luciferase assay (Promega) according to the manufacturer's instructions. The on-target reporter plasmid contained a single site in the 3'-untranslated region (3'UTR) of Renilla luciferase that was perfectly complementary to the antisense strand. The off-target reporter plasmid contained four tandem seed-complementary sites in the 3'-UTR of Renilla luciferase, separated by a 19-nucleotide spacer (TAATATTACATAAATAAAA). Both plasmids co-expressed firefly luciferase as a transfection control.

[0381] In vivo gene expression silencing: All studies were conducted using protocols that complied with local, state, and federal regulations, where applicable, and were approved by Alnylam Pharmaceuticals' Institutional Animal Care and Use Committee (IACUC). Six- to eight-week-old female C56BL / 6 mice (Charles River Laboratories) were subcutaneously administered 1 mg / kg GalNAc-siRNA formulated in 1x PBS. A control cohort received the same volume (200 μL) of 1x PBS as the siRNA-treated animals. Because prealbumin / Ttr may be present in the circulation, blood samples were collected immediately before treatment administration and 7 and 14 days after siRNA administration. Collected serum samples were stored at -80°C until further analysis. Two weeks after compound administration, animals were euthanized, and livers were removed and cryopreserved. RNA isolation from liver was performed on a PerkinElmer Chemagic system (Waltham, MA) according to the supplier's guidelines, followed by cDNA preparation and multiplex RT-qPCR analysis to assess Ttr transcript levels (Taqman probe Mm00443267_m1; mouse Gapdh 4351309 from ABI). Quantification of Ttr protein levels in serum was performed spectrophotometrically with a Mouse Prealbumin / Ttr ELISA kit (41-PALMS-E01) according to the manufacturer's protocol (ALPCO, Salem, NH).

[0382] Nuclease stability assay: To assess stability against 3'-specific or 5'-specific exonucleases, modified oligonucleotides were prepared at a final concentration of 0.1 mg / mL in either 50 mM Tris (pH 7.2) containing 10 mM MgCl2 or 50 mM sodium acetate (pH 6.5) containing 10 mM MgCl2, respectively. Exonuclease (150 mU / mL SVPDE or 500 mU / mL phosphodiesterase II) was added immediately before analysis using an IEX HPLC (dionex DNAPac PA200, 4 x 250 mm) with a 37-52% mobile phase gradient over 7.5 min at a flow rate of 1 mL / min (1 M NaBr, 20 mM sodium phosphate, pH 11, 15% MeCN; stationary phase: 20 mM sodium phosphate, 15% MeCN, pH 11). Samples were injected at given time points and monitored for up to 24 h. The amount of full-length ON was measured using A. 260 The percent full-length ON was determined as the area under the curve at t = 0. The percent full-length ON was calculated by dividing the area under the curve at t = 0 and multiplying by 100. Terminal phosphorothioate bonds (5'-T for 3'- or 5'-exonuclease activity, respectively) were used. 19 T or 5'-T T 19 Enzyme activity was confirmed in each experiment by including oligo-2'-deoxythymidylate with 2'-diaminobenzyl 2'-deoxythymidylate. Each aliquot of enzyme was thawed immediately before the experiment. Half-lives were determined by fitting to first-order kinetic equations.

[0383] X-ray crystallography: Two ANA-modified oligonucleotides were subjected to crystallization experiments. Crystals of the dodecamer CGCGA(ANA-A)BrUUAGCG were in space group I222 with unit cell dimensions a = 47.26 Å, b = 81.71 Å, and c = 125.62 Å and diffracted to 4.4 Å resolution. Crystals of the octamer BrCGA(ANA-A)UUCG were in space group P6522 with unit cell dimensions a = b = 96.14 Å, c = 125.99 Å, and γ = 120° and diffracted to 2.1 Å resolution. Single-wavelength anomalous dispersion data at the bromine edge (Br-SAD) of the latter oligonucleotide were collected on beamline 21-ID-D of the Life Sciences Collaborative Access Team (LS-CAT) at the Advanced Photon Source (APS, Argonne National Laboratory, Argonne, IL). Data were processed using the program... (continued if the phase of the structure can be determined).

[0384] Molecular modeling: Crystal structure of the ANA:RNA hybrid duplex a(CCGUAAUGCC-P):r(GGCAUUACGG) [see Ovaere et al. NAR 2012] with PDB ID code 3OK2 and microRNA miR-20a bound to human Argonaute 2 (Ago2) with PDB ID 4F3T 20 The coordinates of the crystal structure of Xrn1 were obtained from the Protein Data Bank at www.rcsb.org. The G residue at position 7 of the RNA in the Ago2 complex was excised and replaced with ANA-G3 from the hybrid duplex with the ANA A strand using the program UCSF Chimera [see Petterson et al. J. Comput. Chem. 2004]. The geometry of the resulting fusion model was locally refined with Amber14 [see Case et al. UCSF 2014], and the glycosidic torsion angles of the ANA purine residues were slightly adjusted to restore stacking of the adjacent residue C8 as seen in the crystal structure of the Ago2 complex with native RNA. The complex between Xrn1 5'-exoribonuclease and 5'P-dTdTdT-3' with PDB ID 2Y35 was also obtained from the Protein Data Bank. 21The crystal structure of the ANA:RNA hybrid duplex was obtained. The dimer 5'P-aC9aC10-3'P was excised from strand A of the crystal structure, and the bases were converted to U using the program UCSF Chimera. This dimer was then superimposed onto the first two nucleotides of the dT trimer in the Xrn1 complex using the match function in UCSF Chimera with selected backbone and base atoms.

[0385] In vitro mRNA knockdown: We assessed the position-dependent impact of ANA modifications on in vitro RNAi activity by systematically substituting each nucleotide of previously studied siRNAs designed to target rodent (mouse and rat) transthyretin (Ttr) mRNA (Figure 1). 12、22~24 This design includes a 21-mer duplex with a two-nucleotide overhang on the antisense strand, modified with 2'-F RNA and 2'-OMe RNA nucleotides. The ends were protected by introducing two phosphorothioate bonds at the ends of each strand, except for the 3' end of the sense strand, which is conjugated to a GalNac moiety.

[0386] ANA nucleotides were walked across nucleotide positions in the sense and antisense strands, and the RNAi activity of the resulting modified siRNAs was studied in primary hepatocytes (Figure 2). 16 ANA at the 5' end (position 1; AS1) of the antisense strand was less effective. This loss of activity is likely due to the inability of cellular kinases to introduce a 5'-monophosphate group into the ANA nucleotide. The 5'-monophosphate is known to play a critical role in RISC loading by interacting with the Ago2 MID domain, and therefore its absence impairs RNAi activity. 25~28 The presence of the ANA modification at the AS2 position was detrimental to RNAi activity.

[0387] Unexpectedly, ANA modifications in the seed (nucleotides 3–8) were better tolerated, and two modified duplexes with ANA nucleotides at AS6 or AS7 exhibited RNAi activity comparable to that of the parent duplex at 10 nM (Figure 2). Additionally, ANA at AS16 also maintained RNAi activity. However, none of these modified duplexes exhibited significantly higher activity compared to the parent siRNA. Similar results were obtained at concentrations as low as 0.1 nM (Figure S1). The in vitro RNAi activity of siRNA duplexes with modifications in the sense strand is shown in Figure 3 and Figure S2. In general, the presence of ANA modifications in the sense strand led to a slight loss of activity. However, no significant changes were observed for the majority of modified duplexes.

[0388] Based on this initial in vitro screening result, some duplexes were selected for recording concentration-dependent RNA activity and their IC 50 In accordance with this initial result, the siRNA duplex with ANA AS7 had an IC value of 0.115 nM. 50 The parent duplex had an IC value of 0.112. 50 values ​​were shown).

[0389] On-target and off-target activity in luciferase reporter assays: Seed-mediated off-target activity has been reported to contribute to siRNA hepatotoxicity in rats 10 To reduce such off-target effects, thermolabile modifications such as GNA have been incorporated into the seed region of the siRNA duplex. 10 GNAs can efficiently mitigate seed-mediated off-target effects while maintaining on-target efficacy. ANAs are well tolerated at positions 6 or 7 of the antisense strand in the seed region. Inspired by this, we measured the effect of ANAs on off-target activity using a luciferase reporter assay, in which four tandem seed matches with siRNAs were cloned into the luciferase 3'-UTR region (Figures 5A and 5B). 29~31ANA in AS6 and AS7 maintained on-target activity, an observation consistent with previous in vitro screening. Off-target activity of the parent and modified duplexes (AS6 and AS7) was comparable. Therefore, ANA does not increase off-target activity.

[0390] In vivo gene expression silencing: Selected siRNA duplexes were also screened for their ability to silence Ttr gene expression in mice. Animals were dosed subcutaneously at 1 mg / kg, and a control cohort received 1x PBS vehicle. Blood samples were collected immediately before dosing and at 7 and 14 days after dosing. Serum Ttr protein levels were measured using a mouse prealbumin / Ttr ELISA kit. The results are shown in Figure 6A. At day 7, duplexes with ANA at the AS6 or AS7 positions showed comparable potency to the parent duplexes. This is consistent with the results of in vitro screening experiments with these duplexes. However, at day 14, only AS7 showed activity, while AS6 was less potent than the parent. The reason for this behavior is currently unknown. Interestingly, duplexes with an ANA modification at AS16 were significantly less active than the parent duplexes. In contrast, the same duplexes demonstrated comparable RNAi activity to the parent duplex in in vitro screening, suggesting inherent intracellular differences between in vitro and in vivo models. Duplexes with an ANA modification at S12 were slightly less active than the parent, but their activity was maintained up to day 14. Consistent with the in vitro data, duplexes with ANA modification at the 5' end of the antisense strand (AS1) were ineffective, most likely due to the inability of ANA to be phosphorylated by endogenous intracellular kinases. Animals were sacrificed on day 14, and RNA was extracted from the liver. cDNA was subsequently prepared, and Ttr transcript levels were assessed by multiplex RT-qPCR analysis (Figure 6B). Results were in good agreement with those obtained from serum samples on day 14.

[0391] To gain insight into the beneficial effect on activity observed in siRNAs in which AS7 was replaced with ANA nucleotides, we investigated the Ago2:miR-20a complex. 20 Based on the crystal structure of , we constructed a model of the modified strand bound to Ago2. The ANA-G residue, which replaces G7 for incorporation into the microRNA strand, is located in the ANA:RNA decameric duplex. 32 The image was taken from the crystal structure of miR-20a. In this hybrid duplex (ANA-strand A paired with RNA-strand E), the intrastrand P–P distance of ANA residues averages 5.53 Å, compared with 5.93 Å for RNA residues. The interphosphate spacing between residues 6, 7, and 8 (P6...P7 and P7...P8) in the seed region of miR-20a is shortened as a result of a pronounced kink in this region. Thus, ANA nucleotides, with their inherently short distance between their 5'- and 3'-phosphate groups, fit quite well into this twisted backbone conformation, as demonstrated by a model of RNA in which G7 has been replaced by ANA-G7 bound to Ago2 (Figure 7).

[0392] Nuclease stability: We incorporated either a single ANA nucleotide or two at the 3' or 5' end of poly(dT) oligonucleotides. For direct comparison, identical sequences with terminal uridines were prepared. ONs with 3'-terminal modifications were incubated with the 3'-specific exonuclease snake venom phosphodiesterase (SVPD), and degradation of the full-length oligonucleotides was monitored using ion-exchange HPLC. Interestingly, both ONs containing uridine or ANA-U exhibited similar stability toward SVPD and thus comparable half-lives. Importantly, incorporation of a second ANA improved half-life by twofold (Figures 8A and 8B).

[0393] The 5'-modified oligonucleotides were then incubated with the 5'-specific exonuclease phosphodiesterase II (PDEII). Importantly, a significant improvement was observed for oligonucleotides bearing an ANA modification at the 5' end. Addition of a second modification at the 5' end rendered the oligonucleotide virtually inert to degradation (Figure 8), as seen in the crystal structure of oligodT bound to the 5'-exoribonuclease Xrna1 [see Jinek et al. Mol. Cell 2011]. 21 When the ANA 5'P-aUaU-3'P dimer is superimposed on two 5'-terminal dTs, the differences in P-P spacing (shorter in ANA) and phosphate orientation, as well as the increased bulkiness of the hexose sugar compared to (2'-deoxy)ribose, are readily apparent (Figure 9).

[0394] conclusion This study unexpectedly and surprisingly demonstrates that nucleotides featuring a six-membered altritol sugar ring induce efficient gene silencing both in vitro and in vivo. In particular, the ANA modification was best suited to the AS7 position. The crystal structure of an ANA-modified RNA octamer demonstrated that the distance between the 5'- and 3'-phosphates of the ANA residue is rather short compared to the average distance between phosphates in ribonucleotides. Thus, the ANA residue can perfectly mimic a ribonucleotide at the AS7 position, where twisting of the siRNA guide strand results in a localized shortening of the interphosphate distance. ONs with ANA at the 5' end were more resistant to nuclease degradation than their natural RNA counterparts. However, this increased stability was not observed in assays testing resistance to degradation by the 3'-exonuclease SVPD. A model of an ON with two ANA-uridines at its 5' end bound to a 5'-exoribonuclease indicates that steric bulk and altered interphosphate spacing compared to DNA and RNA are the primary reasons for the increased protection from nuclease degradation.

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[0396] All U.S. patents, U.S. patent application publications, foreign patents, foreign patent applications, and non-patent articles referenced herein are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified as necessary to utilize concepts from various patents, applications, and publications to provide further embodiments.

[0397] These and other variations to the embodiments may be made in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. 1. A double-stranded RNA (dsRNA) molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, the dsRNA molecule comprising a hexopyranose nucleoside, and the dsRNA molecule comprising a ligand.

2. 1. A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, and wherein the antisense strand comprises at least one hexopyranose nucleoside in a seed region of the antisense strand, or the sense strand comprises at least one altritol nucleotide in a central region of the sense strand.

3. 1. A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, and the dsRNA comprises at least one hexopyranose nucleoside, at least one 2'-fluoro nucleotide, and at least one 2'-OMe nucleotide.

4. 1. A double-stranded RNA molecule capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference, the dsRNA comprises at least one hexopyranose nucleoside, and the dsRNA comprises at least four phosphorothioate internucleotide linkages.

5. The double-stranded RNA molecule of any one of claims 1 to 4, wherein the antisense strand comprises a hexopyranose nucleoside in the seed region of the antisense strand.

6. The double-stranded RNA molecule of claim 5, wherein the antisense strand comprises a hexopyranose nucleoside at least one of positions 3 to 8 counting from the 5' end of the antisense strand.

7. The double-stranded RNA molecule of claim 6 , wherein the antisense strand comprises a hexopyranose nucleoside at at least one of positions 6, 7, 9, 12, 16, 21, and 22, counting from the 5′ end of the antisense strand.

8. The double-stranded RNA molecule of claim 7 , wherein the antisense strand comprises a hexopyranose nucleoside at at least one of positions 6, 7, and 16.

9. The double-stranded RNA molecule of claim 8 , wherein the antisense strand comprises a hexopyranose nucleoside at at least one of positions 6 and 7.

10. The double-stranded RNA molecule of claim 9 , wherein the antisense strand comprises a hexopyranose nucleoside at position 7.

11. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises at least two or more consecutive independently selected hexopyranose nucleosides.

12. The double-stranded RNA molecule of claim 11, wherein the sense strand comprises at least three or more consecutive independently selected hexopyranose nucleosides.

13. The double-stranded RNA molecule of any one of claims 1 to 4, wherein the sense strand comprises a hexopyranose nucleoside in the central region of the sense strand.

14. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises a hexopyranose nucleoside at at least one of positions 3 and 12.

15. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the antisense strand comprises at least two or more consecutive independently selected hexopyranose nucleosides.

16. The double-stranded RNA molecule of claim 15, wherein the antisense strand comprises at least three or more consecutive independently selected hexopyranose nucleosides.

17. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises a hexopyranose nucleoside at the 5' end of the sense strand.

18. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises a hexopyranose nucleoside at the 5' end of the sense strand.

19. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises a 5'-vinylphosphonate (VP) group.

20. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the hexopyranose nucleotide is selected from the group consisting of allopyranose nucleotides, altritol nucleotides, glucopyranose nucleotides, mannopyranose nucleotides, gulopyranose nucleotides, idopyranose nucleotides, galactopyranose nucleotides, talopyranose nucleotides, fucopyranose nucleotides, rhamnopyranose nucleotides, quinovopyranose nucleotides, pneumopyranose nucleotides, and any combination thereof.

21. 21. The double-stranded RNA molecule of claim 20, wherein the hexopyranose nucleotides are selected from the group consisting of altritol nucleotides, glucopyranose nucleotides, mannopyranose nucleotides, galactopyranose nucleotides, fucopyranose nucleotides, and any combination thereof.

22. The double-stranded RNA molecule according to any one of claims 2 to 4, comprising a ligand.

23. The double-stranded RNA molecule of claim 1 or 22, wherein the ligand is an ASGPR ligand.

24. The ASGPR ligand is 【Chemistry 1】 The double-stranded RNA molecule of claim 23,

25. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages.

26. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end).

27. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the antisense strand comprises 1, 2, 3, or 4 phosphorothioate internucleotide linkages.

28. The double-stranded RNA molecule of any one of claims 1 to 4, wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end).

29. The double-stranded RNA molecule of any one of claims 1 to 4, wherein the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end).

30. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand and the antisense strand are independently 19 to 25 nucleotides in length.

31. 31. The double-stranded RNA molecule of claim 30, wherein the sense strand is 21 nucleotides in length.

32. 32. The double-stranded RNA molecule of claim 31, wherein the antisense strand is 23 nucleotides in length.

33. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

34. The double-stranded RNA molecule according to any one of claims 1 to 4, comprising a single-stranded overhang at the 3' end of the antisense strand.

35. The double-stranded RNA molecule according to any one of claims 1 to 4, comprising a blunt end at the 5' end of the antisense strand.

36. The double-stranded RNA molecule according to any one of claims 1 to 4, comprising a 2'-fluoro or 2'-OMe nucleotide at a position complementary to the hexopyranose nucleotide.

37. The double-stranded RNA molecule according to any one of claims 1 to 4, which does not contain any nucleotides other than hexopyranose, 2'-fluoro and 2-OMe nucleotides.

38. 5. The double-stranded RNA molecule of any one of claims 1 to 4, comprising a nucleotide selected from the group consisting of acyclic nucleotides, locked nucleic acids (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, and 2'-deoxy.

39. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein the sense strand comprises a 5'-morpholino modification, a 5'-dimethylamino modification, a 5'-deoxy modification, an inverted abasic modification, or an inverted abasic locked nucleic acid modification at the 5' end.

40. The double-stranded RNA molecule according to any one of claims 1 to 4, which has a melting temperature in the range of about 40°C to about 80°C.

41. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 40, alone or in combination with a pharmaceutically acceptable carrier or excipient.

42. A gene silencing kit comprising the dsRNA molecule of any one of claims 1 to 40.

43. 41. A method for silencing a target gene in a cell, comprising the step of introducing into said cell a dsRNA molecule according to any one of claims 1 to 40.

44. 44. The method of claim 43, wherein the dsRNA agent is administered via subcutaneous or intravenous administration.

45. 41. A method for silencing a target gene in a cell, comprising expressing in said cell a dsRNA molecule according to any one of claims 1 to 40.

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