Modified double-stranded oligonucleotides

Abstract

One aspect of the present invention relates to double-stranded RNA (dsRNA) agents capable of inhibiting the expression of target genes. Another aspect of the present invention relates to pharmaceutical compositions comprising these dsRNA molecules suitable for therapeutic use, and to methods of inhibiting the expression of target genes by administering these dsRNA molecules, for example, to treat various disease conditions. TIFF2024504694000123.tif92156

Description

[Technical Field]

[0001] Cross-reference of related applications This application is based on U.S. Provisional Application No. 63 / 140,714, filed on 22 January 2021; Provisional Patent Application No. 63 / 146,115, filed on 5 February 2021; Provisional Patent Application No. 63 / 148,991, filed on 12 February 2021; Provisional Patent Application No. 63 / 153,983, filed on 26 February 2021; and Provisional Patent Application No. 63 / 156,47, filed on 4 March 2021, pursuant to Section 119(e) of the U.S. Patent Act. Claiming the benefits of U.S. Provisional Patent Application No. 63 / 161,313 filed on 15 March 2021, U.S. Provisional Patent Application No. 63 / 164,467 filed on 22 March 2021, U.S. Provisional Patent Application No. 63 / 179,607 filed on 26 April 2021, and U.S. Provisional Patent Application No. 63 / 141,748 filed on 29 April 2021, the contents of each of these U.S. Provisional Patent Applications are incorporated herein by reference in their entirety.

[0002] Field of Invention This invention relates to dsRNA molecules having specific motifs advantageous for inhibiting target gene expression, and dsRNA agent compositions suitable for therapeutic use. In addition, this invention provides a method for inhibiting target gene expression by administering these dsRNA agents, for example, for the treatment of various diseases. [Background technology]

[0003] background RNA interference, or "RNAi," is a term first coined by Fire and his collaborators to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811, Elbashir et al. (2001) Genes Dev. 15, 188-200 (Non-patent Literature 1)). Short dsRNAs direct gene-specific post-transcriptional silencing in many organisms, including vertebrates, and have become a new tool for studying gene function. RNAi is mediated by RNA-induced silencing complexes (RISCs), which are sequence-specific multicomponent nucleases that disrupt messenger RNA homologous to the silencing trigger. While it is known that RISCs contain short RNAs (approximately 22 nucleotides) derived from double-stranded RNA triggers, the protein components of this activity remained unknown.

[0004] In this field, there is still a need for effective nucleotide or chemical motifs for dsRNA molecules that are advantageous for inhibiting target gene expression. This invention is directed toward that endeavor. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Fire et al. (1998) Nature 391, 806-811, Elbashir et al. (2001) Genes Dev. 15, 188-200 [Overview of the project]

[0006] overview The present invention provides effective nucleotide motifs or chemical motifs for dsRNA molecules that are advantageous for inhibiting target gene expression, and RNAi compositions suitable for therapeutic use.

[0007] The inventors have discovered, in particular, that double-stranded RNA (dsRNA) molecules having a 2'-fluoronucleotide at position 10 of the sense strand have, unexpectedly and surprisingly, improved in vitro efficacy, i.e., increased RNA interference (RNAi) activity. Therefore, in one aspect, the present invention provides a double-stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, wherein the sense strand contains a 2'-fluoronucleotide at position 10, counted from the 5' end of the sense strand.

[0008] It should be noted that the sense strand may further contain one or more additional 2'-fluoronucleotides, for example, 1, 2, 3, 4, or 5. Thus, in some embodiments, the sense strand contains 1, 2, 3, 4, or 5 additional 2'-fluoronucleotides. The additional 2'-fluoronucleotides can be located anywhere in the sense strand. Thus, in some embodiments, the sense strand further contains 2'-fluoronucleotides at positions 8, 9, 11, or 12, counting from the 5' end of the sense strand. For example, the sense strand further contains a 2'-fluoronucleotide at position 9, counting from the 5' end of the sense strand. In other words, the sense strand contains 2'-fluoronucleotides at positions 9 and 10, counting from the 5' end of the sense strand. In another example, the sense strand further contains a 2'-fluoronucleotide at position 11, counting from the 5' end of the sense strand. For example, the sense strand contains 2'-fluoronucleotides at positions 10 and 11, counting from the 5' end of the sense strand.

[0009] In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions 8, 9, and 10, counting from the 5' end of the sense strand. In yet another embodiments, the sense strand contains 2'-fluoronucleotides at positions 10, 11, and 12, counting from the 5' end of the sense strand.

[0010] In any one or several embodiments of the above aspects, the sense strand does not contain a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand. For example, the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

[0011] The antisense strand of the dsRNA molecule described herein may contain one or more 2'-deoxynucleotides, e.g., 2'-H. For example, the antisense strand may contain 1, 2, 3, 4, 5, 6 or more 2'-deoxynucleotides. In some embodiments, the antisense strand may contain 2, 3, 4, 5 or 6 2'-deoxynucleotides. The 2'-deoxynucleotides can be located anywhere in the antisense strand. For example, the antisense strand may contain 2'-deoxynucleotides at 1, 2, 3, 4, 5 or 6 of the positions 2, 5, 7, 12, 14 and 16, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand may contain 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand may contain 2'-deoxynucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand.

[0012] In some embodiments, the antisense can contain one or more 2'-fluoronucleotides, for example, 1, 2, 3, 4, 5 or more. For example, the antisense chain can contain a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense chain.

[0013] In some embodiments, the antisense strand contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense strand, and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16. For example, the antisense strand contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense strand, and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16.

[0014] In some embodiments, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, and a 2'-fluoronucleotide at position 14. In some embodiments, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, a 2'-fluoronucleotide at position 14, and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16. For example, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, a 2'-fluoronucleotide at position 14, and a 2'-OMe nucleotide at position 16.

[0015] In some embodiments, the antisense strand contains a 2'-deoxynucleotide at position 14, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than 2'-fluoro at position 7, counting from the 5' end of the sense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 12, and 14, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand, and a nucleotide other than 2'-fluoro at position 7.

[0016] In various embodiments, dsRNA molecules have a double-stranded region of 19–25 base pairs. For example, dsRNA molecules have a double-stranded region of 20, 21, 22, 23, or 24 base pairs. In some specific embodiments, dsRNA molecules have a double-stranded region of 20, 21, or 22 base pairs.

[0017] In some embodiments, dsRNA molecules contain ligands. For example, the sense strand of a dsRNA molecule contains a ligand. Exemplary ligands include, but are not limited to, ASGPR ligands and ligands containing lipophilic groups.

[0018] A dsRNA molecule may contain one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate ligatures. The phosphorothioate ligatures may be present on only one or both strands of the dsRNA. For example, the sense strand may contain 1, 2, 3, or 4 phosphorothioate ligatures. In another, less restrictive example, the antisense strand may contain 1, 2, 3, 4, 5, or 6 phosphorothioate ligatures. In some embodiments, the sense strand may contain 1, 2, 3, or 4 phosphorothioate ligatures, and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate ligatures. For example, the sense strand may contain 1 or 2 phosphorothioate ligatures, and the antisense strand may contain 1, 2, 3, or 4 phosphorothioate ligatures.

[0019] In some embodiments, the sense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand, the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand, and the antisense further includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 3' end of the antisense strand. For example, the sense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the sense strand, and the antisense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the antisense strand, as well as between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 3' end of the antisense strand.

[0020] In some embodiments, the remaining nucleotides in dsRNA are 2'-OMe nucleotides. For example, all remaining nucleotides in the sense strand are 2'-OMe nucleotides. In other words, the sense strand alone contains both 2'-fluoronucleotides and 2'-OMe nucleotides.

[0021] The antisense strand is understood to have sufficient complementarity to the target sequence in order to mediate RNA interference. In other words, the dsRNA molecule of the present invention can inhibit the expression of the target gene.

[0022] In another aspect, the present invention further provides a method for delivering the dsRNA molecule of the present invention to a specific target in a subject by subcutaneous or intravenous administration. The present invention further provides the dsRNA molecule of the present invention for use in a method for delivering the agent to a specific target in a subject by subcutaneous or intravenous administration.

[0023] This patent document or application document includes at least one drawing drawn in color. A copy of this patent or patent application publication containing the color drawing will be provided by the United States Patent and Trademark Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0024] [Figure 1] Figures 1A and 1B are graphs showing that dsRNA according to exemplary embodiments of the present invention exhibits improved in vitro potency compared to the parent dsRNA molecule when administered at 10 nM (Figure 1A) or 1 nM (Figure 1). [Figure 2] Figures 2A-2D are graphs showing that dsRNA molecules according to embodiments of the present invention have improved in vivo efficacy compared to the parent molecules. The parent double helix molecules are AD-1181401 (sequence 1, Figure 2A), AD-1181410 (sequence 2, Figure 2B), AD-1181426 (sequence 3, Figure 2C), and AD-1181451 (sequence 4, Figure 2D). [Figure 3]Figures 3A-3D are graphs showing that dsRNAs according to exemplary embodiments of the present invention, targeting various targets, exhibit improved in vitro potency compared to the parent dsRNA molecule when administered at 1 nM (Figures 3A and 3B) or 0.1 nM (Figure 3C). [Figure 4] Figures 4A-4C are graphs showing that the presence of a 2'-fluoronucleotide at position 10 of the sense strand (counting from the 5' end of the sense strand) enhances the RNAi effectiveness of the dsRNA molecule compared to the parent. [Figure 5A] This is a schematic diagram of several exemplary designs of a dsRNA molecule according to an aspect of the present invention. [Figure 5B] This is a schematic diagram of several exemplary designs of a dsRNA molecule according to an aspect of the present invention. [Figure 6] Figures 6A and 6B are graphs showing the in vitro targeted knockdown (Figure 6A) and log2 activity (Figure 6B) of exemplary dsRNAs according to several aspects of this disclosure, compared to exemplary parental dsRNA molecules. [Figure 7] Figures 7A and 7B are graphs showing the in vitro targeted AGT knockdown (Figure 6A) and log2 activity (Figure 6B) of exemplary dsRNAs according to several aspects of this disclosure, compared to exemplary parental dsRNA molecules that target AGT. [Figure 8A] Similar or improved metabolic stability of the sense strands (Figures 8A-8D) and antisense strands (Figures 8E-8H) of exemplary dsRNA molecules in mouse liver homogenates (Figures 8A and 8E), rat liver homogenates (Figures 8B and 8F), rat brain homogenates (Figures 8C and 8G), and cynomolgus monkey liver homogenates (Figures 8D and 8H). The parent double helix are AD-1181401 (TTR Seq 1), AD-1181410 (TTR Seq 2), and AD-74210 (F12). [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 8E] See the explanation in Figure 8A. [Figure 8F] See the explanation in Figure 8A. [Figure 8G] See the explanation in Figure 8A. [Figure 8H] See the explanation in Figure 8A. [Figure 9] Figures 9A and 9B show similar or improved metabolic stability of exemplary dsRNA molecules in mice. The parental double helix molecules are AD-1181401 (TTR Seq 1), AD-1181410 (TTR Seq 2), and AD-74210 (F12). [Figure 10] Figures 10A-10D are graphs showing that dsRNA molecules according to embodiments of the present invention have improved in vivo efficacy and / or duration compared to the parent molecules AD-74210 (Figures 10A and 10C) and AD-75885 (Figures 10B and 10D) in non-human primates, mice (Figures 10A and 10B), and cynomolgus monkeys (Figures 10C and 10D). [Modes for carrying out the invention]

[0025] Detailed explanation In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent of the present invention can be used in place of a dsRNA molecule and can be used in RNA interference-based gene silencing techniques, including, but not limited to, in vitro or in vivo applications.

[0026] Generally, a dsRNA molecule contains a sense strand (also called a passenger strand) and an antisense strand (also called a guide strand). Each strand of a dsRNA molecule can be in the range of 15 to 35 nucleotides in length. For example, each strand can be 17 to 35 nucleotides long, 17 to 30 nucleotides long, 25 to 35 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long. The sense strand and antisense strand may or may not be of equal length. For example, the sense strand and antisense strand may independently have lengths of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0027] In some embodiments, the antisense chain is 15–35 nucleotides long. In some embodiments, the antisense chain is 15–35, 17–35, 17–30, 25–35, 27–30, 17–23, 17–21, 17–19, 19–25, 19–23, 19–21, 21–25, 21–25, or 21–23 nucleotides long. For example, the antisense chain can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long. In some embodiments, the antisense chain is 19, 20, 21, 22, 23, 24, or 25 nucleotides long. For example, the antisense chain is 21, 22, 23, 24, or 25 nucleotides long. In some specific embodiments, the antisense strand is 22, 23, or 24 nucleotides long. For example, the antisense strand is 23 nucleotides long.

[0028] Similar to the antisense strand, the sense strand can be 15–35 nucleotides long in some embodiments. In some embodiments, the sense strand is 15–35, 17–35, 17–30, 25–35, 27–30, 17–23, 17–21, 17–19, 19–25, 19–23, 19–21, 21–25, 21–25, or 21–23 nucleotides long. For example, the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. For example, the sense strand is 19, 20, 21, 22, or 23 nucleotides long. In some specific embodiments, the sense strand is 20, 21, or 22 nucleotides long. For example, the sense strand is 21 nucleotides long.

[0029] In some embodiments, the sense chain can be 15–35 nucleotides long, and the antisense chain can be 15–35 nucleotides long independently of the sense chain. In some embodiments, the sense chain is 15–35, 17–35, 17–30, 25–35, 27–30, 17–23, 17–21, 17–19, 19–25, 19–23, 19–21, 21–25, 21–25, or 21–23 nucleotides long, and the antisense chain is independently 15–35, 17–35, 17–30, 25–35, 27–30, 17–23, 17–21, 17–19, 19–25, 19–23, 19–21, 21–25, 21–25, or 21–23 nucleotides long. For example, the sense strand and antisense strand can independently be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides long. In some embodiments, the sense strand and antisense strand can independently be 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. For example, the sense strand is 19, 20, 21, 22, or 23 nucleotides long, and the antisense strand is 21, 22, 23, 24, or 25 nucleotides long. In some specific embodiments, the sense strand is 20, 21, or 22 nucleotides long, and the antisense strand is 22, 23, or 24 nucleotides long. For example, the sense strand is 21 nucleotides long, and the antisense strand is 23 nucleotides long.

[0030] The sense strand and antisense strand typically form a double-stranded or dual-stranded region. The dual-stranded region of the dsRNA agents described herein may be 12–35 nucleotide (or base) pairs long, but is not limited to these. For example, the dual-stranded region may be 14–35 nucleotide pairs long, 17–30 nucleotide pairs long, 25–35 nucleotide pairs long, 27–35 nucleotide pairs long, 17–23 nucleotide pairs long, 17–21 nucleotide pairs long, 17–19 nucleotide pairs long, 19–25 nucleotide pairs long, 19–23 nucleotide pairs long, 19–21 nucleotide pairs long, 21–25 nucleotide pairs long, or 21–23 nucleotide pairs long. In another example, the dual-stranded region may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs long. In some embodiments, the double-stranded region is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs long. For example, the double-stranded region is 19, 20, 21, 22, or 23 nucleotide pairs long. In some embodiments, the double-stranded region is 20, 21, or 22 nucleotide pairs long. For example, a dsRNA molecule has a 21-base-pair double-stranded region.

[0031] As described herein, the dsRNA molecule of the present invention may further contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-deoxyribonucleotides, e.g., 2'-H nucleotides. For example, dsRNA may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-deoxyribonucleotides, e.g., 2'-H nucleotides. The 2'-deoxyribonucleotides may be present at any position of any nucleotide on the sense strand, the antisense strand, or both strands. In a non-limiting example, the sense strand does not contain a 2'-deoxyribonucleotide at position 11, counting from the 5' end of the sense strand.

[0032] In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, or 6 2'-deoxynucleotides. For example, the antisense strand may contain 2, 3, 4, 5, or 6 2'-deoxynucleotides. The 2'-deoxynucleotides can be located anywhere in the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at 1, 2, 3, 4, 5, or 6 of the positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand. In a non-limiting example, the antisense strand contains 2'-deoxynucleotides at 1, 2, 3, or 4 of the positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand.

[0033] In some embodiments, the antisense contains 2'-deoxynucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 5, 7, and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, and 7, counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand.

[0034] In some embodiments, the antisense contains a 2'-deoxynucleotide at position 2 or 12, counting from the 5' end of the antisense strand. For example, the antisense contains a 2'-deoxynucleotide at position 12, counting from the 5' end of the antisense strand.

[0035] In some embodiments, the sense strand contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand, and the antisense strand contains a 2'-deoxynucleotide at positions 5 and 7, counting from the 5' end of the antisense strand. For example, the sense strand contains a 2'-fluoronucleotide at positions 9 and 10, counting from the 5' end of the sense strand, and the antisense strand contains a 2'-deoxynucleotide at positions 5 and 7, counting from the 5' end of the antisense strand. In another example, the sense strand contains a 2'-fluoronucleotide at positions 8, 9 and 10, counting from the 5' end of the sense strand, and the antisense strand contains a 2'-deoxynucleotide at positions 5 and 7, counting from the 5' end of the antisense strand.

[0036] In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 10 and 11 counting from the 5' end of the sense strand, and the antisense strand contains 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand. For example, the sense strand contains 2'-fluoronucleotides at positions 10, 11 and 12 counting from the 5' end of the sense strand, and the antisense strand contains 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand.

[0037] In some embodiments of any one of the above aspects, the sense strand does not contain a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand. For example, the sense strand contains a 2'-fluoronucleotide at at least one of positions 9, 10, and 11, e.g., 1, 2, or 3, counting from the 5' end of the sense strand, but does not contain a 2'-fluoronucleotide at position 7. In some embodiments, the sense strand contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand, and contains a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand, and contains a 2'-OMe nucleotide at position 7.

[0038] In some embodiments, the sense strand contains a 2'-fluoronucleotide at positions 9 and 10, counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains a 2'-fluoronucleotide at positions 9 and 10, counting from the 5' end of the sense strand, and a 2'-OMe nucleotide at position 7.

[0039] In some embodiments, the sense strand contains a 2'-fluoronucleotide at positions 10 and 11, counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains a 2'-fluoronucleotide at positions 10 and 11, counting from the 5' end of the sense strand, and a 2'-OMe nucleotide at position 7.

[0040] In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, and a 2'-OMe nucleotide at position 7.

[0041] In some embodiments, the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand, and the remaining nucleotides in the sense strand are 2'-OMe nucleotides.

[0042] 2'-Fluoromodification (antisense chain) The dsRNA molecule of the present invention comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limited to these, all 2'-fluoronucleotides can be present in a single strand. In some embodiments, both the sense strand and the antisense strand contain at least one 2'-fluoronucleotide. 2'-fluoro modifications can be present on any nucleotide of the sense strand or the antisense strand. For example, 2'-fluoro modifications can be present on all nucleotides on the sense strand and / or the antisense strand, or each 2'-fluoro modification can be present in an alternating pattern on the sense strand or the antisense strand, or the sense strand or antisense strand contains both 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0043] In some embodiments, the antisense strand contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. However, the 2'-fluoro modifications in the antisense strand can be located at any position.

[0044] In some embodiments, the antisense comprises one or more 2'-fluoronucleotides, e.g., 1, 2, 3, 4, 5 or more. For example, the antisense chain comprises 1, 2, 3, 4 or 5 or more 2'-fluoronucleotides. In some embodiments, the antisense chain comprises 1, 2 or 3 2'-fluoronucleotides. For example, the antisense chain comprises a single 2'-fluoronucleotide. Note that the 2'-fluoronucleotide can be located anywhere in the antisense chain. For example, the 2'-fluoronucleotide can be at position 2 or 14, counting from the 5' end of the antisense chain. In some embodiments, the antisense comprises a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense chain.

[0045] In some embodiments, the antisense contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense chain, and a 2'-deoxynucleotide at positions 5 and 7. For example, the antisense contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense chain, and a 2'-deoxynucleotide at positions 5, 7 and 12. In a further example, the antisense contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense chain, and a 2'-deoxynucleotide at positions 2, 5, 7 and 12.

[0046] In some embodiments, the antisense strand contains a 2'-deoxynucleotide at positions 2 and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand further contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense strand. For example, the antisense strand contains a 2'-deoxynucleotide at positions 2 and 12, counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14.

[0047] In some embodiments, the antisense strand contains a nucleotide other than a 2'-deoxynucleotide at position 16, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand contains a nucleotide other than a 2'-fluoronucleotide at position 16, counting from the 5' end of the antisense strand. For example, the antisense strand contains a 2'-deoxynucleotide at positions 2 and 12, counting from the 5' end of the antisense strand, and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16. In some embodiments, the antisense strand contains a 2'-deoxynucleotide at positions 2 and 12, counting from the 5' end of the antisense strand, and a 2'-OMe nucleotide at position 16.

[0048] In some embodiments, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, a 2'-fluoronucleotide at position 14, and a nucleotide other than 2'-deoxy at position 16. In some embodiments, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, a 2'-fluoronucleotide at position 14, and a nucleotide other than 2'-fluoro at position 16. For example, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, a 2'-fluoronucleotide at position 14, and a nucleotide other than 2'-deoxy or 2'-fluoronucleotide at position 16. In some embodiments, the antisense chain contains a 2'-deoxynucleotide at positions 2 and 12 counting from the 5' end of the antisense chain, a 2'-fluoronucleotide at position 14, and a 2'-OMe nucleotide at position 16.

[0049] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14, with the remaining nucleotides in the antisense strand being 2'-OMe nucleotides.

[0050] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14, and the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, and the remaining nucleotides in the antisense strand and the sense strand are 2'-OMe nucleotides.

[0051] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand, with the remaining nucleotides in the antisense strand being 2'-OMe nucleotides.

[0052] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14 counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, with the remaining nucleotides in the antisense and sense strands being 2'-OMe nucleotides.

[0053] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16, counting from the 5' end of the antisense strand, with the remaining nucleotides in the antisense strand being 2'-OMe nucleotides.

[0054] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, with the remaining nucleotides in the antisense and sense strands being 2'-OMe nucleotides.

[0055] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand, with the remaining nucleotides in the antisense strand being 2'-OMe nucleotides.

[0056] In some embodiments, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16 counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, with the remaining nucleotides in the antisense and sense strands being 2'-OMe nucleotides.

[0057] The remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the sense strand and / or antisense strand, can be unmodified or modified nucleotides. Therefore, in some embodiments, the remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the sense strand, are either unmodified or modified nucleotides. For example, the remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the sense strand, are 2'-OMe, 2'-F, 2'-H, and 2'-OC. 10~30 It can be a modified nucleotide selected from the group consisting of aliphatic groups, and is optional, except for 2'-OC. 10~30 The modified nucleotides are assumed to have one or fewer aliphatic groups.

[0058] In some embodiments, the remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the antisense strand, are either unmodified or modified nucleotides. For example, the remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the antisense strand, can be modified nucleotides. In some embodiments, the remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the antisense strand, can be selected from the group consisting of 2'-OMe, 2'-F, 2'-H, GNA, and 3'-RNA, where 3'-RNA is optionally 3'-OH, and there is no more than one modified nucleotide that is either GNA or 3'-RNA.

[0059] In some embodiments, the remaining nucleotides in the sense strand and / or antisense strand are 2'-OMe nucleotides.

[0060] As described herein, dsRNA agents may contain one or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, that contain modified sugars. "Modified sugar" means a sugar other than 2'-deoxy (i.e., 2'-H), 2'-OH, 2'-F, or 2'-OMe ribose sugar. Some exemplary nucleotides containing modified sugars include located nucleic acid (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), and 2'-ara-F. Therefore, in some embodiments, the dsRNA agent may contain one or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, independently 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), and 2'-ara-F. Nucleotides containing modified sugars can be present anywhere in the dsRNA molecule. For example, nucleotides containing modified sugars can be present in the sense strand, or nucleotides containing modified sugars can be present in the antisense strand. If there are two or more nucleotides containing modified sugars in the dsRNA molecule, they can all be present in the sense strand, the antisense strand, or both the sense and antisense strands.

[0061] In some embodiments, unmodified nucleotides are unmodified nucleic acid bases, i.e., 2'-OH nucleotides containing adenine, guanine, cytosine, or uracil.

[0062] In some embodiments, dsRNA may contain nucleotides comprising one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural nucleic acid bases. “Non-natural nucleic acid bases” means nucleic acid bases other than adenine, guanine, cytosine, uracil, or thymine. Exemplary non-natural nucleic acid bases include inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil, and 5-halositol Syn, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothimine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and 8-substituted guanines, 5-trifluoromethyl and other 5-substituted uracils and 5-substituted cytosine N, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl -Uracil, N3-methyluracil, substituted 1,2,4-triazole, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4Examples of purines and pyrimidines include, but are not limited to, acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases. Further purines and pyrimidines are disclosed in U.S. Patent No. 3,687,808, in Concise Encyclopedia of Polymer Science and Engineering, edited by J.I. Kroschwitz, John Wiley & Sons, 1990, pp. 858-859, and in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0063] In some aspects, non-natural nucleic acid bases include inosine, xanthine, hypoxanthine, nubularin, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, and 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4 -(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudracil, 4-(thio)pseudracil, 2,4-(dithio)pseudracil, 5-(alkyl)pseudracil, 5-(methyl)pseudracil, 5-(alkyl)-2-(thio)pseudracil, 5-(methyl)-2-(thio)pseudracil, 5-(alkyl)-4-(thio)pseudracil, 5-(methyl)-4-(thio)pseudracil, 5-(alkyl)-2,4-(dithio)pseudracil Douracil, 5-(methyl)-2,4-(dithio)pseudracil, 1-substituted pseudouracil, 1-substituted 2(thio)pseudracil, 1-substituted 4-(thio)pseudracil, 1-substituted 2,4-(dithio)pseudracil, 1-(aminocarbonylethylenyl)pseudracil, 1-(aminocarbonylethylenyl)-2(thio)pseudracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudracil Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiadin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl,7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl,7-substituted 1,3-(diaza)-2-(oxo)-phenthiadin-1-yl,7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(guanidinium alkyl (Hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularin, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-ino Synyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrillyl, 5-(methyl)isocarbostyrillyl, 3-(methyl)-7-(propynyl)isocarbostyrillyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrillyl, 7-(propynyl)isocarbostyrillyl, propynyl-7-(aza)indolyl Lu, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenantracenyl, pyrenyl, stilbenyl, tetracerenyl, pentaceryl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N, 2 - Substitution purine, N6 - Substitution purine, O 6 -The group can be selected from substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

[0064] Nucleotides containing non-natural nucleic acid bases can be located anywhere within a dsRNA molecule. For example, a nucleotide containing a non-natural nucleic acid base can be located in the sense strand, or in the antisense strand. If there are two or more nucleotides containing non-natural nucleic acid bases in a dsRNA molecule, they can all be located in the sense strand, the antisense strand, or both the sense and antisense strands.

[0065] The dsRNA molecule of the present invention may further include at least one phosphorothioate or methylphosphonate internucleotide junction. The phosphorothioate internucleotide junction modification or the methylphosphonate internucleotide junction modification may be present at any position on the chain, on any nucleotide of the sense strand, the antisense strand, or both. For example, the internucleotide junction modification may be present on all nucleotides on the sense strand and / or the antisense strand, or each internucleotide junction modification may be present in an alternating pattern on the sense strand or the antisense strand, or the sense strand or antisense strand may contain both internucleotide junction modifications in an alternating pattern. The alternating pattern of internucleotide junction modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide junction modifications on the sense strand may be shifted relative to the alternating pattern of internucleotide junction modifications on the antisense strand.

[0066] In some embodiments, the dsRNA molecule includes phosphorothioate internucleotide junction modifications or methylphosphonate internucleotide junction modifications in the overhang region. For example, the overhang region includes two nucleotides having a phosphorothioate internucleotide junction or a methylphosphonate internucleotide junction between the two nucleotides. Internucleotide junction modifications may be made to link the overhang nucleotides to terminal paired nucleotides in the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked by phosphorothioate internucleotide junctions or methylphosphonate internucleotide junctions, and optionally, additional phosphorothioate internucleotide junctions or methylphosphonate internucleotide junctions may be present linking the overhang nucleotides to the paired nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide junctions between the three terminal nucleotides, where two of those three nucleotides are overhang nucleotides and the third is a paired nucleotide adjacent to the overhang nucleotide. Preferably, these three terminal nucleotides may be located at the 3' end of the antisense strand.

[0067] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate nucleotide junctions or methylphosphonate nucleotide junctions, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide junctions, one of which is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand containing any combination of phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, or phosphate nucleotide junctions.

[0068] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of phosphorothioate nucleotide junctions or methylphosphonate nucleotide junctions, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate nucleotide junctions, one of which is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, or phosphate nucleotide junctions.

[0069] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of three phosphorothioate nucleotide junctions or methylphosphonate nucleotide junctions, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide junctions, one of which is positioned at any location in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, or phosphate nucleotide junctions.

[0070] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of four phosphorothioate nucleotide junctions or methylphosphonate nucleotide junctions, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate nucleotide junctions, one of which is positioned at any location in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, or phosphate nucleotide junctions.

[0071] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of five phosphorothioate internucleotide junctions or methylphosphonate internucleotide junctions, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate nucleotide junctions, one of which is positioned at any location in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, or phosphate nucleotide junctions.

[0072] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of six phosphorothioate internucleotide junctions or methylphosphonate internucleotide junctions, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate nucleotide junctions, one of which is positioned at any location in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, or phosphate nucleotide junctions.

[0073] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate internucleotide junctions or methylphosphonate internucleotide junctions, separated by one, two, three, four, five, six, seven, or eight phosphate nucleotide junctions, one of which is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, or phosphate nucleotide junctions.

[0074] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate internucleotide junctions or methylphosphonate internucleotide junctions, separated by one, two, three, four, five, or six phosphate nucleotide junctions, one of which is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate internucleotide junctions, methylphosphonate internucleotide junctions, or phosphate nucleotide junctions.

[0075] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate nucleotide junctions or methylphosphonate nucleotide junctions separated by one, two, three, or four phosphate nucleotide junctions, one of which is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, and phosphate nucleotide junctions, or with an antisense strand containing either phosphorothioate nucleotide junctions, methylphosphonate nucleotide junctions, or phosphate nucleotide junctions.

[0076] In some embodiments, the dsRNA molecule of the present invention further comprises one or more phosphorothioate nucleotide junction modifications or methylphosphonate nucleotide junction modifications within 1 to 10 of the terminal positions of the sense strand and / or antisense strand. For example, at one or both ends of the sense strand and / or antisense strand, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphorothioate nucleotide junctions or methylphosphonate nucleotide junctions.

[0077] In some embodiments, the dsRNA molecule of the present invention contains one or more phosphorothioate nucleotide junction modifications or methylphosphonate nucleotide junction modifications within 1 to 10 of the internal regions of each duplex of the sense strand and / or antisense strand. For example, at positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphorothioate-methylphosphonate nucleotide junctions, and the dsRNA molecule may optionally further contain one or more phosphorothioate nucleotide junction modifications or methylphosphonate nucleotide junction modifications within 1 to 10 of the terminal positions.

[0078] In some embodiments, the dsRNA molecule of the present invention further comprises 1 to 5 phosphorothioate internucleotide junction modifications or methylphosphonate internucleotide junction modifications within positions 1 to 5 (counting from the 5' end) of the sense strand, and 1 to 5 phosphorothioate internucleotide junction modifications or methylphosphonate internucleotide junction modifications within the last 3 positions, and 1 to 5 phosphorothioate internucleotide junction modifications or methylphosphonate internucleotide junction modifications within positions 1 and 2 (counting from the 5' end) of the antisense strand, and 1 to 5 phosphorothioate internucleotide junction modifications or methylphosphonate internucleotide junction modifications within the last 6 positions.

[0079] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate internucleotide junction modification within positions 1 to 5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide junction modification or methylphosphonate internucleotide junction modification within the last six positions, and one phosphorothioate internucleotide junction modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate internucleotide junction modifications or methylphosphonate internucleotide junction modifications within the last six positions.

[0080] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate nucleotide junction modification within the last six positions, as well as one phosphorothioate nucleotide junction modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate nucleotide junction modifications within the last six positions.

[0081] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1 to 5 (counting from the 5' end) of the sense strand, and two phosphorothioate nucleotide junction modifications within the last four positions, as well as one phosphorothioate nucleotide junction modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate nucleotide junction modifications within the last six positions.

[0082] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate nucleotide junction modifications within the last four positions, as well as one phosphorothioate nucleotide junction modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate nucleotide junction modification within the last six positions.

[0083] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate nucleotide junction modification within positions 1 to 5 (counting from the 5' end) of the sense strand, and one phosphorothioate nucleotide junction modification within the last four positions, as well as two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate nucleotide junction modifications within the last six positions.

[0084] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate nucleotide junction modification within positions 1 to 5 (counting from the 5' end) of the sense strand, and one within the last six positions, as well as two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one within the last six positions.

[0085] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate nucleotide junction modification within positions 1 to 5 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate nucleotide junction modification within the last 6 positions.

[0086] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1-5 (counting from the 5' end) of the sense strand, one phosphorothioate nucleotide junction modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate nucleotide junction modifications within the last six positions.

[0087] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1-5 (counting from the 5' end) of the sense strand and one within the last six positions, and two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and one within the last six positions.

[0088] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate nucleotide junction modification within the last six positions, and two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate nucleotide junction modifications within the last six positions.

[0089] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate nucleotide junction modification within the last six positions, as well as one phosphorothioate nucleotide junction modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate nucleotide junction modifications within the last six positions.

[0090] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide junction modifications at positions 20 and 21, and one phosphorothioate nucleotide junction modification at position 1 (counting from the 5' end) and one at position 21 of the antisense strand.

[0091] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate nucleotide junction modification at position 1 (counting from the 5' end) of the sense strand, and one phosphorothioate nucleotide junction modification at position 21, and two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate nucleotide junction modifications at positions 20 and 21.

[0092] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide junction modifications at positions 21 and 22, and one phosphorothioate nucleotide junction modification at position 1 (counting from the 5' end) and one phosphorothioate nucleotide junction modification at position 21 of the antisense strand.

[0093] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate nucleotide junction modification at position 1 (counting from the 5' end) of the sense strand, and one phosphorothioate nucleotide junction modification at position 21, and two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate nucleotide junction modifications at positions 21 and 22.

[0094] In some embodiments, the dsRNA molecule of the present invention further comprises two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the sense strand, two phosphorothioate nucleotide junction modifications at positions 22 and 23, and one phosphorothioate nucleotide junction modification at position 1 (counting from the 5' end) of the antisense strand, and one phosphorothioate nucleotide junction modification at position 21.

[0095] In some embodiments, the dsRNA molecule of the present invention further comprises one phosphorothioate nucleotide junction modification at position 1 (counting from the 5' end) of the sense strand, and one phosphorothioate nucleotide junction modification at position 21, as well as two phosphorothioate nucleotide junction modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate nucleotide junction modifications at positions 22 and 23.

[0096] In some embodiments, the sense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand. For example, the sense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the sense strand.

[0097] In some embodiments, the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand. For example, the antisense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the antisense strand.

[0098] In some embodiments, the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 3' end of the antisense strand. For example, the antisense strand includes phosphorothioate junctions between nucleotides n and n-1 and between nucleotides n-1 and n-2, where n is the length of the antisense strand, i.e., the number of nucleotides in the antisense strand. In other words, the antisense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3, counted from the 3' end of the antisense strand.

[0099] In some embodiments, the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand. For example, the antisense strand includes phosphorothioate junctions between nucleotides 1 and 2, and between nucleotides 2 and 3, counted from the 5' end of the antisense strand, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counted from the 3' end of the antisense strand.

[0100] In some embodiments, the sense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand, and the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand. For example, the sense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the sense strand, and the antisense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the antisense strand.

[0101] In some embodiments, the sense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand, and the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 3' end of the antisense strand. For example, the sense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the sense strand, and the antisense strand includes phosphorothioate junctions between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 3' end of the antisense strand.

[0102] In some embodiments, the compounds of the present invention include a backbone chiral center in a certain pattern. In some embodiments, the common pattern of the backbone chiral center includes at least five Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least six Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least seven Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least eight Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least nine Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least ten Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least eleven Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least twelve Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 13 Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 14 Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 15 Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 16 Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 17 Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 18 Sp-configured internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 19 Sp-configured internucleotide junctions.In some embodiments, the common pattern of backbone chiral centers contains eight or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains seven or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains six or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains five or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains four or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains three or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains two or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains one or fewer nucleotide junctions of the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains eight or fewer non-chiral internucleotide junctions (phosphodiesters, as an unrestricted example). In some embodiments, the common pattern of backbone chiral centers contains seven or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of backbone chiral centers contains six or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of backbone chiral centers contains five or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of backbone chiral centers contains four or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of backbone chiral centers contains three or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of backbone chiral centers contains two or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of backbone chiral centers contains one or fewer non-chiral internucleotide junctions.In some embodiments, the common pattern of the backbone chiral center includes at least 10 Sp-configured internucleotide junctions and 8 or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 11 Sp-configured internucleotide junctions and 7 or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 12 Sp-configured internucleotide junctions and 6 or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 13 Sp-configured internucleotide junctions and 6 or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of the backbone chiral center includes at least 14 Sp-configured internucleotide junctions and 5 or fewer non-chiral internucleotide junctions. In some embodiments, the common pattern of the backbone chiral centers includes at least 15 Sp-configured internucleotide junctions and 4 or fewer non-chiral internucleotide junctions. In some embodiments, the Sp-configured internucleotide junctions may be optionally continuous or non-contiguous. In some embodiments, the Rp-configured internucleotide junctions may be optionally continuous or non-contiguous. In some embodiments, the non-chiral internucleotide junctions may be optionally continuous or non-contiguous.

[0103] In some embodiments, the compounds of the present invention include a block which is a stereochemical block. In some embodiments, the block is an Rp block where each nucleotide junction of the block is an Rp block. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the block is an Sp block where each nucleotide junction of the block is an Sp block. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, the oligonucleotide provided includes both Rp blocks and Sp blocks. In some embodiments, the oligonucleotide provided includes one or more Rp blocks but does not include any Sp blocks. In some embodiments, the oligonucleotide provided includes one or more Sp blocks but does not include any Rp blocks. In some embodiments, the oligonucleotide provided includes one or more PO blocks where each nucleotide junction is a native phosphate junction.

[0104] In some embodiments, the compounds of the present invention include a 5'-block, which is an Sp block in which each sugar portion contains a 2'-fluoro modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide junction is a modified internucleotide junction and each sugar portion contains a 2'-fluoro modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide junction is a phosphorothioate junction and each sugar portion contains a 2'-fluoro modification. In some embodiments, the 5'-block contains four or more nucleoside units. In some embodiments, the 5'-block contains five or more nucleoside units. In some embodiments, the 5'-block contains six or more nucleoside units. In some embodiments, the 5'-block contains seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar portion contains a 2'-fluoro modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide junction is a modified internucleotide junction and each sugar portion contains a 2'-fluoro modification. In some embodiments, the 3'-block is an Sp-block in which each internucleotide junction is a phosphorothioate junction and each sugar moiety contains a 2'-fluoro modification. In some embodiments, the 3'-block contains four or more nucleoside units. In some embodiments, the 3'-block contains five or more nucleoside units. In some embodiments, the 3'-block contains six or more nucleoside units. In some embodiments, the 3'-block contains seven or more nucleoside units.

[0105] In some embodiments, the compounds of the present invention include a nucleoside of a certain type in a region or oligonucleotide, followed by a specific type of internucleotide junction, such as a natural phosphate junction, a modified internucleotide junction, an Rp chiral internucleotide junction, an Sp chiral internucleotide junction, and the like. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate junction (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate junction (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate junction (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate junction (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0106] Various publications describe multimeric siRNAs, all of which can be used with the dsRNA of the present invention. Such publications include WO2007 / 091269, U.S. Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are incorporated herein by reference in their entirety.

[0107] Ligand A wide variety of entities can be coupled to the dsRNA agents described herein. The preferred part is the ligand, which is preferably covalently coupled directly or indirectly via an intervening tether. Generally, the ligand modifies the distribution, targeting, or lifespan of the dsRNA described herein into which it is incorporated. In some embodiments, the ligand provides enhanced affinity with respect to a selected target, such as a molecule, cell or cell type, compartment, receptor, such as a compartment in a cell or organ, tissue, organ, or region of the body, compared to a molecular species lacking such a ligand. A ligand that provides enhanced affinity with respect to a selected target is also referred to herein as a targeting ligand.

[0108] Some ligands may possess endosomal lysis properties. Endosomal lysis ligands promote the lysis of endosomes and / or the transport of the composition or its components from endosomes to the cytoplasm of cells. Endosomal lysis ligands may be polyanionic peptides or peptidomimetic compounds exhibiting pH-dependent membrane activity and membrane fusion properties. In some embodiments, endosomal lysis ligands adopt their active conformation at endosomal pH. The “active” conformation is the conformation in which the endosomal lysis ligand promotes the lysis of endosomes and / or the transport of the composition or its components from endosomes to the cytoplasm of cells. Examples of endosomal soluble ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972, this document is incorporated herein by reference in its entirety), EALA peptide (Vogel et al., J.Am.Chem.Soc., 1996, 118:1581-1586, this document is incorporated herein by reference in its entirety), and their derivatives (Turk et al., Biochem.Biophys.Acta, 2002, 1559:56-68, this document is incorporated herein by reference in its entirety). In some embodiments, the endosomal soluble components may contain chemical groups (e.g., amino acids) that undergo a change in charge or protonation in response to a change in pH. The endosomal soluble components may be linear or branched.

[0109] Ligands can improve the transport, hybridization, and specificity properties of the resulting natural or modified oligoribonucleotides, or polymer molecules containing any combination of the monomers and / or natural or modified ribonucleotides described herein, and can also improve nuclease resistance.

[0110] Ligands can generally include therapeutic modifiers, such as those that enhance uptake; diagnostic compounds or reporter groups, such as those that monitor distribution; crosslinking agents; and nuclease resistance-constituting moieties. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetic compounds.

[0111] Ligands may include natural substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids, or 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-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0112] Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, such as antibodies, nanobodies, or parts of antibodies or nanobodies that bind to specified cell types, such as kidney cells or blood-brain barrier cells. Targeting groups can be thyrotropins, melanotropins, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin targeting groups, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, RGD peptides, RGD peptide mimetics, or aptamers.

[0113] Other examples of ligands include dyes, inserts (e.g., acridines), crosslinking agents (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyllin, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantane acetate, 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-( Examples include oleoyl(colentic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, or AP.

[0114] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for a co-ligand, or antibodies, such as antibodies that bind to a specified cell type, such as cancer cells, endothelial cells, or osteocytes. Ligands may also include hormones and hormone receptors. They may also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0115] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of that cell, for example, by disrupting the cellular microtubules, microfibrils and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoservin.

[0116] Ligands can increase the uptake of dsRNA into cells, for example, by activating the inflammatory response. Exemplary ligands that may have such effects include tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or gamma interferon.

[0117] In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues, for example, to non-renal target tissues of the body. For example, the target tissue can be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Lipid-based ligands can be used to modulate, for example, control the binding of the conjugate to target tissues. For example, the more strongly a lipid or lipid-based ligand binds to HSA, the less likely it will be targeted to the kidney and therefore less likely to be removed from the body. Lipid or lipid-based ligands that bind weakly to HSA can be used to target the conjugate to the kidney.

[0118] In a preferred embodiment, a lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the conjugate is preferably distributed to non-renal tissue. However, the affinity is preferably not so strong that the HSA-ligand binding becomes irreversible.

[0119] In another preferred embodiment, the lipid-based ligand either weakly binds to the HSA or does not bind at all, such that the conjugate is preferably distributed in the kidney. Other moieties that target kidney cells can be used instead of, or in addition to, the lipid-based ligand.

[0120] In some embodiments, the ligand is a portion of the target cell, such as a proliferating cell, e.g., a vitamin. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as cancer cells, whether malignant or non-malignant. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include the B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. HAS, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) are also included.

[0121] In another aspect, the ligand is a cell permeability agent, preferably a helix cell permeability agent. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or Antennapedia. If the agent is a peptide, it can be modified, including the use of peptidyl mimetic, invertomer, non-peptide or pseudopeptide linkers, and D-amino acids. The helix agent is preferably an alpha-helix agent, preferably having a lipophilic phase and oleophobicity.

[0122] The ligand can be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a distinct three-dimensional structure similar to that of natural peptides. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptidomimetic can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., one consisting mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. Another option is that the peptide moiety can contain a hydrophobic membrane translocation sequence (MTS). An example of a hydrophobic MTS-containing peptide is an amino acid sequence This is an RFGF containing TIFF0007882858000001.tif4128. It is an RFGF analog containing hydrophobic MTS (e.g., amino acid sequence). TIFF0007882858000002.tif4128) can also be a targeting region. The peptide portion can be a "delivery" peptide that carries large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, a sequence derived from the HIV Tat protein: Sequences derived from TIFF0007882858000003.tif4128 and Drosophila Antennapedia protein: TIFF0007882858000004.tif4128 has been shown to function as a delivery peptide. Peptides or peptidomimetic peptides can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-94, 1991; this document is incorporated herein by reference in its entirety). The peptide or peptidomimetic peptide linked to the iRNA agent via incorporated monomer units is preferably a cell-targeting peptide such as an arginine-glycine-aspartate (RGD) peptide or RGD mimic. The length of the peptide portion can range from approximately 5 to approximately 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or to direct conformational properties. Any of the structural modifications described below are available. The RGD peptide portion 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; this document is incorporated herein by reference in its entirety). The RGD peptide can facilitate the targeting of iRNA agents to tumors in various other tissues, including the lungs, kidneys, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001; this document is incorporated herein by reference in its entirety). Preferably, the RGD peptide will facilitate the targeting of iRNA agents to the kidneys. The RGD peptide can be linear or cyclic and may be modified, e.g., glycosylated or methylated, to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can target iRNA agents to α VIt can be delivered to tumor cells expressing β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001; this document is incorporated herein by reference in its entirety). Peptides that target markers enriched in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly those exhibiting integrins. Thus, RGD peptides, RGD-containing cyclic peptides, RGD peptides containing D-amino acids, and synthetic RGD mimetic can be used. In addition to RGD, other moieties that target integrin ligands can also be used. In general, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type have ligands that target PECAM-1, VEGF, or other oncogenes, for example, the oncogenes described herein.

[0123] "Cell-permeable peptides" can permeate cells, such as microbial cells like bacterial or fungal cells, or mammalian cells like human cells. Microbial cell-permeable peptides can be, for example, α-helix linear peptides (e.g., LL-37 or ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides overwhelmingly containing only one or two amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides can also contain nuclear localization signals (NLS). For example, a cell-permeable peptide can be a bipartite amphiphilic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003; this document is incorporated herein by reference in its entirety).

[0124] In some embodiments, the targeted peptide can be an amphiphilic α-helix peptide. Exemplary amphiphilic α-helix peptides include, but are not limited to, secropine, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidine, ceratotoxin, S. clava peptide, slugfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaceptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin. Several factors will preferably be considered to maintain the integrity of helix stability. For example, the maximum number of helix-stabilizing residues will be utilized (e.g., leu, ala, or lys), and the minimum number of helix-destabilizing residues will be utilized (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 extra H bonds to stabilize the helix). Formation of salt bridges between residues with opposite charges separated by i±3 or i±4 can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine, or histidine can form salt bridges with anionic residues such as glutamic acid or aspartic acid.

[0125] Peptides and peptidomimetic ligands include native or modified peptides, such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides in which one or more amide linkers, i.e., peptide linkers, are replaced by one or more urea, thiourea, carbamate, or sulfonylurea linkers; or cyclic peptides.

[0126] A targeted ligand can be any ligand capable of targeting a specific receptor. Examples include folic acid, GalNAc, galactose, mannose, mannose-6P, sugar clusters, e.g., GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeted ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Ligands can also be nucleic acid-based, such as aptamers. Aptamers can be unmodified or have any combination of modifications disclosed herein.

[0127] Examples of endosomal release agents include imidazoles, poly- or oligoimidazoles, PEIs, peptides, fusion peptides, polycarboxylates, polycations, shielded oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with shielded or unshielded cationic or anionic charges, and dendrimers with shielded or unshielded cationic or anionic charges.

[0128] PK modifiers refer to pharmacokinetic modifiers. Examples of PK modifiers include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, and vitamins. Exemplary PK modifiers include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. It is also known that oligonucleotides containing several phosphorothioate linkages can bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in their backbone, such as oligonucleotides of about 5, 10, 15, or 20 bases, can also be applied to the present invention as ligands (e.g., as PK modulating ligands).

[0129] In addition, aptamers that bind to serum components (e.g., serum proteins) can also be applied to the present invention as PK-modulating ligands.

[0130] Other ligand conjugates applicable to the present invention are described in the following U.S. patent applications, each incorporated herein by reference in its entirety for all purposes: U.S. Patent Application No. 10 / 916,185 filed August 10, 2004; U.S. Patent Application No. 10 / 946,873 filed September 21, 2004; U.S. Patent Application No. 10 / 833,934 filed August 3, 2007; U.S. Patent Application No. 11 / 115,989 filed April 27, 2005; and U.S. Patent Application No. 11 / 944,227 filed November 21, 2007.

[0131] In some embodiments, a dsRNA molecule may contain two or more ligands, for example, two, three, four, or five. If two or more ligands are present, they 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, ligands may have targeting properties, endosomal lysis activity, or PK modulating properties. In one preferred embodiment, all ligands have different properties.

[0132] In some embodiments, a dsRNA molecule contains two ligands. For example, the sense strand of a dsRNA molecule contains a first ligand attached to the 3' end of the sense strand and a second ligand attached to the 5' end of the sense strand. In some embodiments, a dsRNA molecule contains two ligands ligated to the sense strand, the first ligand containing an inverted debasalized nucleotide (i.e., a debasalized nucleotide linked by a 3'→3' junction), and the second ligand containing an ASGPR ligand.

[0133] Ligands can be coupled to dsRNA at various locations, for example, at the 3' end, 5' end, and / or internal position of the sense and / or antisense strands. In a preferred embodiment, the ligand is attached to the sense and / or antisense strands of the dsRNA via a linker or tether. The ligand or linked ligand can be present on the monomer when the monomer is incorporated into the growing chain. In some embodiments, the ligand can be incorporated into the “precursor” monomer after the “precursor” monomer has been incorporated into the growing chain. For example, monomers having a tether with an amino group at the end (i.e., those without a ligand), such as TAP-(CH2) n NH2 can be incorporated into the growing oligonucleotide chain. In subsequent operations, i.e., after the incorporation of the precursor monomer into the chain, an electrophilic ligand, such as a pentafluorophenyl ester or aldehyde group, can be attached to the precursor monomer by coupling the electrophile of the ligand with the terminal nucleophile of the tether of the precursor monomer.

[0134] In another example, monomers having chemical groups suitable for participating in click chemistry reactions, such as azide or alkyne terminal tethers / linkers, can be incorporated. In subsequent operations, i.e., after the incorporation of the precursor monomer into the chain, a ligand having complementary chemical groups, such as alkynes or azides, can be attached to the precursor monomer by coupling the alkyne and azide together.

[0135] The ligand can be attached to one or both strands. In some embodiments, the dsRNA described herein includes a ligand conjugated to the sense strand. In some embodiments, the dsRNA described herein includes a ligand conjugated to the antisense strand.

[0136] In some embodiments, the ligand is conjugated to the sense strand. As described herein, the ligand can be conjugated at the 3' end, 5' end, or internal position of the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand. In some embodiments, the ligand is conjugated to the 5' end of the sense strand. In some embodiments, the ligand is conjugated at an internal position of the sense strand. In other words, the ligand is conjugated to a non-terminal nucleotide of the sense strand. Note that the ligand can be conjugated to a nucleic acid base, sugar moiety, or internucleotide junction of the sense strand.

[0137] In some embodiments, the ligand is conjugated at the 2' position of a nucleotide in the sense strand. For example, the ligand is conjugated at an internal position in the sense strand, i.e., at the 2' position of a nucleotide at a non-terminal position.

[0138] In some embodiments, ligands can be conjugated to nucleic acid bases, sugar moieties, or internucleoside junctions of nucleic acid molecules. Conjugation to purine nucleic acid bases or their derivatives can occur at any position, including intraring and extraring atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of purine nucleic acid bases are attached to the conjugate moiety. Conjugation to pyrimidine nucleic acid bases or their derivatives can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of pyrimidine nucleic acid bases can be substituted by the conjugate moiety. Conjugation to the sugar moiety of nucleosides can occur at any carbon atom. Examples of carbon atoms in the sugar moiety that can be attached to the conjugate moiety include the 2', 3', and 5' carbon atoms. In the case of debasic residues, the 1' position can also be attached to the conjugate. Internucleoside junctions can also hold the conjugate moiety. For phosphorus-containing conjugates (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates, etc.), the conjugate portion can be attached directly to the phosphorus atom or to the O, N, or S atoms bonded to the phosphorus atom. In the case of nucleoside conjugates containing amines or amides (e.g., PNAs), the conjugate portion can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0139] In some embodiments, the ligand is conjugated to the sense strand. As described herein, the ligand can be conjugated at the 3' end, 5' end, or internal position of the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand. Furthermore, it should be noted that the ligand can be conjugated to the nucleic acid base, sugar moiety, or internucleotide junction of the sense strand.

[0140] Any suitable ligand in the field of RNA interference can be used, but ligands are typically carbohydrates, such as monosaccharides (e.g., GalNAc), disaccharides, trisaccharides, tetrasaccharides, and polysaccharides.

[0141] Examples of linkers that conjugate ligands to nucleic acids include those mentioned above. For example, the ligand can be one or more carbohydrates, such as a GalNAc (N-acetylgalactosamine) derivative attached by a monovalent, divalent, or trivalent branched linker.

[0142] In some embodiments, the dsRNA of the present invention is conjugated to bivalent and trivalent branched linkers comprising structures represented by any of formulas (IV) to (VII): TIFF0007882858000005.tif82161In formula, q 2A , q 2B , q 3A , q 3B q4 A , q 4B , q 5A , q 5B and q 5C Each occurrence independently represents a number from 0 to 20, and the repeating unit can be the same or different. P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C Each of these terms is either absent or, in each instance, is CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O. Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5CEach instance is independently either absent or an alkylene, a substituted alkylene, where one or more methylene groups are O, S, S(O), SO2, N(R) N The process may be interrupted or terminated at one or more of the following: 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 Each of these terms is independent of its occurrence and either does not exist, or is represented by NH, O, S, CH2, C(O)O, C(O)NH, or NHCH(R). a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, TIFF0007882858000006.tif18153 or heterocycline, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C Each of these terms independently represents a ligand, i.e., a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. R a This is either H or an amino acid side chain.

[0143] For example, equation (VII): Trivalent galNAc derivatives, such as TIFF0007882858000007.tif40128, are particularly useful for use in the dsRNA agents described herein for inhibiting the expression of target genes. In the formula, L 5A , L 5B and L 5C These are monosaccharides such as GalNAc derivatives.

[0144] Suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the following compounds: TIFF0007882858000008.tif58128TIFF0007882858000009.tif202147TIFF0007882858000010.tif118128.

[0145] In some embodiments, the dsRNA described herein includes ligand 1, i.e., a ligand having the following structure: TIFF0007882858000011.tif50128.

[0146] In some embodiments, the dsRNAs described herein include ligands described in U.S. Patent No. 5,994,517 or U.S. Patent No. 6,906,182, the contents of which are incorporated herein by reference in their entirety.

[0147] In some embodiments, the ligand may be a triantennae-type ligand as shown 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 triantennae-type ligands: TIFF0007882858000012.tif199170.

[0148] In some aspects of any one of the above aspects, the antisense chain contains a phosphoryl analog or phosphate mimic at its 5' end. In some aspects, the antisense chain contains an alkenyl phosphonate, i.e., a vinyl phosphonate, at its 5' end. For example, the antisense chain contains a 5'-E-vinyl phosphonate.

[0149] In some embodiments, the antisense chain contains a cyclopropylphosphonate at its 5' end. For example, the antisense chain has a cyclopropylphosphonate at its 5' end. The formula includes TIFF0007882858000013.tif20128, where * represents the attachment of the nucleotide at the 5' end to the C5 position.

[0150] In any one or several embodiments of the above aspects, at least one of the strands, for example, the sense strand and / or antisense strand of a double-stranded RNA, comprises a monomer or ligand selected from the following: In formula TIFF0007882858000014.tif26128, * represents a bond to the 5',3' terminal hydroxyl group of the chain; In formula TIFF0007882858000015.tif30128, * represents the bond to the 2'-hydroxyl group of a nucleotide in the chain; In formula TIFF0007882858000016.tif42135, * represents a bond to the 5' or 3' end of the chain; In formula TIFF0007882858000017.tif42128, * represents a bond to the 5' or 3' end of the chain; In formula TIFF0007882858000018.tif85128, -O-* is a connection to the 5' or 3' end of the chain; and / or In formula TIFF0007882858000019.tif65128, -P-* represents a connection to a 5' or 3'-hydroxyl group in the chain.

[0151] In some embodiments, the ligand includes a lipophilic group. For example, the ligand is C 6~30 Aliphatic group or C 10~30 It may be an aliphatic group. As used herein, “aliphatic” means a saturated or unsaturated linear, branched and / or cyclic hydrocarbon having a specified number of carbon atoms, examples of which include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl and cycloalkylalkynyl groups having a specified number of carbon atoms. In some embodiments, the ligand is C 10~30It is an alkyl group. For example, the ligand is a linear or branched hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl, or tetracosyl group. For example, the ligand is a linear hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl, or tetracosyl group. For example, the ligand is a linear hexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, eicosyl, or docosyl group. For example, the ligand is a linear hexadecyl group. For example, the ligand is a linear docosyl group.

[0152] In certain embodiments, the ligand is conjugated at the 2' position of a nucleotide located inside the sense strand, i.e., at a non-terminal position, and is a linear or branched tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand is conjugated at the 2' position of a nucleotide located inside the sense strand, i.e., at a non-terminal position, and is a linear or branched hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand is conjugated at the 2' position of a nucleotide located inside the sense strand, i.e., at a non-terminal position, and is a linear hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand is conjugated at the 2' position of a nucleotide in the sense strand, i.e., at a non-terminal position, and is a linear hexadecyl, octadecyl, icosyl, or docosyl group.

[0153] The internal sense strand nucleotide position can be any position on the sense strand except for the three terminal positions from each end. In some embodiments, the sense strand cleavage region is excluded from the internal position. In some embodiments, positions 9-12 or 11-13 are excluded from the internal position, counting from the 5' end of the sense strand. For example, the internal nucleotide position can be one or more of positions 4-8 and 13-18 on the sense strand, counting from the 5' end of the sense strand, for example, one or more of positions 5, 6, 7, 15, and 17 on the sense strand. In one embodiment, the internal nucleotide position can be one of positions 5, 6, 7, or 8 on the sense strand, counting from the 5' end. For example, in each of these embodiments, the internal nucleotide position is position 6 or 7 on the sense strand, counting from the 5' end. For example, in each of these embodiments, the internal nucleotide position is position 6 on the sense strand, counting from the 5' end. For example, in each of these embodiments, the internal nucleotide position is position 7 on the sense strand, counting from the 5' end. In a certain embodiment, the internal nucleotide containing the ligand is given by formula: The formula has TIFF0007882858000020.tif58128, where B is a nucleotide base or nucleotide base analog, and optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0154] In some embodiments, the ligand comprises an inverted nucleotide or an inverted debasalized nucleotide. For example, the ligand comprises a debasalized nucleotide ligated to the strand of the dsRNA molecule via a 5'→5' or 3'→3' ligation site. In some embodiments, the ligand comprises a debasalized nucleotide ligated to the 3' end of the sense strand via a 3'→3' ligation site.

[0155] In another embodiment, the ligand comprises a lipophilic group including a steroid condensed ring system. For example, the ligand may include cholesterol or corticosterone. Examples of such ligands include, for example, This includes TIFF0007882858000021.tif27170. For example, such ligands can be attached to the 5' and / or 3' ends of the dsRNA molecule strand. In some embodiments, the ligand can be attached to the 5' or 3' end of the sense strand. In some embodiments, the ligand can be attached to the 5' end of the sense strand. In some embodiments, the ligand can be attached to the 3' end of the sense strand. Attachment to the dsRNA molecule can be done by bonding to the oxygen atom illustrated above, which has an empty valence, or by bonding formed using the 4-hydroxyl group of pyrrolidine.

[0156] The ligand may be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one “backbone attachment point,” preferably two “backbone attachment points,” and (ii) at least one “tethering attachment point.” As used herein, “backbone attachment point” refers to a functional group, such as a hydroxyl group, or more broadly, a bond, which can be used and is suitable for incorporating the carrier into a ribonucleic acid backbone, such as a phosphate backbone, or a modified phosphate, such as a sulfur-containing backbone. “Tethering attachment point” (TAP) refers, in some embodiments, to a ring atom of a cyclic carrier, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment points), which connects a selected portion. The portion may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected portion is connected to the cyclic carrier by an intervening tether. Therefore, cyclic supports often contain, or more broadly, provide, functional groups suitable for the incorporation or attachment of other chemical entities, such as ligands, to the constituent ring, such as amino groups.

[0157] In one embodiment, the dsRNA molecule of the present invention is conjugated to a ligand via a carrier, the carrier can be a cyclic or acyclic group, preferably the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl and decalin, and preferably the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0158] Ligands can be attached to the sense strand, antisense strand, or both strands at the 3' end, 5' end, or both ends. For example, a ligand can be conjugated to the sense strand, particularly at the 3' end of the sense strand.

[0159] A ligand can be conjugated to a sense strand or antisense strand via a linker containing a cleavable linker. A cleavable linker is a linker that is sufficiently stable extracellularly but is cleaved upon entering a target cell, releasing the two parts linked together by the linker. In a preferred embodiment of the dsRNA molecule of the present invention, the cleavable linker is cleaved at least 10 times, preferably at least 100 times faster, in the target cell or under a first reference condition (which can be selected to mimic or express intracellular conditions, for example) than in the target blood or under a second reference condition (which can be selected to mimic or express conditions found in blood or serum, for example).

[0160] Cleavable linkers are sensitive to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are found more prevalent, at higher levels, or with greater activity inside cells than in serum or blood. Examples of such degradable agents include redox agents selected for specific substrates or those without substrate specificity, such as oxidases or reductases, or reducing agents present in cells, such as mercaptans, that can degrade redox cleavable linkers by reduction; esterases; agents that can create endosomes or acidic environments, such as those resulting in a pH of 5 or less; and enzymes, peptidases (which can be substrate-specific) and phosphatases, that can hydrolyze or degrade acid cleavable linkers by acting as general acids.

[0161] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes are even more acidic, with a pH range of 5.5 to 6.0, and lysosomes are even more acidic, around 5.0. Some linkers may have cleavable linking groups that, upon cleavage at a favorable pH, release cationic lipids from their ligands into the cell or into a desired compartment of the cell.

[0162] Linkers can contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, liver-targeting ligands can be linked to cationic lipids via linkers containing ester groups. Since liver cells are rich in esterases, linkers will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0163] Linkers containing peptide bonds can be used when targeting peptidase-rich cell types, such as liver cells and synovial cells.

[0164] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linker. It would also be desirable to test the candidate cleavable linker for its resistance to cleavage in blood or in contact with other non-target tissues. Thus, the relative sensitivity to cleavage can be determined between a first condition selected to demonstrate cleavage in target cells and a second condition selected to demonstrate cleavage in other tissues or biological fluids, such as blood or serum. Evaluations can be performed in cell-free systems, in cells, in cell cultures, in organs or tissues, or in whole animals. It would be useful to perform the initial evaluation under cell-free or cell culture conditions and confirm it with further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at least 2, 4, 10, or 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0165] One class of cleavable linkers is the redox cleavable linker, which can be used in the dsRNA molecule of the present invention that is cleaved upon reduction or oxidation. An example of a reductively cleavable linker is the disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable “reductively cleavable linker” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent, one can rely on the methods described herein. 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 that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved by at most 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2-fold, 4-fold, 10-fold, or 100-fold faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using a standard enzyme kinetic assay under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0166] The phosphate-based cleavable linking group usable in the dsRNA molecule of the present invention is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate-based linking groups 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-, -OP(S)(Rk)-S-, where Rk can be hydrogen, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl, depending on the occurrence. 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-, and -OP(S)(H)-S-. One preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using a method similar to the one described above.

[0167] The acid-cleavable linking groups that can be used in the dsRNA molecule of the present invention are linking groups that are cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. In cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable linking groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the oxygen-attached carbon (alkoxy group) of the ester 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.

[0168] The ester-based cleavable linking groups that can be used in the dsRNA molecule of the present invention are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkylylene groups. Ester-based 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.

[0169] The peptide-based cleavable linkers usable in the dsRNA molecule of the present invention are cleaved in cells by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linkers do not contain amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. Peptide-based cleavable linkers are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and do not include the entire amide functional group. The general formula for peptide-based cleavable linkers is -NHCHR A C(O)NHCHR B It contains C(O)-, in the formula, R A and R B These are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above. As used herein, "carbohydrate" means a carbohydrate itself, which is composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) and an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound which has as part a carbohydrate portion composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) and an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical hydrocarbons include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars with 5 or more C5 (preferably C5 to C8), and disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (preferably C5 to C8).

[0170] In some embodiments, the dsRNA molecule of the present invention includes one or more overhang regions and / or capping groups at the 3' or 5' end or both ends of the strand. The overhang can be 1 to 10 nucleotides long. For example, the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In some embodiments, the overhang is 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides long. The overhang can be the result of one strand being longer than the other, or the result of two strands of equal length being offset from each other. The overhang can form a mismatch with the target sequence, be complementary to the target gene sequence, or be another sequence. The first and second strands can also be joined, for example, by an additional base to form a hairpin, or by another non-base linker.

[0171] In some embodiments, the nucleotides in the overhang region of the dsRNA molecule of the present invention can each be independently modified or unmodified nucleotides, for example, but not limited to, 2'-sugar modifications such as 2'-fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof. For example, dTdT can be an overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, be complementary to the target gene sequence, or be another sequence.

[0172] The present invention allows for phosphorylation of the sense strand, antisense strand, or the 5'-overhang or 3'-overhang of both strands of the dsRNA molecule. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between them, and these two nucleotides may be the same or different. In some embodiments, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In some embodiments, this 3'-overhang is located on the antisense strand. In some embodiments, this 3'-overhang is located on the sense strand.

[0173] The dsRNA molecule of the present invention may contain only one overhang, which can enhance its interference activity without affecting the overall stability of the dsRNA. For example, the single-stranded overhang may be located at the 3' end of the sense strand or at the 3' end of the antisense strand. The dsRNA may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa.

[0174] Generally, the antisense strand of a dsRNA has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Without being bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are advantageous for the guide strand loading process into RISC. For example, a single overhang is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In some embodiments, the dsRNA has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0175] The dsRNA of the present invention may contain one or more modified nucleotides. For example, all nucleotides in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modifications, and these modifications may include one or more alterations of one or both of the unbound phosphate oxygens and / or one or more of the bound phosphate oxygens; alteration of components of the ribose sugar; replacement of the ribose sugar; complete replacement of the phosphate moiety with a "dephospho" linker; modification or substitution of native bases; and substitution or modification of the ribose-phosphate backbone.

[0176] Since nucleic acids are polymers of subunits, most modifications occur at repeating positions within the nucleic acid, such as modifications to bases, phosphate moieties, or unbound oxygen atoms in phosphate moieties. In some cases, modifications may be present at all target positions in the nucleic acid, but this is not the case in most cases. For example, modifications may be present only at the 3' or 5' end, or only in the central region, or only in the non-terminal region, or within the terminal region, for example, at a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications can be present in double-stranded regions, single-stranded regions, or both. Modifications may be present only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, unbound O-position phosphorothioate modifications may be present at one or both ends, or within the terminal region, for example, at a position on the terminal nucleotide, or within the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in the double-stranded and single-stranded regions, especially at the ends. One or more 5' ends may be phosphorylated.

[0177] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, such as the 5' overhang or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or part of the bases in the 3' overhang or 5' overhang may be modified, for example, by modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar by modifications known in the art, such as the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modifiers instead of the ribose sugar of the nucleic acid base, and modifications in the phosphate group, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0178] In some embodiments, the dsRNA molecule of the present invention includes alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. As used herein, the terms “alternating motif” or “alternating pattern” refer to a motif having one or more modifications, where each modification lies on an alternating nucleotide of a single strand. Alternating nucleotides can refer to patterns such as alternating nucleotides or alternating nucleotides. For example, if A, B, and C each represent a type of modification to a nucleotide, then the alternating motif could be “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AAABBBAAABBB…”, or “ABCABCABCABC…”.

[0179] The types of modifications included in the alternating motif may be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternating pattern, i.e., the alternating nucleotide modifications, may be the same, but each of the sense strand or antisense strand can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0180] In some embodiments, the dsRNA molecule of the present invention includes a modification pattern relating to the alternating motif on the sense strand that is shifted relative to the modification pattern relating to the alternating motif on the antisense strand. The shift may be such that modified groups of nucleotides on the sense strand correspond to differently modified groups of nucleotides on the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, within the double helix region, the alternating motif in the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from 3' to 5' of the strand. As another example, within the double helix region, the alternating motif in the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from 3' to 5' of the strand, so that a complete or partial shift of the modification patterns occurs between the sense and antisense strands.

[0181] 5'-modified In some embodiments, the dsRNA molecule of the present invention may be 5'-phosphorylated or may contain a phosphoryl analog or phosphate mimic at its 5' end. Examples of 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'); and 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 nucleotide cap structure 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'-monoditithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5' Examples include phosphorothiolates ((HO)2(O)PS-5'); any further combinations of oxygen / sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phospholamidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (e.g., RP(OH)(O)-O-5'-, R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl, (OH)2(O)P-5'-CH2-), cyclopropylphosphonates, and 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). For example, modifications can be placed on the antisense strand of a dsRNA molecule.

[0182] In some aspects of any one of the above aspects, the antisense chain contains a phosphoryl analog or phosphate mimic at its 5' end. In some aspects, the antisense chain contains an alkenyl phosphonate, i.e., a vinyl phosphonate, at its 5' end. For example, the antisense chain contains a 5'-E-vinyl phosphonate. In an exemplary embodiment, the 5' vinyl phosphonate-modified nucleotide at the 5' end may have the following structure: In formula TIFF0007882858000022.tif35128, X is O or S, and R is hydrogen, hydroxyl, fluoro, or C 1~20 It is an alkoxy (e.g., methoxy), and R 5' A is = C(H)-P(O)(OH)2, the double bond between the C5' carbon and R5' is E-oriented or Z-oriented (e.g., E-oriented), B is a nucleic acid base or modified nucleic acid base, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0183] In some embodiments, the antisense chain contains a cyclopropylphosphonate at its 5' end. For example, the antisense chain has a cyclopropylphosphonate at its 5' end. Includes TIFF0007882858000023.tif20128 (which, for example, can replace the 4' group in the preceding structure.)

[0184] The dsRNA agent of the present invention may include thermal destabilization modifications in the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. Without being bound by theory, a dsRNA having an antisense strand containing at least one double-strand thermal destabilization modification within the first nine nucleotide positions counting from the 5' end of the antisense strand has reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five or more) double-strand thermal destabilization modifications within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, the double-strand thermal destabilization modifications are located at positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modifications are located at positions 5, 6, 7, or 8, from the 5' end of the antisense strand.

[0185] In some further embodiments, the thermal destabilization modification of the double chain is located at position 7, from the 5' end of the antisense chain.

[0186] The term "thermally destabilizing modification" encompasses modifications that result in a lower overall melting temperature (Tm) of dsRNA (preferably 1, 2, 3, or 4 degrees lower than the Tm of dsRNA without such modification). In some embodiments, the double-stranded thermally destabilizing modification is located at positions 2, 3, 4, 5, 6, 7, 8, or 9 from the 5' end of the antisense strand.

[0187] Thermal destabilization modifications may include, but are not limited to, debasing modifications; mismatches with opposing nucleotides in opposing strands; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, e.g., unlocked nucleic acids (UNAs) or glycolic nucleic acids (GNAs), or 5'-2' linked nucleotides (e.g., those having 3'-OMe, 3'-F, 3'-H, or 3'-OH, as defined herein, "3'-RNA"). Examples of thermal destabilization modifications include, but are not limited to, the following mUNA and GNA building blocks: TIFF0007882858000024.tif244166TIFF0007882858000025.tif175165.

[0188] In some embodiments, the destabilization modification is a 5'-2' linkage nucleotide (e.g., having 3'-OMe, 3'-F, 3'-H, or 3'-OH). In some embodiments, the destabilization modification is a 5'-2' linkage nucleotide having 3'-OMe, 3'-F, 3'-H, or 3'-OH. In some embodiments, the destabilization modification is a 5'-2' linkage nucleotide having 3'-OMe. In some embodiments, the destabilization modification is a 5'-2' linkage nucleotide having 3'-F. In some embodiments, the destabilization modification is a 5'-2' linkage nucleotide having 3'-H. In some embodiments, the destabilization modification is a 5'-2' linkage nucleotide having 3'-OH, for example, the following formula: In formula TIFF0007882858000026.tif30128, B is a nucleotide base or nucleotide base analogue, and optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0189] In some embodiments, the destabilization modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA.

[0190] In some embodiments, the destabilization-modified mUNA is TIFF0007882858000027.tif87152R=H,OH;OMe;Cl,F;OH;O-(CH2)2OMe;SMe,NMe2;NH2;Me;CCH(alkyne),O-nPr;O-alkyl;O-alkylamino; R'=H, Me; B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles;psudouracil;isoC;isoG;2,6-diaminopurine;pseudocytosine;2-aminopurine;xanthosine;N6-alkyl-A;O6-alkyl-G;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;7-deazapurine, phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles Selected from the group consisting of the following, the stereochemistry is R or S, or a combination of R and S, for chiral centers that are not specified.

[0191] In some embodiments, the destabilization-modified mUNA is TIFF0007882858000028.tif56158R=H,OH;OMe;Cl,F;OH;O-(CH2)2OMe;SMe,NMe2;NH2;Me;CCH(alkyne),O-nPr;O-alkyl;O-alkylamino; R'=H, Me; B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles;psudouracil;isoC;isoG;2,6-diaminopurine;pseudocytosine;2-aminopurine;xanthosine;N6-alkyl-A;O6-alkyl-G;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;7-deazapurine, phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles Selected from the group consisting of the following, the stereochemistry is R or S, or a combination of R and S, for chiral centers that are not specified.

[0192] In some embodiments, the destabilization-modified mUNA is TIFF0007882858000029.tif85156R=H, OMe;F;OH;O-(CH2)2OMe;SMe,NMe2;NH2;Me;O-nPr;O-alkyl;O-alkylamino; R'=H, Me; B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles;pseudracil;isoC;isoG;2,6-diaminopurine;pseudocytosine;2-aminopurine;xanthosine;N6-alkyl-A;O6-alkyl-G;7-deazapurine Selected from the group consisting of the following, the stereochemistry is R or S, or a combination of R and S, for chiral centers that are not specified.

[0193] In some embodiments, the destabilization-modified mUNA is TIFF0007882858000030.tif86151R=H,OH;OMe;Cl,F;OH;O-(CH2)2OMe;SMe,NMe2;NH2;Me;CCH(alkyne),O-nPr;O-alkyl;O-alkylamino; R'=H, Me; B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles;psudouracil;isoC;isoG;2,6-diaminopurine;pseudocytosine;2-aminopurine;xanthosine;N6-alkyl-A;O6-alkyl-G;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;7-deazapurine, phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles Selected from the group consisting of, The stereochemistry of chiral centers that are not specified is R or S, or a combination of R and S.

[0194] In some embodiments, the destabilization-modified mUNA is TIFF0007882858000031.tif51147R=H,OH;OMe;Cl,F;OH;O-(CH2)2OMe;SMe,NMe2;NH2;Me;CCH(alkyne),O-nPr;O-alkyl;O-alkylamino; R'=H, Me; B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles;psudouracil;isoC;isoG;2,6-diaminopurine;pseudocytosine;2-aminopurine;xanthosine;N6-alkyl-A;O6-alkyl-G;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;7-deazapurine, phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles Selected from the group consisting of the following, the stereochemistry is R or S, or a combination of R and S, for chiral centers that are not specified.

[0195] In some embodiments, the modified mUNA is TIFF0007882858000032.tif84156R=H, OMe;F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;O-nPr;O-alkyl;O-alkylamino; R'=H, Me; B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G-clamp;non-standard monocyclic, bicyclic and tricyclic heterocycles;pseudracil;isoC;isoG;2,6-diaminopurine;pseudocytosine;2-aminopurine;xanthosine;N6-alkyl-A;O6-alkyl-G;7-deazapurine Selected from the group consisting of the following, the stereochemistry is R or S, or a combination of R and S, for chiral centers that are not specified.

[0196] Examples of debasement modifications include, but are not limited to, the following: In formula TIFF0007882858000033.tif60128, R = H, Me, Et or OMe, R' = H, Me, Et or OMe, R” = H, Me, Et or OMe In formula TIFF0007882858000034.tif45135, B is a modified or unmodified nucleic acid base, and the asterisk in each structure represents either R, S, or racemic.

[0197] Examples of sugar modifications include, but are not limited to, the following: In formula TIFF0007882858000035.tif84132, B is a modified or unmodified nucleic acid base, and the asterisk in each structure represents either R, S, or racemic.

[0198] In some embodiments, the double-strand thermal destabilization modification is selected from the mUNA and GNA building blocks described in Examples 1-3 of this specification. In some embodiments, the destabilization modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments thereof, the dsRNA molecule further comprises at least one thermal destabilization modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (Mod A-Mod K).

[0199] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, and / or at least one of the ribose carbons or ribose oxygens (e.g., C1', C2', C3', C4', or O4') is deleted from the nucleotide independently or in combination. In some embodiments, acyclic nucleotides are, The formula is TIFF0007882858000036.tif35148, where B is a modified or unmodified nucleic acid base, R1 and R2 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 bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) is removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985) and Fluiter et al., Mol. Biosyst., 10:1039 (2009). These references are incorporated herein by reference in their entirety). Acyclic derivatives increase the flexibility of the backbone without affecting Watson-Crick pairings. Acyclic nucleotides can be linked by 2'-5' or 3'-5' ligatures.

[0200] The term "GNA" refers to glycol nucleic acids, polymers similar to DNA or RNA, but differing in that their "backbone" is composed of repeating glycerol units linked by phosphodiester bonds. This refers to TIFF0007882858000037.tif57128.

[0201] Double-strand thermal destabilization modifications can be mismatches (i.e., non-complementary base pairs) between thermally destabilized nucleotides in the dsRNA double-strand and their corresponding nucleotides in the reverse 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 can also be applied to the present invention. Mismatches can occur between nucleotides that are either native or modified nucleotides. That is, mismatch base pairings can occur between the nucleic acid bases of each nucleotide, independently of modifications on the ribose sugar of the nucleotides. In certain embodiments, a dsRNA molecule contains at least one nucleic acid base that is a 2'-deoxynucleotide in the mismatch pairing, for example, the 2'-deoxynucleotide being in the sense strand.

[0202] In some embodiments, thermal destabilization modification of the double helix in the seed region of the antisense strand results in a nucleotide in which the WHC bond to the complementary base on the target mRNA is impaired, for example, Includes TIFF0007882858000038.tif72135.

[0203] Further examples of debasic nucleotide modifications, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0204] Thermal destabilization modifications may include universal bases in which the ability to form hydrogen bonds with the opposing base is reduced or lost, as well as phosphate modifications.

[0205] In some embodiments, double-strand thermal destabilization modifications include nucleic acid base modifications in which nucleotides have non-standard bases, for example, but whose ability to form hydrogen bonds with bases in the reverse strand is impaired or completely lost. These nucleic acid base modifications have been evaluated for destabilization of the central region of dsRNA double helices, as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleic acid base modifications are: The filename is TIFF0007882858000039.tif63157.

[0206] In some embodiments, double-strand thermal destabilization modification in the seed region of the antisense strand involves the addition of one or more α-nucleotides complementary to the base on the target mRNA, for example, The formula includes TIFF0007882858000040.tif20146, where R is H, OH, OCH3, F, NH2, NHMe, NMe2, or O-alkyl.

[0207] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA double helix compared to natural phosphodiester ligatures are: The filename is TIFF0007882858000041.tif30139.

[0208] The alkyl group used as the R group can be C1-C6 alkyl. Specific examples of alkyl groups used as the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0209] It should be noted that thermal destabilization modifications can replace 2'-deoxynucleotides in the antisense strand. For example, 2'-deoxynucleotides at positions 2, 5, 7, 12, 14 and / or 16, counting from the 5' end of the antisense strand, can be replaced with the thermal destabilization modifications described herein. Thus, in some embodiments, the antisense strand contains thermal destabilization modifications at 1, 2, 3, 4, 5 and / or 6 of positions 2, 5, 7, 12, 14 and / or 16, counting from the 5' end of the antisense strand. For example, the antisense strand contains thermal destabilization modifications at positions 5 and 7, counting from the 5' end of the antisense strand.

[0210] In addition to the antisense strand containing thermal destabilization modifications, the dsRNA may also contain one or more stabilization modifications. For example, the dsRNA may contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilization modifications. However, all stabilization modifications can be present on a single strand. In some embodiments, both the sense strand and the antisense strand contain at least two stabilization modifications. Stabilization modifications can be present on any nucleotide of the sense strand or the antisense strand. For example, a stabilization modification may be present on all nucleotides on the sense strand and / or the antisense strand, or each stabilization modification may be present in an alternating pattern on the sense strand or the antisense strand, or the sense strand or antisense strand may contain both stabilization modifications in an alternating pattern. The alternating pattern of stabilization modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of stabilization modifications on the sense strand may be shifted relative to the alternating pattern of stabilization modifications on the antisense strand.

[0211] In some embodiments, the antisense chain includes at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Stabilizing modifications in the antisense chain can be located at any position, but are not limited to these. In some embodiments, the antisense includes stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In some yet another embodiment, the antisense includes stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0212] In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification may be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., a nucleotide at a position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand includes stabilizing modifications at both the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 from the position of the destabilizing modification.

[0213] In some embodiments, the antisense chain includes at least two stabilizing modifications at the 3' end of the destabilizing modifications, i.e., at least two stabilizing modifications at positions +1 and +2 from the position of the destabilizing modifications. In some embodiments, the sense chain includes at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Stabilizing modifications in the sense chain can be located at any position, but are not limited to these. In some embodiments, the sense chain includes stabilizing modifications at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense chain includes stabilizing modifications at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense chain includes stabilizing modifications at positions relative to or complementary to positions 11, 12 and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some other embodiments, the sense chain includes stabilizing modifications at positions relative to or complementary to positions 11, 12, 13 and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some embodiments, the sense chain includes a block of two, three, or four stabilization modifications.

[0214] In some embodiments, the sense chain does not contain stabilization modifications in positions relative to or complementary to the thermal destabilization modifications of the double chain in the antisense chain.

[0215] Exemplary thermal stabilization modifications include, but are not limited to, 2'-fluoro modifications. Other thermal stabilization modifications include, but are not limited to, LNA.

[0216] Note that thermal stabilization modifications can replace 2'-fluoronucleotides in the sense and / or antisense strands. For example, the 2'-fluoronucleotides at positions 8, 9, 10, 11 and / or 12 counting from the 5' end of the sense strand can be replaced by thermal stabilization modifications. Similarly, the 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand can be replaced by thermal stabilization modifications.

[0217] For a dsRNA molecule to be more effective in vivo, the antisense strand must be metabolically stable to some extent. In other words, for a dsRNA molecule to be more effective in vivo, a certain amount of the antisense strand may need to be present in vivo after a certain period following administration. Therefore, in some embodiments, on day 5 after in vivo administration, at least 40% of the dsRNA antisense strand, 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%, is present in vivo, for example, in mouse liver. In some embodiments, on day 6 after in vivo administration, at least 40% of the dsRNA antisense strand, 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%, is present in vivo, for example, in mouse liver. In some embodiments, on day 7 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in mouse liver. In some embodiments, on day 8 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in mouse liver. In some embodiments, on day 9 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in mouse liver. In some embodiments, on day 10 after in vivo administration, 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, for example, in the liver of a mouse.In some embodiments, on day 11 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in mouse liver. In some embodiments, on day 12 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in mouse liver. In some embodiments, on day 13 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in mouse liver. In some embodiments, on day 14 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in the liver of a mouse. In some embodiments, on day 15 after in vivo administration, at least 40% of the antisense strand of the dsRNA, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, is present in vivo, for example, in the liver of a mouse.

[0218] Use of dsRNA The present invention further relates to the use of dsRNA molecules as defined herein for inhibiting the expression of a target gene. In some embodiments, the present invention further relates to the use of dsRNA molecules for inhibiting the expression of a target gene in vitro.

[0219] The present invention further relates to dsRNA molecules, as defined herein, for use in inhibiting the expression of target genes in a subject. The subject may be any animal, such as a mammal, such as a mouse, rat, sheep, cattle, dog, cat, or human.

[0220] In some embodiments, the dsRNA molecule of the present invention is administered in a buffer.

[0221] In some embodiments, the siRNA compounds described herein may be formulated for administration to a subject. Formulated siRNA compositions can exist in a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in the aqueous phase, for example, in a solution containing water.

[0222] The aqueous or crystalline composition can be incorporated into a delivery medium, such as liposomes (particularly for the aqueous phase) or particles (e.g., fine particles that may be suitable for the crystalline composition). Generally, siRNA compositions are formulated in a manner suitable for the intended method of administration, as described herein. For example, in certain embodiments, the composition is prepared by at least one of the following methods: spray drying, freeze-drying, vacuum drying, evaporation, fluidized bed drying, or a combination of these techniques; or sonication, freeze-drying, condensation, and other self-assembly with lipids.

[0223] dsRNA preparations can be formulated in combination with other agents, such as other therapeutic agents, or with agents that stabilize dsRNA, such as proteins that complex with dsRNA to form iRNPs. Further other agents include chelating agents, such as EDTA (e.g., Mg 2+ Examples of solutions include salts and RNAse inhibitors (such as broad-specific RNAse inhibitors like RNAsin) used to remove divalent cations.

[0224] In some embodiments, the dsRNA preparation comprises another dsRNA compound, e.g., a second dsRNA capable of mediating RNAi with respect to a second gene or with respect to the same gene. Further, another preparation can comprise at least 3, 5, 10, 20, 50 or 100 or more different siRNA species. Such dsRNAs can mediate RNAi with respect to a similar number of different genes.

[0225] In some embodiments, the dsRNA preparation comprises at least a second therapeutic agent (e.g., an agent other than RNA or DNA). For example, a dsRNA composition for treating a viral disease, e.g., HIV, may comprise a known antiviral agent (e.g., a protease inhibitor or a reverse transcriptase inhibitor). In another example, a dsRNA composition for treating cancer may further comprise a chemotherapeutic agent.

[0226] Exemplary formulations that can be used to administer the dsRNA molecules of the invention are discussed below.

[0227] Liposomes. The dsRNA preparation can be formulated into membrane molecular assemblies, such as liposomes or micelles, for delivery. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one bilayer or multiple bilayers. Liposomes include unilamellar vesicles and multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the siRNA composition, but in some examples may contain the siRNA composition. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to the biological membrane, when liposomes are applied to tissues, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the association between the liposome and the cell progresses, the inner aqueous contents containing dsRNA 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 the dsRNA to a specific cell type.

[0228] Liposomes containing dsRNA can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed from the lipid components. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. Next, the dsRNA preparation is added to the micelles containing the lipid components. The cationic groups on the lipid interact with the siRNA and condense around the dsRNA to form liposomes. After condensation, the detergent is removed, such as by dialysis, to obtain the liposome preparation of dsRNA.

[0229] If necessary, a support compound to aid condensation can be added during the condensation reaction, for example, by controlled addition. For instance, the support compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0230] Further descriptions of methods for producing stable polynucleotide delivery media incorporating polynucleotide / cationic lipid complexes as components of the delivery media are provided, for example, in WO96 / 37194. Liposome formation has been described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, U.S. Patent No. 4,897,355, U.S. Patent 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 This may also include one or more aspects of the exemplary methods described in al. Endocrinol. 115:757, 1984, which are incorporated herein by reference in their entirety. Common techniques for preparing lipid aggregates of a size suitable for use as a delivery medium include sonication and freeze-thaw and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, which is incorporated herein by reference in its entirety). If consistently small (50–200 nm) and relatively uniform aggregates are desired, microfluidic techniques can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, which is incorporated herein by reference in its entirety). These methods can be readily adapted for packaging siRNA preparations into liposomes.

[0231] pH-sensitive or negatively charged liposomes encapsulate nucleic acids rather than form complexes with them. Since both nucleic acid molecules and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acid molecules are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding thymidine kinase genes to a monolayer of cultured cells. Exogenous gene expression was detected in target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274; this document is incorporated herein by reference in its entirety).

[0232] One of the main types of liposome compositions contains phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions can be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic membrane-fused liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Yet another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0233] Other examples of methods for introducing liposomes into cells in vitro include U.S. Patents 5,283,185, 5,171,678, WO94 / 00569, WO93 / 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.

[0234] In some embodiments, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes, although they cannot efficiently fuse with the plasma membrane, can be taken up by macrophages in vivo and used to deliver siRNA to macrophages.

[0235] Further advantages of liposomes include the biocompatibility and biodegradability of liposomes derived from natural phospholipids, their ability to encapsulate a wide range of water-soluble and lipid-soluble drugs, and their ability to protect siRNA encapsulated in their inner compartment from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposomal formulations include lipid surface charge, vesicle size, and liposome water volume.

[0236] 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. These liposomes fuse with negatively charged lipids on the cell membranes of tissue culture cells, thereby enabling the delivery of siRNA (for DOTMA and its use with DNA, see, for example, Felgner, Plet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355, which are incorporated herein by reference in their entirety).

[0237] By using the DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) in combination with phospholipids, DNA-complexed vesicles can be formed. Lipofectin® (Bethesda Research Laboratories, Gaythersburg, Maryland) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells and contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. With sufficient positively charged liposomes, the net charge of the resulting complex is also positive. The positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces and fuse with the plasma membrane to efficiently deliver functional nucleic acids, for example, into 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 portion is linked by an ester rather than an ether linkage.

[0238] Other reported cationic lipid compounds include those conjugated to various parts, such as carboxyspermine conjugated to one of two types of lipids, including compounds such as 5-carboxyspermylglycine dioctaoleylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, for example, U.S. Patent No. 5,171,678).

[0239] Another cationic lipid conjugate includes cholesterol-based lipid derivatization ("DC-Chol") formulated in liposomes in combination with DOPE (see, e.g., Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991; this document is incorporated herein by reference in its entirety). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and result in more efficient transfection 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., Gaythersburg, Maryland). Other cationic lipids suitable for oligonucleotide delivery are described in WO98 / 39359 and WO96 / 37194.

[0240] Liposome formulations are particularly well-suited for topical administration. Liposomes offer several advantages compared to other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver siRNA to the skin. In some embodiments, liposomes are used to deliver siRNA to epidermal cells and to enhance siRNA penetration into dermal tissue, such as the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes has been described in detail (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, R. J. 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. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983, Wang, C. and Huang, L., Proc. Natl. Acad. Sci. USA) See 84:7851–7855, 1987. (These documents are incorporated herein by reference in their entirety.)

[0241] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in drug delivery to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs to the dermis of mouse skin. Such formulations containing the dsRNA described herein are useful for treating skin disorders.

[0242] Liposomes containing dsRNA as described herein can be made highly deformable. Such deformability can allow the liposomes to penetrate through pores smaller than the average radius of the liposome. Transfersomes, for example, are a type of deformable liposome. Transfersomes can be prepared by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing dsRNA as described herein can be delivered to keratinocytes in the skin, for example, by subcutaneous infection. To traverse intact mammalian skin, the lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transcutaneous gradient. In addition, due to the properties of lipids, these transfersomes can be self-optimizing (adaptable to the shape of pores, e.g., skin pores), self-repairing, often reaching their respective targets without fragmentation, and often self-loading.

[0243] Other formulations suitable for the present invention are described in U.S. Provisional Applications No. 61 / 018,616, filed on January 2, 2008; No. 61 / 018,611, filed on January 2, 2008; No. 61 / 039,748, filed on March 26, 2008; No. 61 / 047,087, filed on April 22, 2008; and No. 61 / 051,528, filed on May 8, 2008. A formulation suitable for the present invention is also described in PCT Application No. PCT / US2007 / 080331, filed on October 3, 2007.

[0244] Surfactants. dsRNA compositions may contain surfactants. In some embodiments, dsRNA is formulated as an emulsion containing a surfactant. The most common method for classifying and ranking the properties of numerous different types of surfactants, both natural and synthetic, is the method using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group are the most useful means of categorizing the various surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms," ​​Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0245] If a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical products and can be used in a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18, depending on their respective structures. 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 alkanol ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, also belong to this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0246] If a surfactant molecule has a negative charge when dissolved or dispersed in water, it is classified as anionic. Anionic surfactants include carboxylates such as soap, acyl lactylates, acylamides of amino acids, sulfuric acid esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0247] If the surfactant molecules have 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.

[0248] If the surfactant molecules can have either a positive or a negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0249] There is a review on the use of surfactants in pharmaceutical products, formulations, and emulsions (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0250] Micelles and other membrane formulations. For simplicity of explanation, the micelles and other formulations, compositions, and methods in this section will generally discuss unmodified siRNA compounds. However, these micelles and other formulations, compositions, and methods can also be implemented with other siRNA compounds, such as modified siRNA compounds, and such implementation can be understood to be within the scope of the present invention. An siRNA compound, such as a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or an siRNA compound, such as a double-stranded siRNA compound, or an ssiRNA compound, or DNA encoding the siRNA compound or its precursor), can be provided as a micelle formulation. As used herein, a "micelle" is defined as a particular type of molecular aggregate in which amphiphilic molecules are arranged in a spherical structure such that all of the hydrophobic portions of the amphiphilic molecules face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. If the environment is hydrophobic, the reverse arrangement exists.

[0251] Mixed micelle formulations suitable for delivery via transdermal membranes include aqueous solutions of dsRNA compositions and alkali metals C8-C8. 22 These can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocolanyglycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogues, polydocanol alkyl ethers and their analogues, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles are formed from a mixture of virtually any type of component, but vigorous mixing is necessary to obtain smaller micelles.

[0252] In one method, a first micelle composition containing a dsRNA composition and at least an alkali metal alkyl sulfate is prepared. 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 dsRNA composition, an alkali metal alkyl sulfate, and at least one of the micelle-forming compounds, and then the remaining micelle-forming compounds are added and vigorously stirred.

[0253] To stabilize the formulation and protect it from bacterial growth, phenol and / or m-cresol may be added to the mixed micelle composition. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the formation of the mixed micelle composition.

[0254] To deliver micelle formulations as a spray, the formulation can be placed in an aerosol dispenser, which is then filled with a propellant. Under pressurization, the propellant is liquid in the dispenser. The ratio of components is adjusted so that there is only one phase, i.e., one phase. If two phases are present, the dispenser needs to be shaken before dispensing a portion of the contents, for example, through a metering valve. The amount of pharmaceutical preparation dispensed is sprayed as a fine mist from the metering valve.

[0255] The propellant may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA134a (1,1,1,2-tetrafluoroethane) may be used.

[0256] The specific concentrations of essential components can be determined by relatively simple experiments. For oral absorption, it is often desirable to increase the dosage by at least two or three times the amount administered by injection or via the gastrointestinal tract.

[0257] Particles. In some embodiments, dsRNA preparations can be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but may also be produced by other methods such as freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination thereof.

[0258] Pharmaceutical composition The dsRNA agents of the present invention can be formulated for pharmaceutically active use. The present invention further relates to pharmaceutically active compositions comprising dsRNA molecules as defined herein. A pharmaceutically acceptable composition comprises one or more dsRNA molecules of any of the above embodiments in a therapeutically effective amount, administered alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipients and / or diluents.

[0259] Pharmaceutical compositions may be specially formulated for administration as solids or liquids, including those suitable for the following administration methods: (1) oral administration, e.g., oral tablets (aqueous or nonaqueous solutions or suspensions), tablets, e.g., those intended for oral mucosal, sublingual, and systemic absorption, boluses, powders, granules, and pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection as sterile solutions or sterile suspensions or sustained-release formulations; (3) topical application, e.g., creams, ointments, or controlled-release patches, or sprays applied to the skin; (4) intravaginal or rectal administration, e.g., as pessaries, creams, or foams; (5) sublingual administration; (6) ocular administration; (7) transdermal administration; or (8) nasal administration. Subcutaneous or intravenous delivery is particularly advantageous.

[0260] As used herein, the term "therapeutic dose" means an amount of a compound, material, or composition containing the compound of the present invention that is effective in producing any desired therapeutic effect in at least a subpopulation of cells in an animal, with a reasonable benefit-to-risk ratio applicable to any medical treatment.

[0261] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within reasonable medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit-risk ratio.

[0262] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or steric acid), or solvent encapsulation material, necessary for transporting or delivering the compound from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers are listed below: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate. (1) state, sodium lauryl sulfate and talc, (8) excipients, e.g., cocoa butter and suppository wax, (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) glycols, e.g., propylene glycol, (11) polyols, e.g., glycerin, sorbitol, mannitol and polyethylene glycol, (12) esters, e.g., ethyl oleate and ethyl laurate, (13) agar, (14) buffering agent Examples include (15) magnesium hydroxide and aluminum hydroxide, (16) alginic acid, (17) pyrogen-free water, (18) isotonic saline, (19) Ringer's solution, (10) ethyl alcohol, (21) pH buffer solution, (22) polyester, polycarbonate and / or polyanhydride, (23) fillers, such as polypeptides and amino acids, (24) serum components, such as serum albumin, HDL and LDL, and (25) other non-toxic compatible substances used in pharmaceutical formulations.

[0263] The formulations can be conveniently presented in unit dosage forms and can be prepared by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will be the amount of the compound that produces the therapeutic effect. Generally, this amount will range from about 0.1 percent to about 99 percent of 100 percent, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent, with the active ingredient being the most common.

[0264] In certain embodiments, the formulation of the present invention comprises a compound of the present invention and an excipient selected from the group consisting of cyclodextrin, cellulose, liposomes, micellar-forming agents such as bile acids, and polymer carriers such as polyesters and polyanhydrides. In certain embodiments, the aforementioned formulation makes the compound of the present invention orally bioavailable.

[0265] dsRNA preparations can be formulated in combination with other agents, such as other therapeutic agents, or with agents that stabilize dsRNA, such as proteins that complex with dsRNA to form iRNPs. Further other agents include chelating agents, such as EDTA (e.g., Mg 2+ Examples of solutions include salts and RNAse inhibitors (such as broad-specific RNAse inhibitors like RNAsin) used to remove divalent cations.

[0266] Methods for preparing these formulations or compositions include the step of mixing the compound of the present invention with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by uniformly and closely mixing the compound of the present invention with a liquid carrier, a pulverized solid carrier, or both, and then molding the product if necessary.

[0267] In some cases, it is desirable to slow down the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effects. This can be achieved by using a suspension of a crystalline or amorphous material with poor water solubility. In this case, the rate of drug absorption depends on its dissolution rate, which may depend on the crystal size and crystal form. Alternatively, delayed absorption of parenterally administered dosage forms can be achieved by dissolving or suspending the drug in an oily medium.

[0268] The compounds of the present invention can be formulated, by analogy with other pharmaceuticals, for administration in any convenient manner for use in medicine or veterinary medicine.

[0269] The term “treatment” shall encompass both therapy and cure. Patients receiving this treatment are any animals in need, such as primates, especially humans, and other mammals, such as horses, cattle, pigs, and sheep, as well as poultry and pets in general.

[0270] Double-stranded RNA agents are produced in vivo in cells, for example, from an exogenous DNA template delivered into the cell. For example, the DNA template can be inserted into a vector and used as a gene therapy vector. The gene therapy vector can be delivered to the target, for example, by intravenous injection, local administration (U.S. Patent No. 5,328,470, which is incorporated herein by reference in its entirety), or by stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057, which is incorporated herein by reference in its entirety). Pharmaceutical preparations of gene therapy vectors may include the gene therapy vector in an acceptable diluent or include a delayed-release matrix in which the gene delivery medium is embedded. The DNA template may include, for example, two transcription units: one transcription unit producing a transcript containing the top strand of a dsRNA molecule, and another transcription unit producing a transcript containing the bottom strand of a dsRNA molecule. When these templates are transcribed, dsRNA molecules are generated and processed into siRNA agent fragments that mediate gene silencing.

[0271] The dsRNA molecules as defined herein, or pharmaceutical compositions comprising the dsRNA molecules as defined herein, can be administered to a subject using a variety of delivery routes. The dsRNA-containing compositions described herein can reach a subject by a variety of routes. Exemplary routes include intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, lung, and ocular delivery.

[0272] The dsRNA molecules of the present invention can be incorporated into a pharmaceutically acceptable composition suitable for administration. Such a composition typically comprises one or more dsRNAs and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” encompasses all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc., that are compatible with pharmaceutically acceptable administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional medium or agent can be considered for use in the present composition, provided that it is not incompatible with the active compound. The present composition may also incorporate auxiliary active compounds.

[0273] The compositions of the present invention can be administered in several ways, depending on whether a topical or systemic treatment is desired, and depending on the area to be treated. Administration may be topical (including ocular, vaginal, rectal, nasal, and percutaneous), oral, or parenteral. Parenteral administration may include intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intravenous administration.

[0274] The route and site of administration can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be 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.

[0275] In one aspect, the present invention is characterized by a method of administering a dsRNA molecule, for example, a dsRNA agent as described herein, to a subject (e.g., a human subject). In another aspect, the present invention relates to a dsRNA molecule as defined herein for use in inhibiting the expression of a target gene in a subject. The method or medical use involves administering a unit dose of a dsRNA molecule, for example, a dsRNA agent as described herein. In some embodiments, the unit dose is less than 10 mg per kg of 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 per kg of body weight, and 200 nmoles of RNA agent per kg of body weight (e.g., about 4.4 × 10⁻¹⁶ mg). 16 The amount of RNA is less than (copy) 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, or 0.00015 nmole per kg of body weight.

[0276] The prescribed dose may be an effective amount to treat or prevent a disease or disorder, such as a disease or disorder associated with a target gene. The unit dose may be administered, for example, by injection (e.g., intravenously, subcutaneously, or intramuscularly), by inhalation, or topically. In some embodiments, the dosage may be less than 10, 5, 2, 1, or 0.1 mg / kg body weight.

[0277] In some embodiments, the unit dose is administered less than once a day, for example, less than once every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a certain frequency (for example, regularly). For example, the unit dose may be administered only once.

[0278] In some embodiments, an effective dose is administered in conjunction with other conventional therapeutic modalities. In some embodiments, the subject has a viral infection, and the modality is an antiviral agent other than a dsRNA molecule, such as an siRNA agent. In another embodiment, the subject has atherosclerosis, and an effective dose of a dsRNA molecule, such as an siRNA agent, is administered afterward, for example, in combination with surgical intervention, such as angioplasty.

[0279] In some embodiments, the subject receives an initial dose and one or more maintenance doses of a dsRNA molecule, such as an siRNA agent (e.g., a precursor, a larger dsRNA molecule that can be processed into an siRNA agent, or DNA encoding a dsRNA molecule, such as an siRNA agent, or its precursor). The one or more maintenance doses may be the same as the initial dose or less, for example, half the initial dose. The maintenance regimen may include treating the subject once or multiple times with a dose in the range of 0.01 μg to 15 mg / kg body weight per day, for example, 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of body weight per day. The maintenance dose is administered at a frequency of no more than once every 2, 5, 10, or 30 days, for example. Furthermore, the treatment regimen is continued for a period of time, which will vary depending on the nature of the specific disease, its severity, and the patient's overall condition. In certain embodiments, the dosage may be delivered at a frequency of no more than once a day, for example, once every 24, 36, or 48 hours or more, for example, once every 5 or 8 days. After treatment, the patient can be monitored for changes in their condition and improvement in the symptoms of the disease state. If the patient does not respond significantly to the current dosage level, the dosage of the compound may be increased, or if improvement in the symptoms of the disease state is observed, the disease state disappears, or undesirable side effects are observed, the dosage may be decreased.

[0280] The effective dose may be administered as a single dose or in two or more doses, depending on the desired outcome or the specific circumstances. If repeated or frequent infusions are desired, implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisional, or intrasacral), or a reservoir may be advisable.

[0281] In some embodiments, the composition comprises multiple dsRNA molecular species. In another embodiment, the dsRNA molecular species have sequences that do not overlap or adjaculate with respect to the native target sequence of another species. In yet another embodiment, the multiple dsRNA molecular species are specific to different native target genes. In yet another embodiment, the dsRNA molecules are allele-specific.

[0282] The dsRNA molecules of the present invention described herein can be administered to mammals, particularly large mammals, such as non-human primates or humans, in several ways.

[0283] In some embodiments, the administration of dsRNA molecules, such as siRNA agents or compositions, is parenteral, e.g., intravenous (e.g., as a bolus or diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, oral mucosa, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ​​or ocular. Administration can be performed by the subject or by another person, e.g., a healthcare provider. Dosage may be in measured doses or by a dispenser that delivers measured doses. Selected modes of delivery are described below.

[0284] The present invention provides methods, compositions, and kits for rectal administration or delivery of dsRNA molecules as described herein.

[0285] In a particular embodiment, the present invention relates to a dsRNA molecule for use in the above-described method.

[0286] Methods for inhibiting the expression of target genes Aspects of the present invention also relate to methods for inhibiting the expression of a target gene. These methods include administering a dsRNA molecule according to any of the above embodiments in an amount sufficient to inhibit the expression of a target gene. The present invention further relates to the use of dsRNA molecules as defined herein for inhibiting the expression of a target gene in target cells. In a preferred embodiment, the present invention further relates to the use of dsRNA molecules for inhibiting the expression of a target gene in target cells in vitro.

[0287] In another aspect, the present invention relates to a method for regulating the expression of a target gene in a cell, comprising the step of providing the cell with the dsRNA molecule of the present invention. In some embodiments, the target gene is factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta 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, VEGF gene, EGFR The mutations are selected from a group consisting of the following: the gene, cyclin A gene, cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivor gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21 (WAF1 / CIP1) gene, mutations in the p27 (KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.

[0288] In a particular embodiment, the present invention relates to a dsRNA molecule for use in the above-described method.

[0289] Exemplary aspects of various situations can be described by the following manner, to which characters are assigned:

[0290] Embodiment A: A dsRNA agent comprising a sense strand and an antisense strand, wherein each strand independently has a length of 15 to 35 nucleotides, the sense strand contains a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand, and the antisense strand contains 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand.

[0291] Embodiment B: The dsRNA agent according to Embodiment A, wherein the sense strand further comprises a 2'-fluoronucleotide at position 11, counting from the 5' end of the sense strand.

[0292] Embodiment C: The dsRNA agent according to Embodiment A or B, wherein the sense strand further comprises a 2'-fluoronucleotide at position 9, counting from the 5' end of the sense strand.

[0293] Embodiment D: The dsRNA according to any one of Embodiments A to C, wherein the sense strand further comprises 2'-fluoronucleotides at positions 9 and 11, counting from the 5th end of the sense strand.

[0294] Embodiment E: A dsRNA agent according to any one of Embodiments A to D, wherein the sense strand contains 2'-fluoronucleotides at positions 8 and 9, counting from the 5th end of the sense strand.

[0295] Embodiment F: A dsRNA agent according to any one of Embodiments A to E, wherein the sense strand contains 2'-fluoronucleotides at positions 11 and 12, counting from the 5th end of the sense strand.

[0296] Embodiment G: A dsRNA agent according to any one of Embodiments A to F, wherein the sense strand contains at least one 2'-OMe nucleotide.

[0297] Embodiment H: A dsRNA agent according to any one of Embodiments A to G, wherein the antisense strand contains a 2'-deoxynucleotide at position 2, counting from the 5' end of the antisense strand.

[0298] Embodiment I: A dsRNA agent according to any one of Embodiments A to H, wherein the antisense strand contains 2'-deoxyribonucleotides at positions 2, 5, and 7, counting from the 5' end of the antisense strand.

[0299] Embodiment J: A dsRNA agent according to any one of Embodiments A to I, wherein the antisense strand comprises at least one 2'-fluoronucleotide.

[0300] Embodiment K: A dsRNA agent according to any one of Embodiments A to J, wherein the antisense strand contains a 2'-fluoronucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand.

[0301] Embodiment L: A dsRNA agent according to any one of Embodiments A to K, wherein the dsRNA agent contains a ligand.

[0302] Embodiment M: A dsRNA agent according to any one of Embodiments A to L, wherein the sense strand contains a ligand.

[0303] Embodiment N: A dsRNA agent according to Embodiment L or M, wherein the ligand is an ASGPR ligand.

[0304] Embodiment O: A dsRNA agent according to any one of Embodiments A to N, comprising at least two phosphorothioate nucleotide junctions.

[0305] Embodiment P: A dsRNA agent according to any one of Embodiments A to O, wherein the sense strand includes at least two phosphorothioate nucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand.

[0306] Embodiment Q: A dsRNA agent according to any one of Embodiments A to P, wherein the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand, and also includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 3' end of the antisense strand.

[0307] Embodiment R: A dsRNA agent according to any one of Embodiments A to Q, wherein the dsRNA has a double-stranded region of 18 to approximately 25 base pairs.

[0308] Embodiment S: A dsRNA agent according to any one of Embodiments A to R, wherein the sense strand is 18 to 23 nucleotides long.

[0309] Embodiment T: A dsRNA agent according to any one of Embodiments A to S, wherein the antisense strand is 18 to 25 nucleotides long.

[0310] Embodiment U: A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long, contains a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand, contains a 2'-fluoronucleotide at position 9 or 11 counting from the 5' end of the sense strand, and the antisense strand is 18 to 25 nucleotides long, contains a 2'-deoxynucleotide at positions 5 and 7 counting from the 5' end of the antisense strand.

[0311] Embodiment V: A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long and contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and the antisense strand is 18 to 25 nucleotides long and contains 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand.

[0312] Further exemplary embodiments may be described by one or more of the following numbered embodiments.

[0313] Embodiment 1: A dsRNA agent comprising a sense strand and an antisense strand, wherein each strand independently has a length of 15 to 35 nucleotides, and each nucleotide independently is modified or unmodified. The sense strand contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand. The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12, counting from the 5' end of the antisense strand. (i) The antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16 counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. The aforementioned dsRNA agent.

[0314] Embodiment 2: The dsRNA agent according to Embodiment 1, wherein the sense strand further comprises a 2'-fluoronucleotide at position 11, counting from the 5' end of the sense strand.

[0315] Embodiment 3: The dsRNA agent according to Embodiment 1 or 2, wherein the sense strand further comprises a 2'-fluoronucleotide at position 9, counting from the 5' end of the sense strand.

[0316] Embodiment 4: The dsRNA according to any one of Embodiments 1 to 3, wherein the sense strand further comprises 2'-fluoronucleotides at positions 9 and 11 counting from the 5th end of the sense strand.

[0317] Embodiment 5: A dsRNA agent according to any one of Embodiments 1 to 4, wherein the sense strand contains 2'-fluoronucleotides at positions 8 and 9, counting from the 5th end of the sense strand.

[0318] Embodiment 6: A dsRNA agent according to any one of Embodiments 1 to 5, wherein the sense strand contains 2'-fluoronucleotides at positions 11 and 12, counting from the 5th end of the sense strand.

[0319] Embodiment 7: A dsRNA agent according to any one of Embodiments 1 to 6, wherein the sense strand contains a nucleotide other than 2'-fluoro at position 7, counting from the 5' end of the sense strand.

[0320] Embodiment 8: The dsRNA according to any one of Embodiments 1 to 7, wherein the sense strand contains a 2'-fluoronucleotide at positions 9, 10, and 11 counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7.

[0321] Embodiment 9: A dsRNA agent according to any one of Embodiments 1 to 8, wherein the sense strand comprises at least one 2'-OMe nucleotide.

[0322] Embodiment 10: A dsRNA agent according to any one of Embodiments 1 to 9, wherein the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

[0323] Embodiment 11: A dsRNA agent according to any one of Embodiments 1 to 10, wherein the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, and contains a 2'-OMe nucleotide at position 7.

[0324] Embodiment 12: The dsRNA agent according to any one of Embodiments 1 to 11, wherein the antisense strand contains a 2'-fluoronucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16.

[0325] Embodiment 13: A dsRNA agent according to any one of Embodiments 1 to 12, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12, counting from the 5' end of the antisense strand, contains a 2'-fluoronucleotide at position 14, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16.

[0326] Embodiment 14: A dsRNA agent according to any one of Embodiments 1 to 13, wherein the antisense strand contains a 2'-OMe nucleotide at position 16, counting from the 5' end of the antisense strand.

[0327] Embodiment 15: A dsRNA agent according to any one of Embodiments 1 to 14, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand, contains a 2'-fluoronucleotide at position 14, and contains a 2'-OMe nucleotide at position 16.

[0328] Embodiment 16: A dsRNA agent according to any one of Embodiments 1 to 11, wherein the antisense strand contains a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0329] Embodiment 17: A dsRNA agent according to any one of Embodiments 1 to 11 or 16, wherein the antisense strand contains a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

[0330] Embodiment 18: A dsRNA agent according to any one of Embodiments 1 to 11 or 16 to 17, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0331] Embodiment 19: A dsRNA agent according to any one of Embodiments 1 to 18, wherein the antisense strand contains a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0332] Embodiment 20: A dsRNA agent according to any one of Embodiments 1 to 19, wherein the antisense strand contains a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

[0333] Embodiment 21: A dsRNA agent according to any one of Embodiments 1 to 20, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0334] Embodiment 22: A dsRNA agent according to any one of Embodiments 1 to 11 or 16 to 21, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12, 14 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0335] Embodiment 23: A dsRNA agent according to any one of Embodiments 1 to 11 or 16 to 22, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12, 14 and 16, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

[0336] Embodiment 24: A dsRNA agent according to any one of Embodiments 1 to 15 or 19 to 20, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and contains 2'-fluoronucleotides at position 14, and the sense strand contains a nucleotide other than 2'-fluoro at position 7, counting from the 5' end of the sense strand.

[0337] Embodiment 25: A dsRNA agent according to any one of Embodiments 1 to 15, 19 to 20, or 24, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 of the antisense strand, counting from the 5' end of the antisense strand, and contains 2'-fluoronucleotides at position 14, and the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

[0338] Aspect 26: The dsRNA agent according to any one of Aspects 1 to 25, comprising a ligand.

[0339] Aspect 27: The dsRNA agent according to any one of Aspects 1 to 26, wherein the sense strand comprises a ligand.

[0340] Aspect 28: The dsRNA agent according to Aspect 27, wherein the ligand is at the 3' end of the sense strand.

[0341] Aspect 29: The dsRNA agent according to Aspect 27, wherein the ligand is at the 5' end of the sense strand.

[0342] Aspect 30: The dsRNA agent according to any one of Aspects 26 to 29, wherein the ligand comprises an ASGPR ligand.

[0343] Aspect 31: The dsRNA agent according to any one of Aspects 26 to 29, wherein the ligand is a lipophilic group.

[0344] Aspect 32: The dsRNA agent according to Aspect 31, wherein the ligand is a C 10~30 aliphatic group.

[0345] Aspect 33: The dsRNA agent according to Aspect 32, wherein the C 10~30 aliphatic group is a C 10~30 salkyl group. Aspect 34: The dsRNA agent according to Aspect 33, wherein the C

[0346] alkyl group is a linear or branched tetradecyl, hexadecyl, octadecyl, icosyl, docosyl or tetracosyl group. 10~30 Aspect 35: The dsRNA agent according to Aspect 34, wherein the ligand is conjugated to a non-terminal nucleotide of the sense strand.

[0347] Aspect 36: The dsRNA agent according to Aspect 35, wherein the ligand is conjugated to the 2'-position of a non-terminal nucleotide of the sense strand and optionally conjugated to one of positions 5, 6, 7 or 8 of the sense strand counted from the 5' end.

[0348] Aspect 37: The dsRNA agent according to Aspect 36, wherein the ligand is conjugated to the 2'-position of a non-terminal nucleotide of the sense strand and optionally conjugated to one of positions 5, 6, 7 or 8 of the sense strand counted from the 5' end.

[0349] Embodiment 37: The dsRNA agent according to any one of Embodiments 26 to 36, wherein the ligand comprises a debasalized nucleotide, and optionally the debasalized nucleotide is an inverted nucleotide ligated to the dsRNA agent chain via a 5'→5' or 3'→3' ligation.

[0350] Embodiment 38: A dsRNA agent according to any one of Embodiments 26 to 37, wherein the ligand is attached to the 3' end of the sense strand.

[0351] Embodiment 39: The dsRNA agent according to Embodiment 38, wherein the ligand is attached to the 3' end of the sense strand via a 3'→3' ligator.

[0352] Embodiment 40: A dsRNA agent according to any one of Embodiments 1 to 39, wherein the dsRNA contains two ligands.

[0353] Embodiment 41: The dsRNA according to Embodiment 40, wherein the sense strand comprises a first ligand attached to the 3' end of the sense strand and a second ligand attached to the 5' end of the sense strand.

[0354] Embodiment 42: The dsRNA according to Embodiment 41, wherein the first ligand comprises a debasalized nucleotide, the second ligand comprises an ASGPR ligand, and optionally the debasalized nucleotide is an inverted nucleotide ligated to the sense strand via a 3'→3' ligation site.

[0355] Embodiment 43: A dsRNA agent according to any one of Embodiments 1 to 42, comprising at least two phosphorothioate nucleotide junctions.

[0356] Embodiment 44: The dsRNA agent according to any one of Embodiments 1 to 43, wherein the sense strand includes at least two phosphorothioate nucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand.

[0357] Embodiment 45: The dsRNA agent according to any one of Embodiments 1 to 44, wherein the antisense strand includes at least two phosphorothioate nucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand, and at least two phosphorothioate nucleotide junctions between the first five nucleotides counted from the 3' end of the antisense strand.

[0358] Embodiment 46: A dsRNA agent according to any one of Embodiments 1 to 45, wherein the dsRNA has a double-stranded region of 18 to approximately 25 base pairs.

[0359] Embodiment 47: A dsRNA agent according to any one of Embodiments 1 to 46, wherein the sense strand is 18 to 23 nucleotides long.

[0360] Embodiment 48: A dsRNA agent according to any one of Embodiments 1 to 47, wherein the antisense strand is 18 to 25 nucleotides long.

[0361] Embodiment 49: A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long, contains a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand, contains a 2'-fluoronucleotide at position 9 or 11 counting from the 5' end of the sense strand, and the antisense strand is 18 to 25 nucleotides long, contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand. (i) The antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16 counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. The aforementioned dsRNA agent.

[0362] Embodiment 50: A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long and contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and the antisense strand is 18 to 25 nucleotides long and contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand. (i) The antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16 counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. The aforementioned dsRNA agent.

[0363] Embodiment 51: A dsRNA agent according to any one of Embodiments 1 to 50, comprising a phosphate mimic at the 5' end of the antisense strand.

[0364] Embodiment 52: The dsRNA agent according to Embodiment 51, wherein the phosphate mimic is a 5'-E-vinylphosphonate.

[0365] Embodiment 53: Phosphate mimic, structure: A dsRNA agent according to embodiment 52, comprising a 5'-cyclopropylphosphonate having TIFF0007882858000042.tif24159, wherein * represents the binding to the C5 position of the nucleotide at the 5' end.

[0366] Embodiment 54: The remaining nucleotides in the sense strand (i.e., nucleotides at positions not otherwise specified) are either unmodified nucleotides or modified nucleotides, optionally 2'-OMe, 2'-F, 2'-H, and 2'-OC. 10~30 A modified nucleotide selected from the group consisting of aliphatic groups, except for 2'-OC. 10~30The dsRNA agent according to any one of Aspects 1 to 53, wherein the number of modified nucleotides that are aliphatic groups is 1 or less.

[0367] Aspect 55: The remaining nucleotides in the sense strand (i.e., the nucleotides at positions not otherwise specified) are modified nucleotides selected from the group consisting of 2'-OMe, 2'-F, 2'-H, and 2'-O-C 10~30 aliphatic groups, provided that 2'-O-C 10~30 The dsRNA agent according to any one of Aspects 1 to 54, wherein the number of modified nucleotides that are aliphatic groups is 1 or less.

[0368] Aspect 56: The remaining nucleotides in the antisense strand (i.e., the nucleotides at positions not otherwise specified) are unmodified nucleotides or modified nucleotides optionally selected from the group consisting of 2'-OMe, 2'-F, 2'-H, GNA, and 3'-RNA, where 3'-RNA is optionally 3'-OH, provided that the number of modified nucleotides that are GNA or 3'-RNA is 1 or less. The dsRNA agent according to any one of Aspects 1 to 55.

[0369] Aspect 57: The remaining nucleotides in the antisense strand (i.e., the nucleotides at positions not otherwise specified) are modified nucleotides selected from the group consisting of 2'-OMe, 2'-F, 2'-H, GNA, and 3'-RNA, where 3'-RNA is optionally 3'-OH, provided that the number of modified nucleotides that are GNA or 3'-RNA is 1 or less. The dsRNA agent according to any one of Aspects 1 to 56.

[0370] Excerpt from definitions Certain terms used in this specification, the examples, and the appended claims are set forth here for convenience. Unless otherwise stated or the context makes clear, the following terms and phrases have the meanings set forth below. Unless otherwise explicitly stated or the context makes clear, the following terms and phrases do not preclude the meanings that the term or phrase has acquired in the relevant art. These definitions are provided to aid in the description of specific embodiments and are not intended to limit the claimed invention, for the scope of the invention is limited only by the claims. Furthermore, unless the context requires otherwise, the singular form includes the plural and the plural form includes the singular.

[0371] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention relates. Known methods, devices, and materials may be used in carrying out or testing the present invention, and such methods, devices, and materials are described herein.

[0372] Furthermore, unless otherwise indicated, the invention may utilize prior art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that falls within the scope of the skills in the art. Such techniques are described in detail in publications such as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RI Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (FMAusubel 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), and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0373] If a range of values ​​is provided, each value interposing between the upper and lower limits of that range (up to one-tenth of its unit unless otherwise clearly indicated in the context), and any other specified or interposing values ​​within that specified range, are understood to be included within the invention. The upper and lower limits of these smaller ranges may be independently contained within those smaller ranges and are similarly included within the invention, except for any limit points specifically excluded within the specified range. If a specified range contains one or both of the limit points, a range excluding one or both of those contained limit points is also included within the invention.

[0374] In this specification, a range is presented as a number preceded by the term “approximately.” In this specification, the term “approximately” is used to literally support the number preceded by it, and any number that is close to or approximates the number preceded by the term. In determining whether a number is close to or approximates a specifically stated number, the close or approximate, unstated number may be a number that is substantially equivalent to the specifically stated number in the context in which it is presented.

[0375] As used herein, the terms “comprising” or “comprises” (including) are used in relation to compositions, methods, and their respective components that are essential to the present invention, but the inclusion of elements not specified, whether essential or not, is also permitted.

[0376] The singular terms “a,” “an,” and “the” encompass multiple referents unless the context makes it clear otherwise. Similarly, the word “or” encompasses “and” unless the context makes it clear otherwise. Furthermore, it should be noted that claims can be drafted in such a way that any optional elements are excluded. Therefore, this statement should serve as a basis for the use of exclusive expressions such as “only” or “negative” limitations in relation to the description of elements of a claim.

[0377] As used herein, the terms “dsRNA,” “siRNA,” and “iRNA agent” are used interchangeably to refer to agents capable of mediating the silencing of target RNA, such as mRNA, or the transcript of a protein-coding gene. For convenience, such mRNA is also referred to herein as the silencing mRNA. Such genes are also called target genes. Generally, the silencing RNA can be an endogenous gene, an exogenous gene, or a pathogenic gene. In addition, RNA other than mRNA, such as tRNA and viral RNA, can also be targeted.

[0378] As used herein, the term "RNAi-mediated" refers to the ability to sequence-specifically silence a target gene, such as mRNA. While we do not wish to be bound by theory, it is assumed that silencing involves an RNAi mechanism or process and a guide RNA, such as the antisense strand of dsRNA, where the antisense strand is 21–23 nucleotides long.

[0379] As used herein, “specifically hybridizable” and “complementary” are terms used to indicate a sufficient degree of complementarity such that stable, specific binding occurs between the present invention and the target RNA molecule. Specific binding requires a sufficient degree of specificity to avoid nonspecific binding of the oligomeric compound to non-target sequences under the conditions under which specific binding is desired, i.e., physiological conditions in the case of an assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay. Non-target sequences typically differ by at least five nucleotides.

[0380] In some embodiments, the dsRNA molecule of the present invention is "sufficiently complementary" to a target RNA, such as a target mRNA, so that the dsRNA molecule silences the production of the protein encoded by the target mRNA. In another embodiment, the dsRNA molecule of the present invention is "strictly complementary" to the target RNA, so that, for example, the target RNA and the dsRNA bistrand agent anneal to form a hybrid consisting exclusively of Watson-Crick base pairs in a strictly complementary region. A "sufficiently complementary" target RNA may contain an internal region (e.g., a region of at least 10 nucleotides) that is strictly complementary to the target RNA. Furthermore, in some embodiments, the dsRNA molecule of the present invention specifically recognizes single-nucleotide differences. In this case, the dsRNA molecule mediates RNAi only when strict complementarity is found in the region where the single-nucleotide difference occurs (e.g., within 7 nucleotides).

[0381] The term "BNA" refers to cross-linked nucleic acids, often called constrained RNA or inaccessible RNA. BNA can contain five-membered, six-membered, or even seven-membered cross-linked structures with "fixed" C3'-end sugar puckering. The cross-linking is typically incorporated at the 2',4' position of ribose, giving 2',4'-BNA nucleotides (e.g., LNA or ENA). Examples of BNA nucleotides include the following nucleosides: TIFF0007882858000043.tif74145.

[0382] The term "LNA" refers to locked nucleic acid, often called restricted RNA or inaccessible RNA. LNA is a modified RNA nucleotide. The ribose portion of an LNA nucleotide is modified by an additional crosslink (e.g., a methylene crosslink or ethylene crosslink) that connects the 2' hydroxyl to the 4' carbon of the same ribose sugar. For example, the crosslink connects the ribose to the 3'-end North conformation: You can "lock" TIFF0007882858000044.tif28128.

[0383] The term "ENA" refers to ethylene-crosslinked nucleic acids, and is often called constrained RNA or inaccessible RNA.

[0384] As used herein, "cleavage site" means a target gene or backbone junction in the sense strand that is cleaved by the RISC mechanism when an iRNA agent is used. The target cleavage site region also includes at least one or at least two nucleotides on both sides of the cleavage site. In the case of the sense strand, if the sense strand itself is the target to be cleaved by the RNAi mechanism, the cleavage site is the backbone junction in the sense strand that is cleaved. The cleavage site can be determined using methods known in the art, such as the 5'-RACE assay described by Soutschek et al., Nature (2004) 432, 173-178, which is incorporated herein by reference in its entirety. As is well understood in the art, in the case of a conical double-stranded RNAi agent containing two 21-nucleotide long chains (where the strands form a double-stranded region of 19 consecutive base pairs with a 2-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.

[0385] The terms “decrease,” “reduce,” “inhibit,” and “inhibit” are used herein to describe a statistically significant reduction. In some embodiments, “reduce,” “inhibit,” or “inhibit” typically mean a reduction of at least 10% compared to a baseline level (e.g., in the absence of a given treatment), and may include reductions of, for example, 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, “reduce” or “inhibit” does not include complete inhibition or complete reduction compared to a baseline level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction may preferably decrease to a level that is acceptable as being within the normal range for an individual without a given disorder.

[0386] As used herein, the “central region” of a chain refers to positions 5–17, counting from the 5' end of the chain, for example, positions 6–16, 6–15, 6–14, 6–13, 6–12, 7–15, 7–14, 7–13, 7–12, 8–16, 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. For example, the central region of a chain means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the chain. The preferred central region of the sense chain is located at positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5' end of the sense chain. The more preferred central region of the sense chain is located at positions 7, 8, 9, 10, 11, 12, and 13, counting from the 5' end of the sense chain. The preferred central region of the antisense chain is located at positions 9, 10, 11, 12, 13, 14, 15, 16, and 17, counting from the 5' end of the antisense chain. The more preferred central region of the antisense chain is located at positions 10, 11, 12, 13, 14, 15, and 16, counting from the 5' end of the antisense chain.

[0387] As will be apparent to those skilled in the art who have read this disclosure, each of the individual aspects described and illustrated herein has separate components and features, and they can be readily separated from or combined with any of the features of any of the other aspects without departing from the scope or spirit of the invention. Any of the methods described may be performed in the order described herein or in any other logically possible order.

[0388] The present invention is further illustrated by the following examples, but these examples should not be construed as further limitations. All references, ongoing patent applications and published patents cited throughout this application are expressly incorporated herein by reference. [Examples]

[0389] Synthesis and purification of oligonucleotides All oligonucleotides were prepared on a 1 μmole scale using a MerMade 192 synthesizer with either a universal or custom support. All phosphoramidites were prepared using the standard protocol for 2-cyanoethyl phosphoramidites at a concentration of 100 mM in 100% acetonitrile or 9:1 acetonitrile:DMF, with the coupling time extended to 400 seconds. Oxidation of the newly formed ligatures was achieved using a 50 mM I2 solution in 9:1 acetonitrile:water for phosphate ligatures and a 100 mM DDTT solution in 9:1 pyridine:acetonitrile for phosphorothioate ligatures. After trityl-off synthesis, the column was incubated with 150 μL of 40% methylamine aqueous solution for 45 minutes, and the solution was drained into a 96-well plate under reduced pressure. After repeated incubation and draining with fresh methylamine aqueous solution, the plate containing the crude oligonucleotide solution was sealed and shaken at room temperature for a further 60 minutes to completely remove all protecting groups. Precipitation of the crude oligonucleotide was achieved by adding 1.2 mL of 9:1 acetonitrile:EtOH to each well, followed by incubation at -20°C overnight. The plate was then centrifuged at 3000 RPM for 45 minutes, the supernatant was removed from each well, and the pellet was resuspended in 950 μL of 20 mM NaOAc aqueous solution. Finally, each crude solution was desalted using a GE Hi-Trap desalting column (Sephadex G25 Superfine), and the final oligonucleotide product was eluted with water. All entities and purities were confirmed by ESI-MS and IEX HPLC, respectively.

[0390] Cell culture and transfection Transfection of primary mouse or cynomolgus monkey hepatocytes (Thermo Fisher Scientific / Gibco) was performed in a 384-well plate by adding 4.9 μL of Opti-MEM + 0.1 μL of Lipofectamine RNAiMax (Invitrogen, catalog no. 13778-150) to 5 μL of siRNA double helix in each well, and incubating at room temperature for 15 minutes. Next, 40 μL of Dulbecco's Modified Eagle Medium (PCH) or William Medium (PMH) containing approximately 5 × 10³ cells was added to the siRNA mixture. The cells were incubated at 37°C for 24 hours and then processed for RNA purification. Experiments were performed with siRNA doses of 10 nM, 1 nM, and 0.1 nM.

[0391] The parental dsRNA molecules are shown in Tables 1-3. The abbreviations used in the sequences are summarized in Table 4.

[0392] (Table 1) Sequence of parental dsRNA molecule TIFF0007882858000045.tif197168

[0393] (Table 2) Further exemplary sequences of parental dsRNA molecules TIFF0007882858000046.tif59169TIFF0007882858000047.tif244169TIFF00078828580 00048.tif244169TIFF0007882858000049.tif244169TIFF0007882858000050.tif106169

[0394] (Table 3) Further exemplary sequences of parental dsRNA molecules TIFF0007882858000051.tif122163TIFF0007882858000052.tif244163TIFF000 7882858000053.tif244163TIFF0007882858000054.tif244163TIFF0007882858 000055.tif244163TIFF0007882858000056.tif244163TIFF0007882858000057. tif244163TIFF0007882858000058.tif244163TIFF0007882858000059.tif27163

[0395] (Table 4) Abbreviations of nucleotide monomers used in the description of nucleic acid sequences * TIFF0007882858000060.tif190156TIFF0007882858000061.tif181156*When present in oligonucleotides, it will be understood that these monomers are linked to each other by a 5'-3' phosphodiester bond. Also, if a nucleotide contains a 2' fluoro modification, it will be understood that the fluoro replaces the hydroxyl at that position on the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoronucleotide).

[0396] Several exemplary dsRNA molecular designs according to aspects of this disclosure are schematically shown in Figures 5A and 5B. Exemplary dsRNA molecules according to aspects of this disclosure are listed in Tables 5 and 6.

[0397] (Table 5) Exemplary dsRNA molecules in some embodiments of the present disclosure TIFF0007882858000062.tif244165TIFF0007882858000063.tif244165TIFF0007882858000064.tif244165TIFF0007882858000065.tif165165

[0398] (Table 6) Further exemplary dsRNA molecules in some aspects of the present disclosure TIFF0007882858000066.tif64166TIFF0007882858000067.tif244166TIFF0007 882858000068.tif244166TIFF0007882858000069.tif244166TIFF00078828580 00070.tif244166TIFF0007882858000071.tif244166TIFF0007882858000072.t if244166TIFF0007882858000073.tif244166TIFF0007882858000074.tif85166

[0399] In some embodiments, the dsRNA molecule is not one of the dsRNA molecules listed in Table 7.

[0400] (Table 7) Further dsRNA molecules TIFF0007882858000075.tif38160

[0401] Total RNA isolation using the DYNABEADS mRNA isolation kit (Invitrogen®, part number 610-12) The cells were lysed in 75 μL of lysis / binding buffer containing 3 μL of beads per well and mixed on an electrostatic shaker for 10 minutes. The washing step was automated using a Biotek EL406 with a magnetic plate support. The beads were washed once in buffer A, once in buffer B, and twice in buffer E (in 90 μL), with aspiration steps in between. Following the final aspiration, a complete 10 μL RT mixture was added to each well as described below.

[0402] cDNA synthesis using the ABI High-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California, catalog number 4368813) For each reaction, a master mix consisting of 1 μL of 10× buffer, 0.4 μL of 25× dNTPs, 1 μL of random primer, 0.5 μL of reverse transcriptase, 0.5 μL of RNase inhibitor, and 6.6 μL of H2O was added to each well. The plate was sealed and stirred with an electrostatic shaker for 10 minutes, then incubated at 37°C for 2 hours. Subsequently, the plate was stirred at 80°C for 8 minutes.

[0403] Real-time PCR Each well of a 384-well plate contained a master mix containing 0.5 μL of human GAPDH TaqMan probe (4326317E), 0.5 μL of human AGT (Hs00174854m1), 2 μL of nuclease-free water, and 5 μL of Lightcycler 480 probe master mix (Roche catalog no. 04887301001). 2 μL of cDNA was added to this master mix (Roche catalog no. 04887301001). Real-time PCR was performed using a LightCycler 480 real-time PCR system (Roche).

[0404] To calculate the relative magnification change, the data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells.

[0405] The results are shown in Figures 1A–1B and 3A–4C, and summarized in Tables 8–14. These results demonstrate consistently improved in vitro activity compared to the parent design in several exemplary designs, as well as in a large set of exemplary sequences, targets, and cell lines.

[0406] (Table 8) In vitro activity of various designs in primary mouse hepatocytes TIFF0007882858000076.tif123170TIFF0007882858000077.tif246170

[0407] (Table 9) In vitro activity of various designs in primary mouse hepatocytes TIFF0007882858000078.tif226160TIFF0007882858000079.tif219160

[0408] (Table 10) In vitro activity of various designs in primary mouse hepatocytes TIFF0007882858000080.tif169164

[0409] (Table 11) In vitro activity of various designs in primary mouse hepatocytes TIFF0007882858000081.tif199160

[0410] (Table 12) In vitro activity of various designs in primary cynomolgus monkey hepatocytes TIFF0007882858000082.tif127156

[0411] (Table 13) In vitro activity of various designs in Hep3B TIFF0007882858000083.tif215154TIFF0007882858000084.tif215159TIFF0007882858000085.tif21515 9TIFF0007882858000086.tif215159TIFF0007882858000087.tif215159TIFF0007882858000088.tif21582

[0412] (Table 14) In vitro activity of various designs targeting Agt in Hep3B TIFF0007882858000089.tif236170TIFF0007882858000090.tif241170TIFF0007882858000091.tif241170TIFF0007882858000092.tif241170 TIFF0007882858000093.tif241170TIFF0007882858000094.tif241170TIFF0007882858000095.tif241170TIFF0007882858000096.tif163170

[0413] In vivo studies in mice and cynomolgus monkeys All studies were conducted using protocols that complied with local, state, and federal regulations, as approved by Alnylam Pharmaceuticals' Institutional Animal Experimentation Committees (IACUCs) as appropriate.

[0414] In the mouse pharmacodynamic study, female C57BL / 6 mice (Charles River Laboratories) were subcutaneously administered either a medium control (1×PBS or 0.9% sodium chloride) or a single dose of siRNA to the upper back. Blood was collected by postorbital collection. Serum was separated at 13,000 rpm for 10 minutes at room temperature. The livers of the mice were collected, immediately rapidly frozen in liquid nitrogen, and stored at -80°C for mRNA and siRNA analysis.

[0415] As shown in Figures 2A–2D and 10A–10D, these results demonstrate improved or similar targeted knockdown in mice and improved or similar targeted knockdown and silencing duration in cynomolgus monkeys.

[0416] Serum protein quantification TTR protein was quantified by ELISA from serum isolated from whole blood. ELISA was performed according to the manufacturer's protocol (ALPCO, 41-PALMS-E01) after a 3025-fold dilution of serum samples. Data were normalized to pre-bleed TTR levels. All samples were dual-assayed. Each data point represents the mean of all mice in each cohort (n=3).

[0417] In some embodiments, the dsRNA molecule is not one of the dsRNA molecules listed in any one of Tables 15-25.

[0418] (Table 15) Exemplary dsRNA molecules TIFF0007882858000097.tif143166

[0419] (Table 16) Exemplary dsRNA molecules TIFF0007882858000098.tif225150TIFF0007882858000099.tif245150TIFF0007882858 000100.tif241150TIFF0007882858000101.tif246150TIFF0007882858000102.tif23150

[0420] (Table 17) Exemplary dsRNA molecules TIFF0007882858000103.tif205164TIFF0007882858000104.tif242164TIFF0007882858000105.tif246164TIFF0007882858000106.tif235164

[0421] (Table 18) Exemplary dsRNA molecules TIFF0007882858000107.tif34168

[0422] (Table 19) Exemplary dsRNA molecules TIFF0007882858000108.tif181165TIFF0007882858000109.tif241165TIFF0007882858000110.tif241165

[0423] (Table 20) Exemplary dsRNA molecules TIFF0007882858000111.tif34170

[0424] (Table 21) Exemplary dsRNA molecules TIFF0007882858000112.tif176161TIFF0007882858000113.tif99161

[0425] (Table 22) Exemplary dsRNA molecules TIFF0007882858000114.tif123147TIFF0007882858000115.tif243147TIFF0007882858000116.tif243147TIFF0007882858000117.tif146147

[0426] (Table 23) Exemplary dsRNA molecules TIFF0007882858000118.tif99155

[0427] (Table 24) Exemplary dsRNA molecules TIFF0007882858000119.tif72151

[0428] (Table 25) Exemplary dsRNA molecules TIFF0007882858000120.tif126161

[0429] All U.S. patents, U.S. patent application publications, foreign patents, foreign patent applications, and non-patent publications referenced herein are incorporated herein by reference in their entirety. Further aspects of the embodiments can be obtained by modifying them to utilize the ideas of various patents, applications, and publications as needed.

[0430] These and other modifications may be made to the present embodiments in light of the detailed description above. In general, the terms used in the appended claims should not be interpreted to limit the claims to the specific embodiments disclosed herein and in the claims, but rather to encompass all conceivable embodiments along the entire scope of equivalents recognized in those claims. Thus, the claims are not limited by the present disclosure.

Claims

1. A dsRNA agent comprising a sense strand and an antisense strand, wherein each strand independently has a length of 15 to 35 nucleotides, and each nucleotide is independently modified or unmodified. The sense strand contains a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand. The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12, counting from the 5' end of the antisense strand. (i) The antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16 counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. The aforementioned dsRNA agent.

2. The dsRNA agent according to claim 1, wherein the sense strand further comprises a 2'-fluoronucleotide at position 11, counting from the 5' end of the sense strand.

3. The dsRNA agent according to claim 1 or 2, wherein the sense strand further comprises a 2'-fluoronucleotide at position 9, counting from the 5' end of the sense strand.

4. The dsRNA according to any one of claims 1 to 3, wherein the sense strand further comprises 2'-fluoronucleotides at positions 9 and 11, counting from the 5th end of the sense strand.

5. A dsRNA agent according to any one of claims 1 to 4, wherein the sense strand contains 2'-fluoronucleotides at positions 8 and 9, counting from the 5th end of the sense strand.

6. A dsRNA agent according to any one of claims 1 to 5, wherein the sense strand contains 2'-fluoronucleotides at positions 11 and 12, counting from the 5th end of the sense strand.

7. A dsRNA agent according to any one of claims 1 to 6, wherein the sense strand contains a nucleotide other than 2'-fluoro at position 7, counting from the 5' end of the sense strand.

8. The dsRNA according to any one of claims 1 to 7, wherein the sense strand contains a 2'-fluoronucleotide at positions 9, 10, and 11 counting from the 5' end of the sense strand, and contains a nucleotide other than a 2'-fluoronucleotide at position 7.

9. A dsRNA agent according to any one of claims 1 to 8, wherein the sense strand comprises at least one 2'-OMe nucleotide.

10. A dsRNA agent according to any one of claims 1 to 9, wherein the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

11. A dsRNA agent according to any one of claims 1 to 10, wherein the sense strand contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and contains a 2'-OMe nucleotide at position 7.

12. A dsRNA agent according to any one of claims 1 to 11, wherein the antisense strand contains a 2'-fluoronucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16.

13. The dsRNA agent according to any one of claims 1 to 12, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12, counting from the 5' end of the antisense strand, contains a 2'-fluoronucleotide at position 14, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16.

14. A dsRNA agent according to any one of claims 1 to 13, wherein the antisense strand contains a 2'-OMe nucleotide at position 16, counting from the 5' end of the antisense strand.

15. A dsRNA agent according to any one of claims 1 to 14, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand, contains a 2'-fluoronucleotide at position 14, and contains a 2'-OMe nucleotide at position 16.

16. A dsRNA agent according to any one of claims 1 to 11, wherein the antisense strand contains a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

17. A dsRNA agent according to any one of claims 1 to 11 or 16, wherein the antisense strand contains a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

18. A dsRNA agent according to any one of claims 1 to 11 or 16 to 17, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

19. A dsRNA agent according to any one of claims 1 to 18, wherein the antisense strand contains a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

20. A dsRNA agent according to any one of claims 1 to 19, wherein the antisense strand contains a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-OMe nucleotide at position 7, counting from the 5' end of the sense strand.

21. A dsRNA agent according to any one of claims 1 to 20, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

22. A dsRNA agent according to any one of claims 1 to 11 or 16 to 21, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12, 14 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

23. A dsRNA agent according to any one of claims 1 to 11 or 16 to 22, wherein the antisense strand contains a 2'-deoxynucleotide at positions 2, 5, 7, 12, 14 and 16, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7, counting from the 5' end of the sense strand.

24. A dsRNA agent according to any one of claims 1 to 15 or 19 to 20, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and contains 2'-fluoronucleotides at position 14, and the sense strand contains a nucleotide other than 2'-fluoro at position 7, counting from the 5' end of the sense strand.

25. A dsRNA agent according to any one of claims 1 to 15, 19 to 20, or 24, wherein the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 of the antisense strand, counting from the 5' end of the antisense strand, and contains 2'-fluoronucleotides at position 14, and the sense strand contains 2'-OMe nucleotides at position 7, counting from the 5' end of the sense strand.

26. A dsRNA agent according to any one of claims 1 to 25, comprising a ligand.

27. A dsRNA agent according to any one of claims 1 to 26, wherein the sense strand contains a ligand.

28. The dsRNA agent according to claim 27, wherein the ligand is located at the 3' end of the sense strand.

29. The dsRNA agent according to claim 27, wherein the ligand is located at the 5' end of the sense strand.

30. A dsRNA agent according to any one of claims 26 to 29, wherein the ligand comprises an ASGPR ligand.

31. A dsRNA agent according to any one of claims 26 to 29, wherein the ligand is a lipophilic group.

32. Ligand is C 10～30 The dsRNA agent according to claim 31, wherein the group is an aliphatic group.

33. C 10～30 The aliphatic group is C 10～30 The dsRNA agent according to claim 32, wherein the alkyl group is an alkyl group.

34. C 10～30 The dsRNA agent according to claim 33, wherein the alkyl group is a linear or branched tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group.

35. The dsRNA agent according to claim 27, wherein the ligand is conjugated to the non-terminal nucleotide of the sense strand.

36. The dsRNA agent according to claim 35, wherein the ligand is conjugated to the 2' position of the non-terminal nucleotide of the sense strand, and optionally conjugated to one of positions 5, 6, 7, or 8 of the sense strand, counting from the 5' end.

37. The dsRNA agent according to any one of claims 26 to 36, wherein the ligand comprises a debasalized nucleotide, and optionally the debasalized nucleotide is an inverted nucleotide ligated to the dsRNA agent chain via a 5'→5' or 3'→3' ligation.

38. A dsRNA agent according to any one of claims 26 to 37, wherein the ligand is attached to the 3' end of the sense strand.

39. The dsRNA agent according to claim 38, wherein the ligand is attached to the 3' end of the sense strand via a 3'→3' ligation site.

40. A dsRNA agent according to any one of claims 1 to 39, wherein the dsRNA comprises two ligands.

41. The dsRNA according to claim 40, wherein the sense strand comprises a first ligand attached to the 3' end of the sense strand and a second ligand attached to the 5' end of the sense strand.

42. The dsRNA according to claim 41, wherein the first ligand comprises a debasalized nucleotide, the second ligand comprises an ASGPR ligand, and optionally the debasalized nucleotide is an inverted nucleotide ligated to the sense strand via a 3'→3' linkage.

43. A dsRNA agent according to any one of claims 1 to 42, comprising at least two phosphorothioate nucleotide linkages.

44. The dsRNA agent according to any one of claims 1 to 43, wherein the sense strand comprises at least two phosphorothioate nucleotide junctions between the first five nucleotides counted from the 5' end of the sense strand.

45. The dsRNA agent according to any one of claims 1 to 44, wherein the antisense strand includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 5' end of the antisense strand, and also includes at least two phosphorothioate internucleotide junctions between the first five nucleotides counted from the 3' end of the antisense strand.

46. A dsRNA agent according to any one of claims 1 to 45, wherein the dsRNA has a double-stranded region of 18 to about 25 base pairs.

47. A dsRNA agent according to any one of claims 1 to 46, wherein the sense strand is 18 to 23 nucleotides long.

48. A dsRNA agent according to any one of claims 1 to 47, wherein the antisense strand is 18 to 25 nucleotides long.

49. A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long, contains a 2'-fluoronucleotide at position 10 counting from the 5' end, and contains a 2'-fluoronucleotide at position 9 or 11 counting from the 5' end, and the antisense strand is 18 to 25 nucleotides long, contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end, (i) The antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16 counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. The aforementioned dsRNA agent.

50. A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long and contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and the antisense strand is 18 to 25 nucleotides long and contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand. (i) The antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand, and contains a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) The antisense strand contains a 2'-deoxynucleotide at position 14 or 16 counting from the 5' end of the antisense strand, and the sense strand contains a nucleotide other than a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. The aforementioned dsRNA agent.

51. A dsRNA agent according to any one of claims 1 to 50, comprising a phosphate mimic at the 5' end of the antisense strand.

52. The dsRNA agent according to claim 51, wherein the phosphate mimic is a 5'-E-vinyl phosphonate.

53. Phosphate mimic, structure: A dsRNA agent according to claim 52, comprising a 5'-cyclopropylphosphonate, wherein * represents binding to the C5 position of the nucleotide at the 5' end.

54. The remaining nucleotides in the sense strand (i.e., nucleotides at positions not otherwise specified) are either unmodified nucleotides or modified nucleotides, optionally 2'-OMe, 2'-F, 2'-H, and 2'-OC. 10～30 A modified nucleotide selected from the group consisting of aliphatic groups, except for 2'-OC. 10～30 A dsRNA agent according to any one of claims 1 to 53, wherein the modified nucleotide is one or less aliphatic group.

55. The remaining nucleotides in the sense strand (i.e., nucleotides at positions not otherwise specified) are 2'-OMe, 2'-F, 2'-H, and 2'-OC. 10～30 A modified nucleotide selected from the group consisting of aliphatic groups, except for 2'-OC. 10～30 A dsRNA agent according to any one of claims 1 to 54, wherein the modified nucleotide is one or less aliphatic group.

56. The dsRNA agent according to any one of claims 1 to 55, wherein the remaining nucleotides in the antisense strand (i.e., nucleotides at positions not otherwise specified) are unmodified nucleotides or modified nucleotides, optionally selected from the group consisting of 2'-OMe, 2'-F, 2'-H, GNA, and 3'-RNA, wherein the 3'-RNA is optionally 3'-OH, and there is one or fewer modified nucleotides that are GNA or 3'-RNA.

57. The dsRNA agent according to any one of claims 1 to 56, wherein the remaining nucleotides in the antisense strand (i.e., nucleotides at positions not otherwise specified) are modified nucleotides selected from the group consisting of 2'-OMe, 2'-F, 2'-H, GNA, and 3'-RNA, the 3'-RNA is optionally 3'-OH, provided that there is one or fewer modified nucleotides that are GNA or 3'-RNA.

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