Endosomal cleavable linker

Abstract

The present disclosure relates generally to cleavable linkers and uses thereof.SOLUTION: A multi-targeted molecule comprising at least two effector molecules, wherein said effector molecules are linked together via an endosomal cleavable linker or a protease cleavable linker, wherein said linker is a carbohydrate linker, and wherein said linker is cleaved at least 1.25 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 447,786, filed January 18, 2017, the contents of which are incorporated herein by reference in their entirety. [Technical Field]

[0002] The present disclosure relates generally to cleavable linkers and uses thereof. [Background technology]

[0003] There is a need in the art for linkers that undergo cleavage, e.g., endosomal cleavage and / or are protease cleavable, and the present disclosure provides some solutions to that need. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, provided herein are cleavable linkers, such as endosomal-cleavable and / or protease-cleavable linkers. In some embodiments, the cleavable linkers described herein can be included in a larger linker. In some embodiments, the cleavable linker is a carbohydrate linker that is cleaved at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions). In some embodiments, the cleavable linker is cleaved less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% faster in blood (or under in vitro conditions selected to mimic extracellular conditions) than in cells (or under in vitro conditions selected to mimic intracellular conditions). In some embodiments, the linker is cleaved at least 10 times faster, preferably at least 100 times faster, within the target cell or under first reference conditions (e.g., which can be selected to mimic or represent intracellular conditions) than in the subject's blood or serum or under second reference conditions (e.g., which can be selected to mimic or represent conditions found in blood or serum). Exemplary compounds for generating cleavable linkers of the invention are described in Schemes 7-11 in Examples 7-11. In some embodiments, the cleavable linker is a linker shown in Figures 1-6.

[0005] Without limitation, the cleavable linkers described herein can be used for any molecule that requires cleavage in the lysosomal compartment. The cleavable linkers described herein can be particularly useful in prodrug approaches, particularly for hydrophobic conjugates, that link endosomally cleavable agents or any other agents that may need to be activated or released in the lysosomal compartment. Thus, the linkers described herein can be used for multiple applications, including, but not limited to, multi-targeted molecules and prodrugs.

[0006] In one aspect, provided herein is a prodrug conjugate comprising a cleavable linker as described herein.

[0007] In another aspect, provided herein is a conjugate comprising an endosomotropic agent attached to a ligand via a cleavable linker described herein.

[0008] In yet another aspect, provided herein is a conjugate comprising a nucleic acid-based effector molecule conjugated to a ligand via a cleavable linker as described herein. Without limitation, any nucleic acid-based effector molecule capable of modulating target gene expression can be included in the conjugate.

[0009] In yet another aspect, the present invention provides a multi-target molecule.Generally, the multi-target molecule comprises at least two nucleic acid-based effector molecules linked together by the cleavable linker described herein.Without limitation, any nucleic acid-based effector molecule that can regulate the gene expression of target can be included in the multi-target molecule disclosed herein.

[0010] "Nucleic acid-based effector molecule" refers to a modified or unmodified single-stranded or double-stranded nucleic acid molecule capable of regulating gene expression of a target gene. Exemplary nucleic acid-based effector molecules capable of regulating gene expression of a target gene include, but are not limited to, double-stranded and single-stranded RNA interfering agents (such as siRNAs and shRNAs, and what are referred to herein as dsRNA agents), antisense oligonucleotides, microRNAs, anti-microRNAs or antimirs, supermirs, antagomirs, ribozymes, triplex-forming oligonucleotides, decoy oligonucleotides, RNA activators, U1 adaptors, guide RNAs (gRNAs) for CRISPR Cas, and the like.

[0011] It should be noted that the at least two effector molecules are two separate effector molecules. In other words, the at least two effector molecules do not overlap with each other. Therefore, the multi-target molecule disclosed herein is different from a molecule in which one effector molecule is directed to two different targets, such as a double-stranded effector molecule in which each strand is directed to a different target, or an effector molecule in which at least a portion of the sequence is complementary to or can hybridize with two different target sequences.

[0012] In some embodiments, the multitargeting molecule or the effector molecule in the multitargeting molecule does not regulate non-specific gene expression by two different mechanisms, for example, the multitargeting molecule or the effector molecule in the multitargeting molecule does not regulate gene expression by RNA interference and targeting the seed region of a microRNA.

[0013] In some embodiments, each nucleic acid-based effector molecule in a multi-target molecule can regulate gene expression of a target nucleic acid. Without limitation, each effector molecule in a multi-target molecule can be directed to the same target gene, different target genes, different locations of the same target gene, or different transcripts of the same target gene. Furthermore, it is noted that the effector molecules included in the multi-target molecules disclosed herein can include any of the nucleic acid modifications, motifs, or structures described herein.

[0014] Furthermore, effector molecules included in the multitargeting molecules described herein have comparable gene expression modulating activity compared to the gene expression modulating activity of the effector molecule when it is not part of the multitargeting molecule. In other words, an effector molecule has similar gene expression modulating activity when it is part of a multitargeting molecule disclosed herein compared to when it is not part of a multitargeting molecule. In some embodiments, effector molecules included in the multitargeting molecules described herein can independently modulate gene expression of their respective target nucleic acids by at least 50% (e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) compared to their modulation of gene expression when not part of a multitargeting molecule. In some embodiments, one effector molecule in a multitargeting molecule modulates gene expression at a higher level than other effector molecules in the multitargeting molecule. In some embodiments, the at least two effector molecules in a multitargeting molecule modulate gene expression at similar levels (e.g., within 10%, 7.5%, 5%, 2.5% or less of each other).

[0015] The present inventors have found that multi-targeting molecules conjugated with a ligand are particularly effective in regulating gene expression. Therefore, in some embodiments, at least one ligand is conjugated to the multi-targeting molecule. Therefore, a multi-targeting molecule conjugated with at least one ligand is also referred to herein as a "conjugated multi-targeting molecule." Without limitation, the ligand may be present on any of the effector molecules in the multi-targeting molecule. Furthermore, the ligand may be present at any position on the effector molecule and / or the multi-targeting molecule. For example, the ligand may be conjugated to the 5'-end, 3'-end, internal position, or a combination thereof, of the effector molecule in the multi-targeting molecule. In some embodiments, at least two ligands are conjugated to the multi-targeting molecule. The at least two ligands may be the same, different, or any combination of the same and different. The two ligands may be independently conjugated to any position in the multi-targeting molecule. In some embodiments, at least two effector molecules in the multi-targeting molecule are bound to at least one ligand. Without intending to be bound by any particular theory, the ligand may improve the delivery or pharmacokinetic profile of the conjugated multitargeting molecule.

[0016] In some embodiments, the cleavable linkers described herein may be used in the multi-targeting single entity conjugates described in PCT Application No. PCT / US2016 / 042498, filed July 15, 2016, the contents of which are incorporated herein by reference in their entirety. [Brief explanation of the drawings]

[0017] [Figure 1-1] 1 shows exemplary cleavable linker structures. 2 shows various carbohydrate and non-carbohydrate linkers used in the bis(siRNA) designs described in Example 28, Table 1. [Figure 1-2] Continued from Figure 1-1. [Figure 2-1]1 shows exemplary cleavable linker structures; 2 shows exemplary monosaccharides of various modified carbohydrates, such as galactose, galactosamine, glucose, glucosamine, mannose, and mannosamine derivatives; 3 shows n=1-12, and m=1-12. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 2-6] Continued from Figure 2-5. [Figure 2-7] Continued from Figure 2-6. [Figure 2-8] Continued from Figure 2-7. [Figure 2-9] Continued from Figure 2-8. [Figure 3-1] 1 shows exemplary cleavable linker structures; 2 shows exemplary monosaccharides of various modified carbohydrates, such as galactose, galactosamine, glucose, glucosamine, mannose, and mannosamine derivatives; 3 shows n=1-12, and m=1-12. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 3-4] Continued from Figure 3-3. [Figure 3-5] Continued from Figure 3-4. [Figure 3-6] Continued from Figure 3-5. [Figure 4-1] 1 shows exemplary cleavable linker structures. 1 shows exemplary disaccharides or trisaccharides of various modified carbohydrates, such as galactose, galactosamine, glucose, glucosamine, mannose, and mannosamine derivatives. 1 is 1-12, and m is 1-12. [Figure 4-2] Continued from Figure 4-1. [Figure 5-1] 1 shows exemplary cleavable linker structures. 1 shows exemplary disaccharides or trisaccharides of various modified carbohydrates, such as galactose, galactosamine, glucose, glucosamine, mannose, and mannosamine derivatives. 1 is 1-12, and m is 1-12. [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 5-4] Continued from Figure 5-3. [Figure 5-5] Continued from Figure 5-4. [Figure 5-6] Continued from Figure 5-5. [Figure 5-7] Continued from Figure 5-6. [Figure 5-8] Continued from Figure 5-7. [Figure 5-9] Continued from Figure 5-8. [Figure 5-10] Continued from Figure 5-9. [Figure 5-11] Continued from Figure 5-10. [Figure 5-12] Continued from Figure 5-11. [Figure 5-13] Continued from Figure 5-12. [Figure 6] 1 shows the structure of an exemplary cleavable linker. 2 shows an exemplary protease-cleavable linker. [Figure 7-1] 1 is a photograph of a gel mobility assay showing the degradation of bis(siRNA) compounds containing exemplary linkers, AM-106 to AM-129, in rats. [Figure 7-2] Continued from Figure 7-1. [Figure 8-1] 1 is a photograph of a gel mobility assay showing the degradation of bis(siRNA) compounds containing exemplary linkers, AM-130 to AM-147, in rats. [Figure 8-2] Continued from Figure 8-1. [Figure 9] 1 is a photograph of a gel mobility assay showing the degradation of bis(siRNA) compounds containing exemplary linkers, AM-148 to AM-154, in rats. [Figure 10] 1 is a photograph of a gel mobility assay showing the degradation of bis(siRNA) compounds containing exemplary linkers, AM-155 to AM-161, in rats. DETAILED DESCRIPTION OF THE INVENTION

[0018] It is to be understood that both the foregoing general description and the following detailed description are intended to be exemplary and explanatory, but are not limiting of the invention, as defined in the claims. As used herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "comprising" and other forms, such as "comprises" and "includes," is not limiting. Also, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include two or more subunits, unless specifically stated otherwise.

[0019] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby incorporated by reference in their entirety for all purposes.

[0020] Effector molecules Those skilled in the art are well aware that double-stranded oligonucleotides containing 20-23, especially 21 base pairs of double helix structure are recognized as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, it has also been found that other double-stranded oligonucleotides, shorter or longer than this, can also be effective.

[0021] As used herein, the term "siRNA" refers to a substance that mediates targeted cleavage of RNA transcripts. These substances bind to a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Substances that are effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or iRNA agents. As used herein, the term siRNA includes microRNAs and pre-microRNAs. As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0022] A double-stranded oligonucleotide comprises two oligonucleotide strands that are sufficiently complementary to hybridize and form a double-helical structure. Typically, the double-helical structure is 15-35, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded oligonucleotides, such as 25-30 base pairs in length, are preferred. In some embodiments, shorter double-stranded oligonucleotides, such as 10-15 base pairs in length, are preferred. In another embodiment, the double-stranded oligonucleotide is at least 21 nucleotides in length.

[0023] In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity that is complementary to at least a portion of a target sequence, and the double-helical region is 14 to 30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14 to 30, more commonly 18 to 25, even more commonly 19 to 24, and most commonly 19 to 21 nucleotides in length.

[0024] As used herein, the term "antisense strand" refers to an oligomeric compound that is substantially complementary or 100% complementary to the intended target sequence. The term "antisense strand" includes the antisense region of both oligomeric compounds formed from two separate strands, as well as unimolecular compounds that can form hairpin or dumbbell structures. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0025] The phrase "sense strand" refers to an oligomeric compound that has all or part of the same nucleoside sequence as a target sequence, such as a sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0026] In some embodiments, the double-stranded region of the double-stranded oligonucleotide is at least equal to or has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotide pairs.

[0027] In some embodiments, the antisense strand of the double-stranded oligonucleotide is at least equal to or has a length of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotide pairs.

[0028] In some embodiments, the sense strand of the double-stranded oligonucleotide is at least equal to or has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotide pairs.

[0029] In some embodiments, one strand has at least one stretch of 1 to 10 single-stranded nucleotides within the double-stranded region. A "single-stranded nucleotide stretch within a double-stranded region" means that there is at least one nucleotide within the double-stranded region that does not base pair with another nucleotide. If the stretch of single-stranded nucleotides is within the double-stranded region, there may be at least one nucleotide base pair at both ends of the single-stranded stretch. If it is at the end of the double-stranded region, the stretch of single-stranded nucleotides may be a single-stranded overhang. The stretch of single-stranded nucleotides within the double-stranded region may be in the form of a bulge or one or more mismatched nucleotides. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. If both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides can be opposite each other (e.g., a mismatched stretch), or they can be positioned such that the second strand does not contain a non-base-paired nucleotide opposite the single-stranded oligonucleotide of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides of either end, e.g., 8, 7, 6, 5, 4, 3, or 2 nucleotides from either the 5' or 3' end of the region of complementarity between the two strands.

[0030] Hairpin and dumbbell-type oligonucleotides can have a double-helical region of at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or more. The double-helical region can be up to 200, 100, or 50 nucleotide pairs in length. In some embodiments, the double-helical region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0031] In some embodiments, the nucleic acid-based effector molecule is a hairpin oligonucleotide, which may have a single-stranded overhang or terminal unpaired region, in some embodiments, at the 3', and in some embodiments, at the antisense side of the hairpin. In some embodiments, the overhang is 1-4, more typically 2-3 nucleotides in length. Hairpin oligonucleotides capable of inducing RNA interference are also referred to herein as "shRNAs."

[0032] In some embodiments, two oligonucleotide strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under the conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0033] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary in various circumstances; the "stringent conditions" under which an antisense compound will hybridize to its target sequence are determined by the nature and composition of the antisense compound and the assay in which it is tested.

[0034] It is understood in the art that the incorporation of nucleotide affinity modifications can achieve a greater number of mismatches compared to unmodified oligonucleotides.Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between oligonucleotides and target nucleic acids, for example, by determining melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA and RNA:RNA double helices by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443).

[0035] In some embodiments, the effector molecule is a double-stranded RNA (dsRNA) agent, i.e., siRNA, for inhibiting expression of a target gene. It is understood that dsRNA, siRNA, and oligonucleotide may be used interchangeably unless otherwise specified. A dsRNA agent includes a sense strand and an antisense strand, each having 14 to 40 nucleotides. A dsRNA agent has the formula (I): [ka] It is expressed by:

[0036] In Formula (I), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In some embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification.

[0037] C1 is a thermolabile nucleotide located at a position opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is located at a position on the sense strand that pairs with nucleotides 2-8 of the 5' end of the antisense strand. The C1 nucleotide carries a thermolabile modification, which may include an abasic modification; a mismatch with the opposite nucleotide in the duplex; and a sugar modification, such as a 2'-deoxy modification or an acyclic nucleotide, e.g., an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In some embodiments, C1 is: i) a mismatch with the opposite nucleotide of the antisense strand; ii) [ka] an abasic modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase; and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In some embodiments, the thermolabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermolabilizing modification in C1 is GNA or [ka] is.

[0038] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that confers steric bulk to the nucleotide equal to or less than that of a 2'-OMe modification. The modification can be at the 2' position of the ribose sugar of the nucleotide, or it can be a non-ribose nucleotide, acyclic nucleotide, or modification to the backbone of the nucleotide, which is similar or equivalent to the 2' position of the ribose sugar and confers steric bulk to the nucleotide equal to or less than that of a 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In some embodiments, T1 is DNA. In some embodiments, T1' is DNA, RNA, or LNA. In some embodiments, T2' is DNA or RNA. In some embodiments, T3' is DNA or RNA.

[0039] n 1 , n 3 and q 1 are independently 4 to 15 nucleotides in length.

[0040] n 5 , q 3 and q 7 are independently 1 to 6 nucleotides in length.

[0041] n 4 , q 2 and q 6 are independently 1 to 3 nucleotides in length.

[0042] q 5 are independently 0 to 10 nucleotides in length.

[0043] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0044] Or, n 4 is 0 to 3 nucleotides in length.

[0045] In some embodiments, n 4can be 0. In one example, n 4 is 0, and q 2 and q 6 is 1. In another example, n 4 is 0, and q 2 and q 6 is 1, which has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0046] In some embodiments, n 4 , q 2 and q 6 are 1 respectively.

[0047] In some embodiments, n 2 , n 4 , q 2 , q 4 and q 6 are 1 respectively.

[0048] In some embodiments, the sense strand is 19 to 22 nucleotides in length, and 4 When C1 is 1, C1 is at positions 14 to 17 of the 5' end of the sense strand.

[0049] In some embodiments, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and 6 is equal to 1.

[0050] In some embodiments, T1' begins at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and 2 is equal to 1.

[0051] In some embodiments, T1' and T3' are separated by a length of 11 nucleotides (i.e., not counting the T1' and T3' nucleotides.

[0052] In some embodiments, T1' is at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and 2 is equal to 1, and the non-ribose, acyclic, or backbone 2'-position modification(s) impart less steric bulk than the 2'-OMe ribose modification.

[0053] In some embodiments, T3' is at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and 6 is equal to 1, and non-ribose, acyclic, or backbone 2'-position modifications impart less steric bulk than 2'-OMe ribose modifications.

[0054] In some embodiments, T1 is at the cleavage site of the sense strand. In one example, the sense strand is 19-22 nucleotides in length and 2 When T1 is 1, T1 is at position 11 from the 5' end of the sense strand.

[0055] In some embodiments, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at positions 6-10 from the 5' end of the antisense strand, and 4 is 1.

[0056] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0057] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0058] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0059] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0060] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0061] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein the sense strand has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0062] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0063] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1, wherein the sense strand has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) and the antisense strand has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0064] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the dsRNA agents of the invention are modified.

[0065] In some embodiments, each of the sense and antisense strands of a dsRNA agent is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0066] In some embodiments, the sense and antisense strands of a dsRNA agent each contain at least two different modifications.

[0067] In some embodiments, the dsRNA agent of Formula (I) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the dsRNA agent of Formula (I) comprises a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In another example, the dsRNA agent has a 5' overhang at the 5' end of the sense strand.

[0068] In some embodiments, a dsRNA agent of the invention does not include any 2'-F modifications.

[0069] In some embodiments, a dsRNA agent of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, an effector molecule of the invention comprises 9 or 10 2'-F modifications.

[0070] In some embodiments, the sense strand and / or antisense strand of a dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0071] In some embodiments, the sense and antisense strands of the dsRNA agent each have 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0072] In some embodiments, the nucleotide at position 1 of the 5' end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, and dT. In some embodiments, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0073] In some embodiments, the antisense strand of a dsRNA agent of the present invention is 100% complementary to the target RNA, hybridizes therewith, and inhibits its expression by RNA interference. In other embodiments, the antisense strand of a dsRNA agent of the present invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0074] In one aspect, the present invention relates to a dsRNA agent capable of inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, where the at least one thermolabile nucleotide is located at or near the site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide is located at positions 14 to 17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids with less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids with less than sterically demanding 2'-OMe modifications are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0075] In certain embodiments, a dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23 and 2'F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0076] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0077] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a desoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23 and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0078] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21 and 2'-F modifications at positions 7, 9, 11, 13, and 15; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23 and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0079] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21 and 2'-F modifications at positions 10 and 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0080] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0081] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21 and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and desoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a four nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0082] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21 and 2'-F modifications at positions 7 and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23 and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0083] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21 and 2'-F modifications at positions 7 and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23 and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0084] In another specific embodiment, the dsRNA agent of the invention comprises: (a) a sense strand having: (i) 19 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19 and 2'-F modifications at positions 5 and 7 to 9; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21 and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end) wherein the dsRNA agent has a two nucleotide overhang on the 3' end of the antisense strand and a blunt end on the 5' end of the antisense strand.

[0085] In one embodiment, a dsRNA agent described herein further comprises a thermodestabilizing modification at position 7, counting from the 5' end of the antisense strand, at position 15, counting from the 5' end of the sense strand, at position 21, counting from the 5' end of the sense strand, or a combination thereof.

[0086] In one aspect, the present invention relates to a dsRNA agent capable of inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, where the at least one thermolabile nucleotide is located at or near the site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). For example, when the sense strand is 21 nucleotides long, the thermolabile nucleotide is located at positions 14 to 17 of the 5' end of the sense strand. The antisense strand includes two modified nucleic acids that are less than sterically demanding 2'-OMe modified, separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0087] In one embodiment, the sense strand of the dsRNA agent further comprises an endonuclease-sensitive modified nucleotide at the cleavage site of the sense strand. In one example, the endonuclease-sensitive modified nucleotide is at position 11 from the 5' end of the sense strand.

[0088] In some embodiments, the effector molecule is a microRNA. MicroRNAs (miRNAs or mirs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals but are not translated into proteins. Pre-microRNAs are processed into miRNAs. Processed microRNAs are single-stranded, approximately 17-25 nucleotide (nt) RNA molecules that are incorporated into RNA-induced silencing complexes (RISCs) and have been identified as key regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in regulating gene expression by binding to the 3'-untranslated regions of specific mRNAs. RISCs mediate downregulation of gene expression through translational inhibition, transcript cleavage, or both. RISCs are also involved in widespread transcriptional silencing within the nuclei of eukaryotic organisms.

[0089] In some embodiments, the effector molecule is a ribozyme. Ribozymes are oligonucleotides with specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci US A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). At least six basic types of natural enzymatic RNAs are currently known. Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding is achieved by the target binding portion of the enzymatic nucleic acid being held in close proximity to the enzymatic portion of the molecule responsible for cleaving the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA through complementary base pairing, and once bound to the appropriate site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA would destroy its ability to direct synthesis of the encoded protein. After an enzymatic nucleic acid binds and cleaves its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0090] Methods for producing ribozymes targeted to any target sequence are known in the art. Ribozymes can be designed as described in International Patent Applications WO 93 / 23569 and WO 94 / 02595 (each of which is expressly incorporated herein by reference) and synthesized for testing in vitro and in vivo as described in these documents.

[0091] In some embodiments, the effector is an aptamer. Aptamers are nucleic acid or peptide molecules that bind to a specific molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA or RNA aptamers have been successfully produced that bind many different entities, from large proteins, to small organic molecules. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5 (2000). Aptamers can be RNA- or DNA-based. Generally, aptamers are engineered to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, through multiple rounds of in vitro selection or, equivalently, SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific for the same target. Furthermore, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers," which contain consensus sequences obtained from comparing two or more known aptamers to a given target.

[0092] Because transcription factors recognize their relatively short binding sequences even in the absence of surrounding genomic DNA, short oligonucleotides with consensus binding sequences of specific transcription factors can be used as tools to manipulate gene expression in living cells.This method involves the intracellular delivery of such "decoy oligonucleotides," which are then recognized and bound by target factors.Therefore, in some embodiments, the effector molecule is a decoy oligonucleotide.

[0093] The occupancy of the DNA binding site of transcription factor by decoy prevents transcription factor from subsequently binding to the promoter region of target gene.Decoy can be used as a therapeutic agent to inhibit the expression of the gene that is activated by transcription factor, or to up-regulate the gene that is suppressed by the binding of transcription factor.An example of the use of decoy oligonucleotide can be found in Mann et al., J.Clin.Invest., 2000,106:1071-1075, which is expressly incorporated herein by reference in its entirety.

[0094] In some embodiments, the effector molecule is an miRNA mimic. MicroRNA mimics (miRNA mimics) represent a class of molecules that can be used to mimic the gene-regulatory activity of one or more miRNAs. Thus, the term "microRNA mimic" refers to a synthetic non-coding RNA (i.e., a miRNA not obtained by purification from a source of endogenous miRNA) that can enter the RNAi pathway and regulate gene expression. miRNA mimics can be designed as mature molecules (e.g., single-stranded) or mimic precursors (e.g., pri- or pre-miRNAs). In one design, miRNA mimics are double-stranded molecules (e.g., having a double-helical region of about 16 to about 31 nucleotides in length) that contain one or more sequences that share identity with the mature strand of a given miRNA. Double-stranded miRNA mimics have designs similar to those described above for double-stranded oligonucleotides.

[0095] In some embodiments, the miRNA mimic comprises a double-helical region of 16-31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide) and all of the Cs and Us; the antisense strand modifications may include 2'F modifications of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages coupled with a two-nucleotide 3' overhang.

[0096] In some embodiments, the effector molecule is a supermir. A supermir refers to, for example, a single-stranded, double-stranded, or partially double-stranded oligonucleotide having a nucleotide sequence that is substantially identical to a miRNA and antisense to its target. This term also includes oligonucleotides that contain at least one non-natural moiety that function similarly. In a preferred embodiment, a supermir does not contain a sense strand, and in another preferred embodiment, a supermir does not self-hybridize to a significant extent. A supermir that is the subject of the present invention may have secondary structure, but is substantially single-stranded under physiological conditions. A substantially single-stranded supermir is single-stranded to the extent that less than about 50% (e.g., less than about 40%, 30%, 20%, 10%, or 5%) of the supermir forms a duplex with itself. Supermirs can comprise hairpin segments, e.g., sequences that self-hybridize, preferably at the 3' end, to form a double-helical region, e.g., a double-helical region of at least 1, 2, 3, or 4, preferably less than 8, 7, 6, or 5, nucleotides, e.g., 5 nucleotides. The double-helical region can be linked by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6 dT, e.g., modified dT. In another embodiment, a supermir forms a duplex with a shorter oligo, e.g., 5, 6, 7, 8, 9, or 10 nucleotides in length, e.g., at one or both of the 3' and 5' ends, or at one end and a non-end or in the middle of the supermir.

[0097] In some embodiments, the effector molecule is an anti-mir. The terms "anti-mir," "microRNA inhibitor," or "miR inhibitor" are synonymous and refer to an oligonucleotide or modified oligonucleotide that interferes with the activity of a specific miRNA. Inhibitors can take a variety of forms, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs. Generally, microRNA inhibitors contain one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the targeted miRNA. Furthermore, miRNA inhibitors may also contain additional sequences located 5' and 3' of the sequence that is reverse-complementary to the mature miRNA. The additional sequences may be reverse-complementary to sequences adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequences may be arbitrary sequences (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences are arbitrary sequences capable of forming a hairpin. Therefore, in some embodiments, the reverse-complementary sequence of the miRNA has a hairpin structure on the 5' and 3' ends. When the microRNA inhibitor is double-stranded, it may contain mismatches between nucleotides in the opposite strands. Furthermore, the microRNA inhibitor may be linked to a conjugate moiety to facilitate the uptake of the inhibitor into cells.

[0098] MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13:723-730 (2007), and WO 2007 / 095387 and WO 2008 / 036825 (each of which is incorporated herein by reference in its entirety). One skilled in the art can select a sequence from a database of the desired miRNA and design an inhibitor useful in the methods disclosed herein.

[0099] In some embodiments, the effector molecule is an antagomir. An antagomir is an RNA-like oligonucleotide with various modifications for pharmacological properties such as RNAse protection and improved tissue and cellular uptake. They differ from normal RNA, for example, by complete 2'-O-methylation of the sugars, phosphorothioate intersugar linkages, and a cholesterol moiety, for example, at the 3' end. In a preferred embodiment, an antagomir contains 2'-O-methyl modifications at all nucleotides, a cholesterol moiety at the 3' end, two phosphorothioate intersugar linkages at the first two positions of the 5' end, and four phosphorothioate linkages at the 3' end of the molecule. An antagomir can be used to efficiently silence endogenous miRNAs by forming a duplex containing the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. One example of antagomir-mediated miRNA silencing is the silencing of miR-122, as described in Krutzfeldt et al., Nature, 2005, 438:685-689, which is expressly incorporated herein by reference in its entirety.

[0100] In some embodiments, the effector molecule is a U1 adaptor. The U1 adaptor is a bifunctional oligonucleotide that blocks the poly(A) site and has a targeting domain complementary to a site in the terminal exon of the target gene and a "U1 domain" that binds to the U1 smaller nuclear RNA component of the U1 snRNP. See, for example, International Patent Application Publication No. WO 2008 / 121963 and Goraczniak, et al., 2008, Nature Biotechnology, 27(3), 257-263 (each of which is expressly incorporated herein by reference in its entirety). The U1 snRNP is a ribonucleoprotein complex that functions primarily to direct the initial steps in spliceosome formation by binding to pre-mRNA exon-intron boundaries (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant MoI Biol 49:77-95).

[0101] In some embodiments, the U1 adaptor comprises at least one annealing domain (targeting domain) linked to at least one effector domain (U1 domain), where the annealing domain hybridizes to a target gene sequence and the effector domain hybridizes to the U1 snRNA of the U1 snRNP. In some embodiments, the U1 adaptor comprises one annealing domain. In some embodiments, the U1 adaptor comprises one effector domain.

[0102] Without intending to be bound by any particular theory, the annealing domain is typically about 10 to about 50 nucleotides in length, more typically about 10 to about 30 nucleotides or about 10 to about 20 nucleotides. In some preferred embodiments, the annealing domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides in length. The annealing domain may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or more preferably 100% complementary to the target gene. In some embodiments, the annealing domain hybridizes to a target site within the 3'-terminal exon of the pre-mRNA, including the terminal coding region and 3' UTR and polyadenylation signal sequence (e.g., via a polyadenylation site). In other embodiments, the target sequence is within about 500 base pairs, about 250 base pairs, about 100 base pairs, or about 50 base pairs of the poly(A) signal sequence of the pre-mRNA. In some embodiments, the annealing domain contains 1, 2, 3, or 4 mismatches with the target gene sequence.

[0103] The effector domain can be about 8 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 10 to about 15 nucleotides in length. The U1 domain can hybridize to the U1 snRNA, particularly to the 5' end, more particularly to nucleotides 2 to 11. In another embodiment, the U1 domain is fully complementary to nucleotides 2 to 11 of the endogenous U1 snRNA. In some embodiments, the U1 domain comprises a nucleotide sequence selected from the group consisting of 5'-GCCAGGUAAGUAU-3', 5'-CCAGGUAAGUAU-3', 5'-CAGGUAAGUAU-3', 5'-CAGGUAAGU-3', 5'-CAGGUAAG-3', and 5'-CAGGUAA-3'. In some embodiments, the U1 domain comprises the nucleotide sequence 5'-CAGGUAAGUA-3'. Without intending to be bound by any particular theory, increasing the length of the Ul domain to include base pairing to stem 1 and / or position 1 of the Ul snRNA improves the affinity of the Ul adaptor for the Ul snRNP.

[0104] The annealing and effector domains of the Ul adapter can be joined such that the effector domain is at the 5' and / or 3' end of the annealing domain. The two domains can be joined such that the 3' end of one domain is joined to the 5' end of the other domain, or the 3' end of one domain is joined to the 3' end of the other domain, or the 5' end of one domain is joined to the 5' end of the other domain. The annealing and effector domains can be joined directly to each other or via a nucleotide-based or non-nucleotide-based linker. If the linker is nucleotide-based, the linker can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15, up to 20, or up to 25 nucleotides.

[0105] In some embodiments, the linker between the annealing domain and the effector domain is a cleavable linker as described herein. In some embodiments, the linker between the annealing domain and the effector domain is multivalent, such as trivalent, tetravalent, or pentavalent. Without intending to be bound by theory, multivalent linkers can be used to link a single annealing domain together with multiple adaptor domains.

[0106] It should be understood that the U1 adaptor can include any of the oligonucleotide modifications described herein. Exemplary modifications for the U1 adaptor include those that improve annealing affinity, specificity, bioavailability in cells and organisms, cellular and / or nuclear transport, stability, and / or resistance to degradation.

[0107] Recent studies have shown that dsRNA can also activate gene expression, a mechanism known as "small RNA-induced gene activation" or RNAa (activating RNA). See, for example, Li, LC et al. Proc Natl Acad Sci USA (2006), 103(46):17337-42 and Li LC (2008). "Small RNA-Mediated Gene Activation." RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. ISBN 978-1-904455-25-7. It has been shown that dsRNA targeting gene promoters induces strong transcriptional activation of associated genes. Endogenous miRNAs that trigger RNAa have also been found in humans. See E. Nature (2007). 448(7156):855-858.

[0108] Another surprising observation is that gene activation by RNAa is long-lasting.The induction of gene expression has been found to last for more than 10 days.The long-term effect of RNAa may be due to epigenetic changes at dsRNA target sites.In some embodiments, RNA activators can increase gene expression.In some embodiments, increased gene expression inhibits survival, growth, development and / or reproduction.

[0109] Thus, in some embodiments, the effector molecule is an activator RNA.

[0110] In some embodiments, the effector molecule is a triplex-forming oligonucleotide (TFO).Recent studies have shown that triplex-forming oligonucleotides can be designed that can recognize and bind to polypurine / polypyrimidine regions in double-stranded DNA in a sequence-specific manner.These recognition rules are outlined in Maher III, LJ, et al., Science (1989) vol.245, pp 725-730; Moser, HE, et al., Science (1987) vol.238, pp 645-630; Beal, PA, et al., Science (1992) vol.251, pp 1360-1363; Conney, M., et al., Science (1988) vol.241, pp 456-459 and Hogan, ME, et al., EP Publication 375408. Modifications of oligonucleotides, such as the introduction of intercalating agents and intersugar bond replacement, and optimization of binding conditions (pH and cation concentration) can help overcome inherent obstacles to TFO activity, such as charge repulsion and instability, and it has recently been shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review, see Seidman and Glazer, J Clin Invest 2003;1 12:487-94). Generally, triplex-forming oligonucleotides have the following sequence correspondences: Oligo 3'-AGGT Double-stranded 5'-AGCT Double-stranded 3'-TCGA

[0111] However, it has been shown that A-AT and G-GC triplets have the greatest triple helix stability (Reither and Jeltsch, BMC Biochem, 2002, Sept. 12, Epub). The same authors demonstrated that TFOs designed according to the A-AT and G-GC rules do not form nonspecific triplexes, indicating that triplex formation is indeed sequence-specific.

[0112] Thus, for any given sequence, triplex-forming sequences can be devised. Triplex-forming oligonucleotides are preferably at least 15, more preferably 25, even more preferably 30 or more nucleotides in length, up to 50 or 100 nucleotides.

[0113] The formation of triple helix structures with target DNA induces conformational and functional changes, preventing transcription initiation and elongation, and allowing the introduction of desired sequence changes in endogenous DNA, resulting in specific downregulation of gene expression. Examples of such suppression of gene expression in TFO-treated cells include knockout of the episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81 and Puri, et al., J Biol Chem, 2001;276:28991-98), as well as sequence-specific and target-specific downregulation of the expression of the Ets2 transcription factor, important in the pathogenesis of prostate cancer (Carbone, et al., Nucl Acids Res. 2003;31:833-43), and the proinflammatory ICAM-I gene (Besch et al., J Biol Chem, 2002;277:32473-79). Furthermore, Vuyisich and Beal recently showed that sequence-specific TFOs can bind to dsRNA and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0114] Furthermore, TFOs designed according to the above principles can induce directed mutagenesis capable of DNA repair, thus providing both down- and up-regulation of endogenous gene expression (Seidman and Glazer, J Clin Invest 2003;112:487-94). Detailed descriptions of the design, synthesis, and administration of effective TFOs can be found in U.S. Patent Application Publication Nos. 2003-017068 and 2003-0096980 to Froehler et al., 2002-0128218 and 2002-0123476 to Emanuele et al., and U.S. Patent No. 5,721,138 to Lawn, the contents of which are incorporated herein in their entireties.

[0115] multi-target molecule In one aspect, the present invention provides a multi-target molecule. Generally, the multi-target molecule comprises at least two nucleic acid-based effector molecules that are covalently or non-covalently linked to each other. Without limitation, any nucleic acid-based effector molecule that can regulate the gene expression of the target can be included in the multi-target molecule disclosed herein.

[0116] In some embodiments, at least one effector molecule in the multi-targeting molecule is siRNA. In some embodiments, the multi-targeting molecule comprises at least two siRNAs. Without limitation, the two siRNAs can be the same or different. For example, the two siRNAs can be directed to the same target or different targets. Furthermore, the two siRNAs can be directed to different regions on the same target.

[0117] In some embodiments, the multi-targeting molecule is assembled from two separate siRNA molecules, where at least one of the siRNAs is bound to at least one ligand. In some other embodiments, the multi-targeting molecule is assembled from two separate siRNA molecules, where each siRNA is bound to at least one ligand.

[0118] In various embodiments of the multi-target molecule, in which at least two siRNAs, each having at least one ligand, are linked to each other, the at least two ligands can be the same or different.Furthermore, the at least two ligands can be independently conjugated at any position of each siRNA.For example, one ligand can be linked to the sense strand of the first siRNA, and the other ligand can be linked to the sense strand of the second siRNA, or one ligand can be linked to the sense strand of the first siRNA, and the other ligand can be linked to the antisense strand of the second siRNA, or one ligand can be linked to the antisense strand of the first siRNA, and the other ligand can be linked to the antisense strand of the second siRNA.Without limitation, the first ligand can be independently linked to the 5'-end, 3'-end, or internal position of one strand (sense or antisense) of the first siRNA.Similarly, the second ligand can be independently linked to the 5'-end, 3'-end, or internal position of one strand (sense or antisense) of the second siRNA.

[0119] In some embodiments, one ligand is conjugated to the 3' end of the sense strand of the first siRNA, and another ligand is conjugated to the 3' end of the antisense strand of the second siRNA.In some embodiments, one ligand is conjugated to the 5' end of the sense strand of the first siRNA, and another ligand is conjugated to the 3' end of the antisense strand of the second siRNA.In some embodiments, one ligand is conjugated to the 3' end of the sense strand of the first siRNA, and another ligand is conjugated to the 5' end of the antisense strand of the second siRNA.In some embodiments, one ligand is conjugated to the 5' end of the sense strand of the first siRNA, and another ligand is conjugated to the 5' end of the antisense strand of the second siRNA.In some embodiments, one ligand is conjugated to the 3' end of the sense strand of the first siRNA, and another ligand is conjugated to the internal position of the antisense strand of the second siRNA. In some embodiments, one ligand is conjugated to the 5' end of the sense strand of the first siRNA, and another ligand is conjugated to the internal position of the antisense strand of the second siRNA.In some embodiments, one ligand is conjugated to the 3' end of the antisense strand of the first siRNA, and another ligand is conjugated to the internal position of the sense strand of the second siRNA.In some embodiments, one ligand is conjugated to the 5' end of the antisense strand of the first siRNA, and another ligand is conjugated to the internal position of the sense strand of the second siRNA.In some embodiments, one ligand is conjugated to the internal position of the antisense strand of the first siRNA, and another ligand is conjugated to the internal position of the sense strand of the second siRNA.

[0120] In some embodiments, one ligand is conjugated to the 3'-end of the first sense strand, and the other ligand is conjugated to the 3'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first sense strand, and the other ligand is conjugated to the 5'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first sense strand, and the other ligand is conjugated to the 3'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first sense strand, and the other ligand is conjugated to the 5'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first sense strand, and the other ligand is conjugated to an internal position of the second sense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first sense strand, and the other ligand is conjugated to an internal position of the second sense strand. In some embodiments, one ligand is conjugated to an internal position of the first sense strand, and the other ligand is conjugated to an internal position of the second sense strand.In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to the 3'-end of the second antisense strand.In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to the 5'-end of the second antisense strand.In some embodiments, one ligand is conjugated to the 5'-end of the first antisense strand, and the other ligand is conjugated to the 3'-end of the second antisense strand.In some embodiments, one ligand is conjugated to the 5'-end of the first antisense strand, and the other ligand is conjugated to the 5'-end of the second antisense strand.In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to the 5'-end of the second antisense strand.In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to the internal position of the second antisense strand. In some embodiments, one ligand is conjugated to the 5' end of the first antisense strand and the other ligand is conjugated to an internal position on the second antisense strand.In some embodiments, one ligand is conjugated at an internal position of the first antisense strand and the other ligand is conjugated at an internal position of the second antisense strand.

[0121] In some embodiments, multi-target molecule is assembled from two siRNAs, wherein the sense strand of the first siRNA is covalently linked to the sense strand of the second siRNA.Without limitation, two sense strands can be linked to each other in any orientation.For example, the 3 ' end of the first sense strand can be linked to the 5 ' end of the second sense strand; the 3 ' end of the first sense strand can be linked to the 3 ' end of the second sense strand; or the 5 ' end of the first sense strand can be linked to the 5 ' end of the second sense strand.

[0122] In some embodiments, multi-target molecule is assembled from two siRNAs, wherein the antisense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA.Without limitation, two antisense strands can be linked to each other in any orientation.For example, the 3 ' end of the first antisense strand can be linked to the 5 ' end of the second antisense strand; the 3 ' end of the first antisense strand can be linked to the 3 ' end of the second antisense strand; or the 5 ' end of the first antisense strand can be linked to the 5 ' end of the second antisense strand.

[0123] In some embodiments, multi-target molecule is assembled from two siRNAs, wherein the sense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA.Without limitation, the sense strand of the first siRNA can be linked to the antisense strand of the second siRNA in any orientation.For example, the 3' end of sense strand can be linked to the 5' end of antisense strand; the 3' end of sense strand can be linked to the 3' end of antisense strand; or the 5' end of sense strand can be linked to the 5' end of antisense strand.

[0124] In some embodiments, the multi-targeting molecule is assembled from two siRNAs, wherein the sense strand of the first siRNA is covalently linked to the sense strand of the second siRNA, and the antisense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA. In some embodiments, the multi-targeting molecule is assembled from two siRNAs, wherein the antisense strand of the first siRNA is covalently linked to the sense strand of the second siRNA, and the sense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA.

[0125] In some embodiments, at least one of the effector molecules in the multi-targeting molecule disclosed herein is an antisense oligonucleotide (ASO).In some embodiments, the multi-targeting molecule is assembled from two antisense oligonucleotides.Without limitation, the antisense oligonucleotides can be the same or different.

[0126] Two antisense oligonucleotides can be linked to each other at either end.For example, the 3' end of the first antisense oligonucleotide can be linked to either the 3' end or the 5' end of the second antisense oligonucleotide.Alternatively, the 5' end of the first antisense oligonucleotide can be linked to either the 3' end or the 5' end of the second antisense oligonucleotide.

[0127] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is an antisense oligonucleotide.

[0128] In some embodiments, at least one of the effector molecules in the multitarget molecule disclosed herein is a microRNA. In some embodiments, the multitarget molecule comprises at least two microRNAs. Without limitation, the microRNAs can be the same or different.

[0129] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a microRNA.

[0130] In some embodiments, at least one of the effector molecules in the multitargeting molecules disclosed herein is a ribozyme. In some embodiments, the multitargeting molecule comprises at least two ribozymes. Without limitation, the ribozymes can be the same or different.

[0131] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a ribozyme.

[0132] In some embodiments, at least one of the effector molecules in the multi-target molecule disclosed herein is an aptamer. In some embodiments, the multi-target molecule comprises at least two aptamers. Without limitation, the aptamers can be the same or different.

[0133] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is an aptamer.

[0134] In some embodiments, at least one of the effector molecules in the multi-targeting molecule disclosed herein is a decoy oligonucleotide. In some embodiments, the multi-targeting molecule comprises at least two decoy oligonucleotides. Without limitation, the decoy oligonucleotides can be the same or different.

[0135] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a decoy oligonucleotide.

[0136] In some embodiments, at least one of the effector molecules in the multi-targeting molecule disclosed herein is an miRNA mimic. In some embodiments, the multi-targeting molecule comprises at least two miRNA mimics. Without limitation, the miRNA mimics can be the same or different.

[0137] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is an miRNA mimic.

[0138] In some embodiments, at least one of the effector molecules in the multitargeting molecules disclosed herein is a supermir. In some embodiments, the multitargeting molecule comprises at least two supermirs. Without limitation, the supermirs can be the same or different.

[0139] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a supermir.

[0140] In some embodiments, at least one of the effector molecules in the multitargeting molecules disclosed herein is an anti-mir. In some embodiments, the multitargeting molecule comprises at least two anti-mirs. Without limitation, the anti-mirs can be the same or different.

[0141] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is an anti-mir.

[0142] In some embodiments, at least one of the effector molecules in the multitargeting molecules disclosed herein is an antagomir. In some embodiments, the multitargeting molecule comprises at least two antagomirs. Without limitation, the antagomirs can be the same or different.

[0143] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is an antagomir.

[0144] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is a U1 adaptor. In some embodiments, the multi-targeting molecule comprises at least two U1 adaptors. Without limitation, the U1 adaptors can be the same or different.

[0145] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a U1 adaptor.

[0146] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an activator RNA. In some embodiments, the multi-targeting molecule comprises at least two activator RNAs. Without limitation, the activator RNAs can be the same or different.

[0147] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is an activator RNA.

[0148] In some embodiments, at least one of the effector molecules in the multi-targeting molecule disclosed herein is a triplex-forming oligonucleotide. In some embodiments, the multi-targeting molecule comprises at least two triplex-forming oligonucleotides. Without limitation, the triplex-forming oligonucleotides can be the same or different.

[0149] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a triplex-forming oligonucleotide.

[0150] Conjugates containing one effector molecule conjugated to a ligand In another aspect, the present invention provides a conjugate comprising one effector molecule conjugated with a ligand via a cleavable linker as described herein.Without limitation, the effector molecule can be selected from the group consisting of double-stranded and single-stranded RNA interference agents (such as siRNA and shRNA, and herein referred to as dsRNA agents), antisense oligonucleotides, microRNAs, anti-microRNAs or anti-mirs, supermirs, antagomirs, ribozymes, triplex-forming oligonucleotides, decoy oligonucleotides, RNA activators, U1 adaptors, and CRISPR Cas guide RNAs (gRNAs).In some embodiments, the effector molecule is siRNA.

[0151] Without limitation, the ligand can be attached at any position of the effector molecule. For example, when the effector molecule is an siRNA, the ligand can be attached at the 5' end, 3' end, or internal position of either the sense strand or the antisense strand of the siRNA.

[0152] Ligand Generally, a ligand modifies one or more properties of the attached molecule (e.g., a multitargeting molecule, an effector molecule, or an endosomotropic agent), including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Ligands are commonly used in the chemical arts and are linked to the parent compound directly or via an optional linking moiety or binding group. A preferred list of ligands includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0153] Preferred ligands suitable for the present invention include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains, such as dodecadiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, such as di-hexadecyl-lacto-glycerol or triethylammonium-1,2-di-O-hexadecyl-lacto-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0154] Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, 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, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamines Quaternary salts, thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomas), isin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., α-helical peptides, amphipathic peptides, RGD peptides, cell-penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen , aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 Activators of MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor α (TNFα), interleukin-1β, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-penetrating agents (e.g., helical cell-penetrating agents).

[0155] Peptide and peptidomimetic ligands include natural or modified peptides, such as D- or L-peptides; α, β, or γ peptides; N-methylpeptides; azapeptides; peptides with one or more amide, i.e., peptides, one or more urea, thiourea, carbamate, or sulfonylurea bonds substituted; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0156] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0157] As used herein, the term "endosomolytic ligand" refers to a molecule that has endosomolytic properties. An endosomolytic ligand promotes lysis of a composition of the invention or its components and / or transport of a composition of the invention or its components from an intracellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular endoplasmic reticulum, into the cytoplasm of a cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear and branched polyamines such as spermine, cationic linear or branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charge, dendrimers with masked or unmasked cationic or anionic charge, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0158] Exemplary endosomolytic / fusogenic peptides include, but are not limited to, the following: AALEALAEALAEALAEALEALAEAAAAGGC (GALA) (SEQ ID NO: 1); AALEALAEALAEALAEALAEALAEALAAAAGGC (EALA) (SEQ ID NO: 2); ALEALAEALEALEALAEA (SEQ ID NO: 3); GLFEAIEGFIENGWEGMIWDYG (INF-7) (SEQ ID NO: 4); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2) (SEQ ID NO: 5); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7) (SEQ ID NO: 6); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3) (SEQ ID NO: 7); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF) (SEQ ID NO: 8); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3) (SEQ ID NO: 9); GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine) (SEQ ID NO: 10); LFEALLELLESLWELLLEA (JTS-1) (SEQ ID NO: 11); GLFKALLKLLKSLWKLLLKA (ppTG1) (SEQ ID NO: 12); GLFRALLRLLRSLWRLLLRA (ppTG20) (SEQ ID NO: 13); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA) (SEQ ID NO: 14); GLFFEAIAEFIEGGWEGLIEGC (HA) (SEQ ID NO: 15); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin) (SEQ ID NO: 16); H5WYG (SEQ ID NO: 17); and CHK6HC (SEQ ID NO: 18).

[0159] Without intending to be bound by any particular theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids typically have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0160] Synthetic polymers with endosomolytic activity suitable for the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the contents of which are incorporated herein by reference in their entireties.

[0161] Exemplary cell-penetrating peptides include, but are not limited to, the following: RQIKIWFQNRRMKWKK (penetratin) (SEQ ID NO: 19); GRKKRRQRRRPPQC (Tat fragment 48-60) (SEQ ID NO: 20); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence-based peptide) (SEQ ID NO: 21); LLIILRRRIRKQAHAHSK (PVEC) (SEQ ID NO: 22); GWTLNSAGYLLKINLKALAALAKKIL (transportan) (SEQ ID NO: 23); KLALKLALKALKAALKLA (amphipathic model peptide) (SEQ ID NO: 24); RRRRRRRRR (Arg9) (SEQ ID NO: 25); KFFKFFKFFK (bacterial cell wall-penetrating peptide) (SEQ ID NO: 26); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNL VPRTES(LL-37) (SEQ ID NO: 27); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 28); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 29); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO: 30); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO: 31); ILPWKWPWWPWRR-NH2 (indolicidin) (SEQ ID NO: 32); AAVALLPAVLLALLAP(RFGF) (SEQ ID NO: 33); AALLPVLLAAP (RFGF analog) (SEQ ID NO: 34); and RKCRIVVIRVCR (bactenecin) (SEQ ID NO: 35).

[0162] Exemplary cationic groups include, but are not limited to, O-AMINE (AMINE = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, e.g., O(CH) nAMINE, (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH2CH2NH) n Protonated amino groups derived from CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino) are included.

[0163] As used herein, the term "targeting ligand" refers to any molecule that confers increased affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0164] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of two or more monosaccharide units. These monosaccharide subunits can be linked to each other or to a scaffold molecule via glycosidic bonds.

[0165] As ligands, several folates and folate analogs suitable for the present invention are described in U.S. Pat. Nos. 2,816,110; 5,552,545; 6,335,434, and 7,128,893, the contents of which are incorporated herein by reference in their entireties.

[0166] As used herein, the terms "PK-modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and transthyretin-binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligomeric compounds containing several phosphorothioate intersugar linkages are also known to bind to serum proteins. Therefore, short oligomeric compounds, e.g., oligonucleotides containing about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., PK-modulating ligands) for the present invention. PK-modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in the PK-modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-modulating ligands for the present invention. Binding to serum components (eg, serum proteins) can be predicted from albumin binding assays such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0167] When there are two or more ligands, the ligands can all have the same properties, or all have different properties, or some ligands can have the same properties and the rest have different properties.For example, the ligands can have targeting properties, endosomolytic activity, or PK regulation properties.In a preferred embodiment, the ligands all have different properties.

[0168] The ligand or tethered ligand may be present on a monomer when the monomer is incorporated into an effector molecule or component of a multitargeting molecule. In some embodiments, the ligand can be incorporated into a "precursor" monomer by coupling after the "precursor" monomer is incorporated into an effector molecule or component of a multitargeting molecule. For example, a monomer having an amino-terminated tether (i.e., no ligand attached), such as Monomer-Linker-NH, can be incorporated into an effector molecule or component of a multitargeting molecule. Subsequent to incorporation of the precursor monomer into the effector molecule or component of a multitargeting molecule, a ligand having an electrophilic group, such as a pentafluorophenyl ester or aldehyde group, can be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the tether of the precursor monomer.

[0169] In another example, one can incorporate monomers bearing chemical groups suitable for participating in click chemistry reactions, such as azide or alkyne terminal tethers / linkers. In a subsequent operation, i.e., after incorporation of the precursor monomer into the chain, a ligand bearing a complementary chemical group, such as an alkyne or azide, can be attached to the precursor monomer by coupling with the alkyne and azide.

[0170] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside bond of the effector molecule or multitargeting molecule. Conjugation to the purine nucleobase or its derivative can be carried out at any position, including the endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the ligand. Conjugation to the pyrimidine nucleobase or its derivative can also be carried out at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be substituted with the ligand. When conjugating the ligand to the nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bond interaction required for base pairing.

[0171] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to the conjugate moiety, such as an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0172] There are many methods for preparing oligonucleotide conjugate.Generally, the reactive group (for example, OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotide is contacted with the reactive group of conjugate moiety, thereby connecting oligonucleotide to conjugate moiety.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0173] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or a thiol. Methods for conjugation of nucleic acids and related oligomeric compounds, with or without linking groups, are well described in the literature, such as, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0174] Ligands can be attached to effector molecules, multitargeting molecules, or endosomotropic agents via carrier monomers, e.g., ligand carriers. The carriers comprise (i) at least one "backbone attachment point," preferably two, and (ii) at least one "tethering attachment point." As used herein, a "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond available and suitable for incorporation of a carrier monomer into a backbone, e.g., the phosphate or modified phosphate (e.g., sulfur-containing) backbone of an oligonucleotide. A "tethering attachment point" (TAP) refers to an atom of a carrier monomer, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), to which a selected moiety is attached. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is linked to the carrier monomer by an intervening tether. Thus, carriers often contain functional groups, e.g., amino groups, or generally provide a bond suitable for incorporation or tethering of a ligand to another chemical entity, e.g., a constituent atom.

[0175] Representative United States patents that teach the preparation of nucleic acid conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; Specification No. 5,138,045; Specification No. 5,414,077; Specification No. 5,486,603; Specification No. 5,512,439; Specification No. 5,578,718; Specification No. 5,608,046; Specification No. 4,587,044; Specification No. 4,605,735 ; Specification No. 4,667,025; Specification No. 4,762,779; Specification No. 4,789,737; Specification No. 4,824,941 Specifications; Specification No. 4,835,263; Specification No. 4,876,335; Specification No. 4,904,582; Specification No. 4,958,013 Specification; Specification No. 5,082,830; Specification No. 5,112,963; Specification No. 5,214,136; Specification No. 5,082,83 Specification No. 0; Specification No. 5,112,963; Specification No. 5,149,782; Specification No. 5,214,136; Specification No. 5,245,0 Specification No. 22; Specification No. 5,254,469; Specification No. 5,258,506; Specification No. 5,262,536; Specification No. 5,272 ,250 specification; 5,292,873 specification; 5,317,098 specification; 5,371,241 specification, 5,39 Specification No. 1,723; Specification No. 5,416,203, Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5, Specification No. 512,667; Specification No. 5,514,785; Specification No. 5,565,552; Specification No. 5,567,810; Specification No. 5 ,574,142 specification; 5,585,481 specification; 5,587,371 specification; 5,595,726 specification; Specification No. 5,597,696; Specification No. 5,599,923; Specification No. 5,599,928; Specification No. 5,672,662;Nos. 5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559,279, the contents of which are incorporated herein by reference in their entirety.

[0176] In some embodiments, the effector molecule or multitargeting molecule is: [ka] The ligand includes a ligand having the structure shown in L G is, independently for each occurrence, a ligand, e.g., a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, polysaccharide; Z', Z'', Z''' and Z'''' are each independently O or S for each occurrence.

[0177] In some embodiments, the effector molecule or multitargeting molecule has formula (II), (III), (IV), or (V): [ka] wherein: q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represents, independently for each occurrence, 0 to 20, and the repeating units may be the same or different; Q and Q' are, for each occurrence, independently absent, -(P 7 -Q 7 -R 7 ) p -T7 -or-T 7 -Q 7 -T 7’ -BT 8’ -Q 8 -T 8 and; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , P 7 , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C , T 7 , T 7’ , T 8 and T 8’ is, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; B is -CH2-N(B L )-CH2-; B L -T B -Q B -T B’ -R x and; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 7 , Q 8 and Q B is, independently for each occurrence, absent, alkylene, or substituted alkylene; one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R'), C≡C, or C(O); T B and T B’ is, independently for each occurrence, absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH, CHNH, or CHO; R x is a lipophilic substance (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine), vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomolytic components, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid), or cationic lipids; R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 is, for each occurrence independently, absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 1 , L 2A , L 2B , L3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is, independently for each occurrence, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide; R' and R'' are each independently H, C 1 ~C6 alkyl, OH, SH or N(R N )2; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; R a is H or an amino acid side chain; Z', Z'', Z''', and Z'''' are each independently for each occurrence O or S; p, independently for each occurrence, is 0 to 20.

[0178] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0179] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0180] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0181] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0182] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0183] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0184] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0185] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0186] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0187] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0188] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0189] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0190] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0191] In some embodiments, the ligand in the effector molecule that is attached to the ligand via a cleavable linker described herein is a ligand described above.

[0192] In some embodiments, the ligand in the endosomoactive agent that is attached to the ligand via a cleavable linker described herein is a ligand described above.

[0193] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0194] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0195] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0196] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0197] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0198] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0199] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0200] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0201] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0202] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0203] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0204] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0205] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0206] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0207] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The ligands include:

[0208] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0209] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0210] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0211] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0212] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0213] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0214] In some embodiments, L 2A and L 2B are all different.

[0215] In some preferred embodiments, L 3A and L 3B are both the same.

[0216] In some embodiments, L 3A and L 3Bare all different.

[0217] In some preferred embodiments, L 4A and L 4B are both the same.

[0218] In some embodiments, L 4A and L 4B are all different.

[0219] In some preferred embodiments, L 5A , L 5B and L 5C are all the same.

[0220] In some embodiments, L 5A , L 5B and L 5C The two are the same.

[0221] In some embodiments, L 5A and L 5B are the same.

[0222] In some embodiments, L 5A and L 5C are the same.

[0223] In some embodiments, L 5B and L 5C are the same.

[0224] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0225] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0226] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0227] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where Y is O or S and n is 1-6.

[0228] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where Y=O or S. n is 1 to 6, R is hydrogen or a nucleic acid, and R' is a nucleic acid.

[0229] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where Y is O or S and n is 1-6.

[0230] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The compound contains at least one, two, three or four monomers of the formula:

[0231] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where X is O or S.

[0232] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0233] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0234] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where R is O or S.

[0235] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0236] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0237] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0238] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0239] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0240] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0241] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0242] In the foregoing monomers, X and Y, for each occurrence, are each independently H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, a -P(Z')(Z")O-nucleoside, a -P(Z')(Z")O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, a nucleoside, or an oligonucleotide; and Z' and Z" are each independently O or S for each occurrence.

[0243] In some embodiments, the effector molecule attached to the ligand via a linker disclosed herein comprises a monomer selected from the group consisting of monomers 1-30.

[0244] In some embodiments, the endosomotropic agent attached to a ligand via a linker disclosed herein comprises a monomer selected from the group consisting of monomers 1-30.

[0245] In some embodiments, the effector molecule, multitargeting molecule, or endosomotropic agent has the structure: [ka] is conjugated with a ligand of

[0246] In some embodiments, the effector molecule, multitargeting molecule, or endosomotropic agent has the structure: [ka] is conjugated with a ligand of

[0247] In some embodiments, the effector molecule or multitargeting molecule has the structure: [ka] The monomers are:

[0248] The synthesis of the aforementioned ligands and monomers is described, for example, in US Pat. No. 8,106,022, the contents of which are incorporated herein by reference in their entirety.

[0249] Target gene Without limitation, target genes for effector molecules include, but are not limited to, genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, gene expression kinases, adaptor protein genes, genes encoding G protein superfamily molecules, genes encoding transcription factors, genes that mediate angiogenesis, viral genes, genes required for viral replication, cellular genes that mediate viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes that mediate unwanted immune responses, genes that mediate pain processing, genes that mediate neurological disorders, alleles found in cells characterized by loss of heterozygosity, or one allele of a polymorphic gene.

[0250] Specific exemplary target genes for effector molecules include, but are not limited to, the following: PCSK-9, ApoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, 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; c-MYC gene; JUN gene; FOS gene; BCL-2 gene; cyclin D gene; VEGF gene; EGFR gene; cyclin A gene; cyclin E gene; WNT-1 gene; β-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; topoisomerase I gene; and topoisomerase II. α gene; p73 gene; p21 (WAF1 / CIP1) gene, p27 (KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor genes; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, such as MLL-AF9, BCR / ABL fusion gene; TEL / AML1 fusion gene; EWS / FLI1 fusion gene; TLS / FUS1 fusion gene; PAX3 / FKHR fusion gene; AML1 / ETO fusion gene; α v-integrin gene; Flt-1 receptor gene; tubulin gene; human papillomavirus genes, genes required for human papillomavirus replication, human immunodeficiency virus (HIV) Virus genes, genes required for human immunodeficiency virus replication, Hepatitis A virus genes, genes required for hepatitis A virus replication, Hepatitis B virus genes, Hepatitis B virus genesGenes required for Hepatitis C virus replication, Hepatitis C virus genes, Genes required for Hepatitis C virus replication, Hepatitis D virus genes, Genes required for Hepatitis D virus replication, Hepatitis E virus genes, Genes required for Hepatitis E virus replication, Hepatitis F virus genes, Genes required for Hepatitis F virus replication, Hepatitis G virus genes, Genes required for Hepatitis G virus replication, Hepatitis H virus genes, Genes required for Hepatitis H virus replication, Respiratory Syncytial Virus (RSV) genes, Genes required for Respiratory Syncytial Virus (RSV) replication, Herpes Simplex Virus genes, genes required for Herpes Simplex Virus replication, Herpes Cytomegalovirus genes, genes required for Herpes Cytomegalovirus replication, Herpes Epstein Barr Virus genes, genes required for Herpes Epstein Barr Virus replication, Kaposi's Sarcoma-associated Herpes Virus genes, genes required for Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus genes, JC Virus genesHuman genes required for HIV replication, myxovirus genes, genes required for myxovirus gene replication, rhinovirus genes, genes required for rhinovirus replication, coronavirus genes, genes required for coronavirus replication, West Nile virus genes, genes required for West Nile virus replication, St. Louis encephalitis genes, genes required for St. Louis encephalitis replication, tick-borne encephalitis virus genes, genes required for tick-borne encephalitis virus replication, Murray Valley encephalitis virus genes, genes required for Murray Valley encephalitis virus replication, dengue virus genes, genes required for dengue virus gene replication, simian virus 40 genes, simian virus Genes required for Human T Cell Lymphotropic Virus 40 replication, Human T Cell Lymphotropic Virus genes, Genes required for Human T Cell Lymphotropic Virus replication, Moloney-Murine Leukemia Virus genes, Genes required for Moloney-Murine Leukemia Virus replication, Encephalomyocarditis virus genes, Genes required for Encephalomyocarditis virus replication, Measles virus genes, Genes required for Measles virus replication, Varicella zoster virus genes, Varicella zoster virus genesGenes required for virus replication, adenovirus genes, genes required for adenovirus replication, yellow fever virus genes, genes required for yellow fever virus replication, poliovirus genes, genes required for poliovirus replication, poxvirus genes, genes required for poxvirus replication, plasmodium genes, genes required for plasmodium replication, Mycobacterium ulcerans genes, genes required for Mycobacterium ulcerans replication, Mycobacterium tuberculosis genes, genes required for Mycobacterium tuberculosis replication, Mycobacterium leprae genes, genes required for Mycobacterium leprae replication, Staphylococcus aureus genes, Staphylococcus Genes required for Streptococcus aureus replication, Streptococcus pneumoniae genes, Genes required for Streptococcus pneumoniae replication, Streptococcus pyogenes genes, Genes required for Streptococcus pyogenes replication, Chlamydia pneumoniae genes, Genes required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, Mycoplasma pneumoniaepneumoniae) replication-related genes, integrin genes, selectin genes, complement system genes, chemokine genes, chemokine receptor genes, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene, proplatelet basic protein gene, MIP-1I gene, MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene, and CMBKR3 gene , CMBKR5v, AIF-1 gene, I-309 gene, genes for components of ion channels, genes for neurotransmitter receptors, genes for neurotransmitter ligands, amyloid family genes, presenilin genes, HD gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, alleles found in loss of heterozygosity (LOH) cells, single alleles of polymorphic genes, and combinations thereof.

[0251] Loss of heterozygosity (LOH) can result in hemizygosis of sequences, e.g., genes, in the region of LOH. This can result in significant genetic differences between normal cells and diseased cells, e.g., cancer cells, which provide useful differentiation between normal cells and diseased cells, e.g., cancer cells. This difference can occur because a gene or other sequence is heterozygous in diploid cells but hemizygous in cells with LOH. Regions of LOH often contain other sequences, including genes whose loss promotes unwanted proliferation, e.g., tumor suppressor genes, as well as other genes, in some cases genes essential for normal function, e.g., growth. The methods of the present invention utilize, in part, the specific regulation of one allele of an essential gene by the compositions of the present invention.

[0252] Nucleic acid modification The effector molecule or multitargeting molecule may contain at least one nucleic acid modification described herein. For example, at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. Without limitation, such a modification may be present anywhere in the effector molecule or multitargeting molecule. For example, the modification may be present in one of the linkers connecting two effector molecules of the effector molecule or multitargeting molecule.

[0253] The naturally occurring base moiety of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. When forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'-5' phosphodiester linkage.

[0254] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art can be used in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomeric modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is replaced with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases, including conjugate moieties, such as the ligands described herein. Preferred conjugate moieties for conjugation to nucleobases contain cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl or amide bonds.

[0255] As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 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 cytosine, 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-(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-diazole-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-(methoxycarbonylmethyl)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)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil Douracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl phenyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, 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 pyrimidines, N, 2 -substituted purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one- Other synthetic or natural nucleic acid bases include ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used.

[0256] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an oligonucleotide duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, and imidizopyridinyl. , 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl and structural derivatives thereof (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0257] Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application PCT / US Patent Publication No. 09 / 038425, filed March 26, 2009; those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30,613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, the contents of all of which are incorporated herein by reference.

[0258] In some embodiments, the modified nucleobase is a nucleobase that is substantially similar in structure to the parent nucleobase, such as, for example, a 7-deazapurine, 5-methylcytosine, or G-clamp. In some embodiments, the nucleobase mimic comprises a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase mimic. Methods for preparing the aforementioned modified nucleobases are well known to those of skill in the art.

[0259] An effector molecule or multitargeting molecule can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers comprising nucleosides or nucleotides having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent group or the bridging of two non-geminal ring atoms to form a locked nucleic acid or a bicyclic nucleic acid. In some embodiments, an oligomeric compound includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers that are LNAs.

[0260] In some embodiments of the locked nucleic acid, the 2' position of the furanosyl is -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -C(=NR1)-, -C(=NR1)- O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) x - and -N(R1)-, linked at the 4' position by a linker independently selected from: x is 0, 1 or 2; n is 1, 2, 3 or 4; each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 cycloaliphatic radical, substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0261] In some embodiments, each of the linkers in the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In other embodiments, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or a C1-C12 alkyl.

[0262] Several LNAs have been prepared and disclosed in the patent and scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al. al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Patent Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0263] Also provided herein are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bond, to form a bicyclic sugar moiety (discussed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, in which case the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; if there is an ethylene group at this position, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy(4'-CH2-O-2')LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0264] An isomer of methyleneoxy(4'-CH2-O-2')LNA that has also been discussed is α-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras, which have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0265] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0266] Analogs of methyleneoxy(4'-CH2-O-2')LNA, phosphorothioate-methyleneoxy(4'-CH2-O-2')LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has also been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been reported.

[0267] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars, including methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars having a 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituent; and 4'-thio modified sugars. Sugars can also be substituted with, among other things, sugar mimetic groups. Methods for preparing modified sugars are well known to those of skill in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; ,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and International Publication No. 2005 / 121371.

[0268] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) nCH2CH2OR, n=1-50; "locked" nucleic acids (LNA) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-amine or O-(CH2) n amines (n=1-10, amine=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0269] "Deoxy" modifications include hydrogen (i.e., the deoxyribose sugar particularly associated with overhanging single strands); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n These include CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which can be optionally substituted, for example with an amino function.

[0270] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0271] The modification at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of the ribose in the same configuration as the 2'-OH in arabinose.

[0272] A sugar can contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar group can also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, an oligomeric compound can contain one or more monomers containing, for example, arabinose as the sugar. The monomer can have an α-linkage at the 1'-position of the sugar, e.g., an α-nucleoside. The monomer can also have the opposite stereoconfiguration at the 4'-position, e.g., C5' and H4', or the substituents replacing them, are swapped. When C5' and H4', or the substituents replacing them, are swapped, the sugar is said to be modified at the 4'-position.

[0273] Effector molecules or multitargeting molecules may also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group in place of the nucleobase at C1'. See, for example, U.S. Pat. No. 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars may also further include modifications to one or more of the constituent sugar atoms. Effector molecules or multitargeting molecules may also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Modifications to the sugar group may also include substitution of the 4'-O with sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0274] Sugar modifications can also include acyclic nucleotides in which the C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, the acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0275] In some embodiments, the sugar modification is selected from the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl, 2'-O-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0276] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be placed at the 3' position of the sugar for that particular nucleotide, for example, the nucleotide linked through its 2' position. The 3' modification can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the 3'-OH of the xylose sugar.

[0277] The hydrogen attached to C4' and / or C1' can be replaced by a straight-chain or branched optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, and the backbone of the alkyl, alkenyl, and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, where R' is hydrogen, acyl, or an optionally substituted aliphatic, and Z' is OR. 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 21 R 31 ,CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is independently for each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 or NR 11 R 11 ' or R 21 and R 31 form a heterocyclic ring together with the atoms to which they are attached; R 41 and R 51 is independently for each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 or CO2R 11 or NR 11 R 11 ' and ;R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is substituted.

[0278] In some embodiments, C4' and C5' together preferably form an optionally substituted heterocycle containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, where M is, independently for each occurrence, an alkylmetal or transition metal with a total charge of +1; and Y is O, S, or NR', where R' is hydrogen or an optionally substituted aliphatic. This modification is preferably at the 5' end of the oligonucleotide.

[0279] In some embodiments, the LNA has the formula: [ka] and bicyclic nucleosides having the formula: Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0280] In some embodiments, each of the substituted groups is mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1).

[0281] In some such embodiments, each of the substituted groups is mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2 (each J1, J2, and J3 is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1).

[0282] In some embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN (each J, J, and J is independently H or C-C alkyl and X is O, S, or NJ). In other embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), substituted alkoxy, or azido.

[0283] In some embodiments, the Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN (each J1, J2, and J3 is independently H or C1-C6 alkyl and X is O, S, or NJ1). In other embodiments, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0284] In some such embodiments, the Z group is in the (R)-configuration: [ka] is.

[0285] In some such embodiments, the Z group is in the (S)-configuration: [ka] is.

[0286] In some embodiments, each of T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and dimethoxytrityl, with a more preferred hydroxyl protecting group being 4,4'-dimethoxytrityl.

[0287] In some embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In some embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0288] In some embodiments, the multi-targeting molecule has the formula: [ka] or the expression: [ka] or the expression: [ka] wherein: Bx is a heterocyclic base moiety; T3 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0289] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1).

[0290] In some embodiments, each of the substituted groups is mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2 (each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O or NJ1).

[0291] In some such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In some embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In some embodiments, at least one Z is C1-C6 alkyl. In some embodiments, each Z is independently C1-C6 alkyl. In some embodiments, at least one Z is methyl. In some embodiments, each Z is methyl. In some embodiments, at least one Z is ethyl. In some embodiments, each Z is ethyl. In some embodiments, at least one Z is substituted C1-C6 alkyl. In some embodiments, each Z is independently substituted C1-C6 alkyl. In some embodiments, at least one Z is substituted methyl. In some embodiments, each Z is substituted methyl. In some embodiments, at least one Z is substituted ethyl. In some embodiments, each Z is substituted ethyl.

[0292] In some embodiments, at least one substituent is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In other embodiments, each substituent is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy).

[0293] In some embodiments, at least one C1-C6 alkoxy substituent is CHO- (e.g., at least one Z is CHOCH-). In other embodiments, each C1-C6 alkoxy substituent is CHO- (e.g., each Z is CHOCH-).

[0294] In some embodiments, at least one substituent is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogens). In some embodiments, each substituent is independently halogen (e.g., each Z is independently C1-C6 alkyl substituted with one or more halogens). In some embodiments, at least one halogen substituent is fluoro (e.g., at least one Z is CH2FCH2-, CHF2CH2-, or CF3CH2-). In some embodiments, each halo substituent is fluoro (e.g., each Z is independently CH2FCH2-, CHF2CH2-, or CF3CH2-).

[0295] In some embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0296] In some embodiments, at least one Z is CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-. In some embodiments, each Z is independently CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-.

[0297] In some embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0298] In some embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0299] In some embodiments, at least one Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or C1-C6 alkyl and X is O, S, or NJ1. In some embodiments, at least one Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0300] In some embodiments, each Z group is independently -CH2Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In other embodiments, each Z group is independently -CH2Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0301] In some embodiments, at least one Z is CH3-. In other embodiments, each Z is CH3-.

[0302] In some embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] It has an (R)-configuration represented by

[0303] In some embodiments, the Z group of each monomer of the above formula is in the (R)-configuration.

[0304] In some embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] The (S)-configuration is represented by

[0305] In some embodiments, the Z group of each monomer of the above formula is in the (S)-configuration.

[0306] In some embodiments, T3 is H or a hydroxyl protecting group. In some embodiments, T4 is H or a hydroxyl protecting group. In other embodiments, T3 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In some embodiments, T4 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In some embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In some embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In some embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In some embodiments, T4 is an internucleoside linking group attached to an oligomeric compound. In some embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from a phosphodiester or a phosphorothioate.

[0307] In some embodiments, the effector molecule or multitargeting molecule has the formula: [ka] or the expression: [ka] or the expression: [ka] The polymer may comprise at least one region of at least two consecutive monomers of

[0308] In some such embodiments, the LNA may include, but is not limited to: [ka] As shown in the table, examples include (A) α-L-methyleneoxy (4'-CH2-O-2') LNA, (B) β-D-methyleneoxy (4'-CH2-O-2') LNA, (C) ethyleneoxy (4'-(CH2)2-O-2') LNA, (D) aminooxy (4'-CH2-ON(R)-2') LNA, and (E) oxyamino (4'-CH2-N(R)-O-2') LNA.

[0309] In some embodiments, an effector molecule or multitargeting molecule can comprise at least two regions of at least two consecutive monomers of the above formula. In some embodiments, a multitargeting molecule comprises a gap motif. In some embodiments, a multitargeting molecule comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In some embodiments, a multitargeting molecule comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0310] In some embodiments, the multi-targeting molecule has the formula: [ka] wherein Bx is a heterocyclic base moiety. and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer.

[0311] In some embodiments, the monomer comprises a sugar mimetic. In some such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while maintaining the nucleobase for hybridization with a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some cases, the mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those skilled in the art.

[0312] Nucleic acid modification (sugar bond) Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming oligomeric compounds, e.g., oligonucleotides. Such linking groups are also referred to as intersugar linkages. Two major classes of linking groups are distinguished by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methyl phosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphodiester bonds, modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In some embodiments, linkages with chiral atoms can be prepared as individual enantiomers or as racemic mixtures. Representative chiral linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0313] The phosphate group in the linking group can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphate triesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced with S, Se, BR3 (R is hydrogen, alkyl, or aryl), C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, or aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or atomic groups makes the phosphorus atom chiral. In other words, the phosphorus atom in such a modified phosphate group is a stereocenter. The stereogenic phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

[0314] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in phosphorodithioates is achiral, which eliminates the formation of oligonucleotide diastereomers. Therefore, without intending to be bound by a particular theory, modifications to both non-bridging oxygens may be desirable, as they eliminate the formation of chiral centers, such as phosphorodithioates, and cannot produce diastereomeric mixtures. Thus, the non-bridging oxygens can independently be any one of O, S, Se, B, C, H, N, or OR (wherein R is alkyl or aryl).

[0315] Phosphate linkers can also be modified by substitution of the bridging oxygen (i.e., the oxygen linking the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). This substitution can be made at either or both linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution with nitrogen is preferred.

[0316] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced or the phosphate group is replaced by a non-phosphorus group are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0317] In some embodiments, the phosphate group can be replaced by a non-phosphorus-containing connector, such as a dephospho linker. Dephospho linkers are also referred to herein as non-phosphodiester linkers. Without intending to be bound by any particular theory, it is believed that because the charged phosphodiester group is the reaction center for nucleic acid degradation, its replacement with a neutral structural mimic confers enhanced nuclease stability. Again, without intending to be bound by any particular theory, in some embodiments, it may be desirable to introduce a modification that replaces the charged phosphate group with a neutral moiety.

[0318] Examples of moieties that can replace the phosphate group include, but are not limited to, amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methylenecarbonylamino, methyl ... Nonionic bonds containing mixed N, O, S, and CH moieties, such as methylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5', and 3'-NHP(O)(OCH3)-O-5') and mixed N, O, S, and CH2 moieties, e.g., Carbohydrate See Modifications in Antisense Research; YS Sanghvi and PDCook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0319] Those skilled in the art will appreciate that, in some cases, substitution of a non-bridging oxygen may result in enhanced cleavage of the intersugar bond by the adjacent 2'-OH, and therefore, in many cases, modification of a non-bridging oxygen may require modification of a 2'-OH, e.g., a modification that does not participate in cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyls, 2'-F, LNA, and ENA.

[0320] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates containing an enantiomeric excess of the Sp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorothioates containing an enantiomeric excess of the Rp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0321] In some embodiments, the multitargeting molecule comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and inclusive, fewer) modified or non-phosphodiester linkage. In some embodiments, the effector molecule or multitargeting molecule may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and inclusive, fewer) phosphorothioate linkage.

[0322] Effector molecules or multitargeting molecules can also be constructed in which the phosphate linker and sugar are replaced by nuclease-resistant nucleoside or nucleotide surrogates. Without intending to be bound by any particular theory, it is believed that the absence of a repeatedly charged backbone weakens binding to proteins that recognize polyanions (e.g., nucleases). Again, without intending to be bound by any particular theory, in some embodiments, it may be desirable to introduce modifications in which the bases are tethered by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and backbone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0323] Effector molecules or multitargeting molecules may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other configurations, which may be defined in terms of absolute stereochemistry as (R) or (S), such as in the case of sugar anomers, or (D) or (L), such as in the case of amino acids. The multitargeting molecules provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0324] The termini of the multitargeting molecule or effector molecule can be modified. Such modifications are possible at one or both termini. For example, the 3' and / or 5' termini of the oligonucleotide can be conjugated with a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entities, such as protecting groups (e.g., sulfur, silicon, boron, or ester-based). The functional molecular entity can be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker can be linked to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker can be linked to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

[0325] When a linker / phosphate-functional molecular entity-linker / phosphate array is placed between the two strands of a double-stranded oligomeric compound, the array can replace a hairpin loop in a hairpin-type oligomeric compound.

[0326] Terminal modifications useful for modulating activity include modification of the 5' end of an oligonucleotide with phosphate or a phosphate analog. In some embodiments, the 5' end of an oligonucleotide is phosphorylated or contains a phosphoryl analog. Exemplary 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. 5' end modifications may also be useful for stimulating or inhibiting the immune system of a subject. In some embodiments, the 5' end of an oligomeric compound is modified [ka] wherein W, X, and Y are each independently O, OR (wherein R is hydrogen, alkyl, or aryl), S, Se, BR3 (wherein R is hydrogen, alkyl, or aryl), BH3 -, C (i.e., alkyl, aryl, etc.), H, NR (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl, or aryl); A and Z, for each occurrence, are each independently absent, O, S, CH, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can include internally and / or terminally one or more of O, S, SS, and NR (R is hydrogen, alkyl, aryl); and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen linked to the 5' carbon of the sugar. When n is 0, W and Y, together with the P to which they are attached, can form an optionally substituted 5-8 membered heterocyclic compound, where W and Y are each independently O, S, NR', or alkylene. Preferably, the heterocyclic compound is substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on the C5' of the 5' terminal nucleotide are replaced with a halogen, eg, F.

[0327] Exemplary 5'-modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); 5'-alpha-thiotriphosphate; 5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkyl phosphonates (R(OH)(O)PO-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)PO-5', R = alkyl ether, e.g., methoxymethyl (CHOMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is an alkyl optionally substituted at least once, e.g., ((HO)2(X)PO[-(CH2) a -OP(X)(OH)-O] b -5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl-terminated phosphates and phosphate mimetics: HO[-(CH2) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b -5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b-5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5', where a and b are each independently 1 to 10. Other embodiments include BH3, BH3 - and / or containing substitution of oxygen and / or sulfur by Se.

[0328] Terminal modifications can also be useful for monitoring distribution; in such cases, preferred groups to add include fluorophores, such as fluorescein, or Alexa dyes, such as Alexa 488. Terminal modifications can also be useful for enhancing uptake; useful modifications for this purpose include targeting ligands. Terminal modifications can also be useful for crosslinking the oligonucleotide to another moiety; useful modifications for this purpose include mitomycin C, psoralens, and their derivatives.

[0329] Effector molecules, such as siRNA or dsRNA agents, can be optimized for RNA interference by increasing the propensity for the dsRNA duplex to dissociate or melt (reducing the free energy of duplex association) by introducing a thermodestabilizing modification into the sense strand at a site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). This modification can increase the propensity for the duplex to dissociate or melt within the seed region of the antisense strand.

[0330] Thermally destabilizing modifications can include abasic modifications; mismatches with the opposite nucleotide on the opposite strand; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, for example, unlocked nucleic acids (UNAs) or glycerol nucleic acids (GNAs).

[0331] Exemplary abasic modifications are as follows: [ka]

[0332] Exemplary sugar modifications are as follows: [ka]

[0333] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is missing from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] where B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR, or alkyl; R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, a UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entireties). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0334] The term "GNA" refers to a molecule that resembles DNA or RNA but has phosphodiester bonds: [ka] Glycerol refers to glycol nucleic acids, which are polymers that differ in the composition of their "backbone" in that they are made up of repeating glycerol units linked by

[0335] The thermodestabilizing modification can be a mismatch (i.e., non-complementary base pair) between the thermodestabilizing nucleotide in the dsRNA double helix and the opposite nucleotide of the opposite strand. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pairings known in the art are also suitable for the present invention. Mismatches can occur between any nucleotide, whether naturally occurring or modified. That is, mismatch base pairing can occur between nucleobases from each nucleotide, regardless of the modification to the ribose sugar of the nucleotide. In some embodiments, an effector molecule, such as an siRNA or dsRNA agent, contains at least one nucleobase in mismatch pairing, which is a 2'-deoxynucleobase; for example, the 2'-deoxynucleobase is present in the sense strand.

[0336] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0337] Thermodestabilizing modifications can include universal bases that have reduced or abolished ability to form hydrogen bonds with the opposing base, as well as phosphate modifications.

[0338] Nucleobase modifications that impair or completely abolish the ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilizing the central region of the dsRNA duplex, as described in WO 2010 / 0011895 (hereby incorporated by reference in its entirety). Exemplary nucleobase modifications include: [ka] is.

[0339] Exemplary phosphate modifications that have been shown to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include: [ka] is.

[0340] In some embodiments, the effector molecules in the multi-targeting molecule may contain 2'-5' linkages (with 2'-H, 2'-OH, and 2'-OMe, and with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense and antisense strands, or such modifications can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0341] In another embodiment, the effector molecule in the multitargeting molecule can contain an L-sugar (e.g., L-ribose, L-arabinose, including 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to promote nuclease resistance or inhibit binding between the sense and antisense strands, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0342] In one embodiment, a dsRNA agent of the invention is conjugated to a ligand via a carrier, which 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, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0343] In some embodiments, at least one strand of at least one effector molecule in the multitargeting molecules disclosed herein is 5' phosphorylated or contains a phosphoryl analog at the 5' prime end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include the following: 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)( any further combinations of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0344] The present invention also includes effector molecules and multitargeting molecules that are chimeric compounds. In the context of the present invention, a "chimeric" compound or "chimera" is a compound that contains two or more chemically distinct regions, each region being composed of at least one monomer unit, e.g., in the case of an oligonucleotide, a modified or unmodified nucleotide. Chimeric compounds can be described as having a specific motif. In some embodiments, motifs include, but are not limited to, alternating motifs, gap motifs, hemimeric motifs, uniformly fully modified motifs, and positionally modified motifs. As used herein, the phrase "chemically distinct region" refers to a region of a multitargeting molecule that differs from other regions by having a modification not present elsewhere in the compound or by the absence of a modification present elsewhere in the compound. A multitargeting molecule can contain two or more chemically distinct regions. As used herein, a region that does not contain a modification is also considered chemically distinct.

[0345] Chemically distinct regions can be repeated within a multi-target molecule compound. Thus, a pattern of chemically distinct regions within a multi-target molecule can be realized such that a first chemically distinct region is followed by one or more second chemically distinct regions. Such sequences of chemically distinct regions can be repeated one or more times. Preferably, the sequences are repeated two or more times. For example, both strands of a double-stranded effector molecule can contain these sequences. Each chemically distinct region may actually contain only a single monomer, e.g., a nucleotide. In some embodiments, each chemically distinct region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 monomers, e.g., nucleotides.

[0346] In some embodiments, alternating nucleotides contain the same modification, e.g., all odd-numbered nucleotides in a strand have the same modification and / or all even-numbered nucleotides in a strand have a modification similar to that in the first strand. In some embodiments, all odd-numbered nucleotides in a double-stranded effector molecule or multi-target molecule have the same modification and all even-numbered nucleotides have a modification that is not present in the odd-numbered nucleotides, or vice versa.

[0347] When both strands of a double-stranded molecule contain alternating modification patterns, nucleotides in one strand may be at complementary positions to similarly modified nucleotides in the second strand. In another embodiment, there is a phase shift between the pattern of modification of the first strand relative to the pattern of similar modification of the second strand. Preferably, this shift is such that the similarly modified nucleotides in the first and second strands are not at complementary positions to each other.

[0348] In some embodiments, the first strand has an alternating modification pattern in which alternating nucleotides contain 2'-modifications, such as 2'-O-methyl modifications. In some embodiments, the first strand contains alternating 2'-O-methyl modifications, and the second strand contains alternating 2'-fluoro modifications. In other embodiments, both strands of the double-stranded oligonucleotide contain alternating 2'-O-methyl modifications.

[0349] When both strands of a double-stranded oligonucleotide contain alternating 2'-O-methyl modifications, such 2'-modified nucleotides can be at complementary positions in the double-helical region, or alternatively, such 2'-modified nucleotides are not at complementary positions in the double-helical region.

[0350] In some embodiments, the oligonucleotides present in the multi-target molecule comprise two chemically distinct regions, where each region is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0351] In other embodiments, the oligonucleotides present in the multi-target molecule comprise three chemically distinct regions. The central region is approximately 5-15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, and each flanking or wing region is independently 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides in length. All three regions may have different modifications, or the wing regions may have similar modifications to each other. In some embodiments, the wing regions are the same length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0352] As used herein, the term "alternating motif" refers to a compound containing a continuous sequence composed of linked monomer subunits having two different types of sugar groups that alternate throughout substantially the entire sequence of the compound. Oligonucleotides having an alternating motif can be represented by the formula: 5'-A(-LBLA)n(-LB)m-3', where A and B are monomer subunits having different sugar groups, each L is an internucleoside linking group, n is from about 4 to about 12, and m is 0 or 1. This allows for compounds having an alternating motif from about 9 to about 26 monomer subunits in length. This length range is not intended to be limiting, as longer and shorter compounds are also suitable for the present invention. In some embodiments, one of A and B is a 2'-modified nucleoside as provided herein.

[0353] As used herein, "type of modification" with respect to a "type" of nucleoside or nucleoside refers to the modification of the nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise specified, a "nucleoside having a first type of modification" can be an unmodified nucleoside.

[0354] As used herein, a "type region" refers to a portion of a compound in which the nucleosides and internucleoside linkages within the region all contain the same type of modification; any adjacent nucleosides and / or internucleoside linkages contain at least one different type of modification. As used herein, the term "homogeneously fully modified motif" refers to an oligonucleotide comprising a contiguous sequence of linked monomer subunits, each having the same type of sugar group. In some embodiments, a homogeneously fully modified motif comprises a contiguous sequence of nucleosides of the present invention. In some embodiments, one or both of the 3' and 5' ends of the contiguous sequence of nucleosides provided herein comprises a terminal group, such as one or more unmodified nucleosides.

[0355] In some embodiments, the 5'-terminal monomer of a compound, e.g., a multitargeting molecule or an effector molecule, comprises a phosphorus-containing moiety at the 5'-terminus. In some embodiments, the 5'-terminal monomer comprises a 2'-modification. In some such embodiments, the 2'-modification of the 5'-terminal monomer is a cationic modification. In some embodiments, the 5'-terminal monomer comprises a 5'-modification. In some embodiments, the 5'-terminal monomer comprises a 2'-modification and a 5'-modification. In some embodiments, the 5'-terminal monomer is a 5'-stabilizing nucleoside. In some embodiments, the modification of the 5'-terminal monomer stabilizes the 5'-phosphate. In some embodiments, compounds comprising a modification of the 5'-terminal monomer are exonuclease resistant. In some embodiments, compounds comprising a modification of the 5'-terminal monomer have improved gene expression modulation properties.

[0356] In some embodiments, the 5'-terminal monomer is attached to the remainder of the compound by a modified linkage, hi some such embodiments, the 5'-terminal monomer is attached to the remainder of the compound by a phosphorothioate linkage.

[0357] In some embodiments, oligomeric compounds of the invention comprise one or more regions of alternating modifications. In some embodiments, oligomeric compounds comprise one or more regions of alternating nucleoside modifications. In some embodiments, oligomeric compounds comprise one or more regions of alternating bond modifications. In some embodiments, oligomeric compounds comprise one or more regions of alternating nucleoside and bond modifications.

[0358] In some embodiments, oligomeric compounds of the present invention comprise a region of one or more alternating 2'-F modified nucleosides and 2'-OMe modified nucleosides. In some such embodiments, such a region of alternating 2'F modified and 2'OMe modified nucleosides also comprises alternating linkages. In some such embodiments, the linkage at the 3' end of the 2'-F modified nucleoside is a phosphorothioate linkage. In some such embodiments, the linkage at the 3' end of the 2'OMe nucleoside is a phosphodiester linkage.

[0359] In some embodiments, such alternating regions include: (2'-F)-(PS)-(2'-OMe)-(PO) is.

[0360] In some embodiments, oligomeric compounds contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 such alternating regions. Such regions may be contiguous or separated by different modified nucleosides or linkages.

[0361] In some embodiments, one or more alternating regions in an alternating motif comprises a plurality of a single nucleoside of a certain type. For example, an oligomeric compound of the invention may comprise the following nucleoside motif: ABA; ABBA; AABA; AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA; ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA; where A is a first type of nucleoside and B is a second type of nucleoside. In some embodiments, A and B are each selected from 2'-F, 2'-OMe, LNA, DNA, and MOE.

[0362] In some embodiments, A is DNA. In some embodiments, B is DNA. In some embodiments, A is 4'-CHO-2'-LNA. In some embodiments, B is 4'-CHO-2'-LNA. In some embodiments, A is DNA and B is 4'-CHO-2'-LNA. In some embodiments, A is 4'-CHO-2'-LNA and B is DNA.

[0363] In some embodiments, A is 2'-OMe. In some embodiments, B is 2'-OMe. In some embodiments, A is 2'-OMe and B is 4'-CHO-2'-LNA. In some embodiments, A is 4'-CHO-2'-LNA and B is 2'-OMe. In some embodiments, A is 2'-OMe and B is DNA. In some embodiments, A is DNA and B is 2'-OMe.

[0364] In some embodiments, A is (S)-cEt. In some embodiments, B is (S)-cEt. In some embodiments, A is 2'-OMe and B is (S)-cEt. In some embodiments, A is (S)-cEt and B is 2'-OMe. In some embodiments, A is DNA and B is (S)-cEt. In some embodiments, A is (S)-cEt and B is DNA.

[0365] In some embodiments, A is 2'-F. In some embodiments, B is 2'-F. In some embodiments, A is 2'-F and B is 4'-CHO-2'-LNA. In some embodiments, A is 4'-CHO-2'-LNA and B is 2'-F. In some embodiments, A is 2'-F and B is (S)-cEt. In some embodiments, A is (S)-cEt and B is 2'-F. In some embodiments, A is 2'-F and B is DNA. In some embodiments, A is DNA and B is 2'-F. In some embodiments, A is 2'-OMe and B is 2'-F. In some embodiments, A is DNA and B is 2'-OMe. In some embodiments, A is 2'-OMe and B is DNA.

[0366] In some embodiments, oligomeric compounds having such alternating motifs also include a 5'-terminal nucleoside that includes a phosphate-stabilizing modification. In some embodiments, oligomeric compounds having such alternating motifs also include a 5'-terminal nucleoside that includes a 2'-cationic modification. In some embodiments, oligomeric compounds having such alternating motifs also include a 5'-terminal modification.

[0367] 2-2-3 motif In some embodiments, the oligonucleotides in the multi-target molecule comprise a region having a 2-2-3 motif. Such a region may have the following motif: 5'-(E) w -(A)2-(B) x -(A)2-(C) y -(A)3-(D) z wherein A is a first type modified nucleoside; B, C, D and E are nucleosides with different modifications than A, but B, C, D and E may have the same or different modifications from each other; w and z are 0 to 15; x and y are 1 to 15.

[0368] In some embodiments, A is a 2'-OMe modified nucleoside. In some embodiments, B, C, D, and E are all 2'-F modified nucleosides. In some embodiments, A is a 2'-OMe modified nucleoside and B, C, D, and E are all 2'-F modified nucleosides.

[0369] In some embodiments, the linkages in the 2-2-3 motif are all modified linkages. In some embodiments, the linkages are all phosphorothioate linkages. In some embodiments, the linkage at the 3' end of each first type of modification is a phosphodiester.

[0370] In some embodiments, Z is 0. In such embodiments, the region of three nucleosides of the first type is at the 3'-end of the oligonucleotide. In some embodiments, such a region is at the 3'-end of the oligomeric compound, and no other group is attached to the 3'-end of the region of three nucleosides of the first type. In some embodiments, an oligomeric compound comprising an oligonucleotide in which Z is 0 can include a terminal group attached to the 3'-terminal nucleoside. Such a terminal group can include another nucleoside. Such another nucleoside is typically a non-hybridizing nucleoside.

[0371] In some embodiments, Z is 1-3. In some embodiments, Z is 2. In some embodiments, the nucleoside of Z is a 2'-MOE nucleoside. In some embodiments, Z represents a non-hybridizing nucleoside. To avoid confusion, it is noted that such a non-hybridizing nucleoside may also be described as a 3'-terminal group with Z=0.

[0372] Combination motifs It should be understood that some of the motifs and modifications described above can be combined. A motif can contain only a small number of nucleotides, so a particular oligonucleotide can contain two or more motifs. As a non-limiting example, in some embodiments, an oligonucleotide in a multi-target molecule can have two or more nucleotide motifs selected from LNA, phosphorothioate bond, 2'-OMe, and conjugate ligand.

[0373] Without limitation, multi-targeting molecules of the present invention having any of the various nucleotide motifs described herein can also have any linkage motif. For example, in oligonucleotides present in a multi-targeting molecule, the first 1, 2, 3, 4, or 5 intersugar linkages at the 5' end can be modified intersugar linkages, and the first 4, 5, 6, 7, or 8 intersugar linkages at the 3' end can be modified intersugar linkages. The central region of such modified oligonucleotides can have intersugar linkages based on any of the other motifs described herein, such as homogeneous, alternating, hemimer, gapmer, etc. In some embodiments, oligonucleotides present in a multi-targeting molecule contain a phosphorothioate linkage between the first and second monomer at the 5' end, alternating phosphorothioate / phosphodiester linkages in the central region, and 6, 7, or 8 phosphorothioate linkages at the 3' end.

[0374] It should be noted that the length of the region defined by the nucleotide motif and the length of the binding motif do not have to be the same.

[0375] In some embodiments, at least one strand of the single-stranded oligonucleotide or double-stranded oligonucleotide comprises at least one of the following motifs: (a) 5'-phosphorothioate or 5'-phosphorodithioate; (b) cationic modifications of 5'-terminal nucleotides 1 and 2, wherein the cationic modifications are located at the C5 position of the pyrimidine and at C2, C6, C8, exocyclic N2 or exocyclic N6 of the purine; (c) at least one G-clamp nucleotide and another nucleotide having a cationic modification in the first two terminal nucleotides of the 5' end, where the cationic modification is located at the C5 position of a pyrimidine or the C2, C6, C8, exocyclic N2 or exocyclic N6 position of a purine; (d) at least one 2'-F modified nucleotide containing a nucleobase modification; (e) at least one gem-2'-O-methyl / 2'-F modified nucleotide comprising a nucleobase modification, preferably wherein the methyl substituent is in the up configuration, e.g., arabinose configuration; (f) a 3'-terminal 5'-PuPu-3' dinucleotide, wherein both nucleotides contain a modified MOE at the 2' position as described in U.S. Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference in their entirety; (g) a 5'-terminal 5'-PuPu-3' dinucleotide, wherein both nucleotides contain a modified MOE at the 2' position as described in U.S. Patent Application Publication No. 20130130378; (h) a 5'-terminal nucleotide comprising a modified MOE at the 2' position as described in U.S. Patent Application Publication No. 20130130378; (i) a 5'-terminal nucleotide with a 3'-F modification; (j) a 5'-terminal nucleotide containing a 4'-substituent; (k) a 5′-terminal nucleotide containing an O4′ modification; (l) a 3'-terminal nucleotide containing a 4'-substituent; and (m) Combinations of these.

[0376] In some embodiments, both strands of a double-stranded oligonucleotide independently comprise at least one of the motifs described above. In some other embodiments, both strands of a double-stranded oligonucleotide comprise at least one of the motifs described above, and these motifs can be the same or different, or some combination of the same and different.

[0377] The foregoing examples are provided merely to illustrate how the described motifs can be used in combination and are not intended to limit the invention to specific combinations or the specific modifications used to exemplify the combinations. Furthermore, specific examples herein, such as, but not limited to, those shown in the table above, are intended to encompass more general embodiments. For example, column A of the table above exemplifies a region of alternating 2'-OMe and 2'-F nucleosides. Accordingly, this same disclosure also exemplifies regions of alternating other 2'-modifications. It also exemplifies regions of alternating 2'-O-alkyl and 2'-halogen nucleosides. It also exemplifies regions of alternating different modified nucleosides. All examples throughout this specification contemplate this inclusive interpretation.

[0378] It is also noted that the length of the oligonucleotides present in a compound, e.g., a multi-target molecule, can be easily manipulated by extending or shortening one or more of the described regions without disrupting the motif.

[0379] In some embodiments, the oligonucleotides in the effector molecule or multitargeting molecule comprise two or more chemically distinct regions and have the structure described in International Application PCT / US 09 / 038433, filed March 26, 2009, the contents of which are incorporated herein in their entirety.

[0380] Synthesis, purification and analysis Oligomerization of modified and unmodified nucleosides and nucleotides can be routinely carried out according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and / or RNA (Scaringe, Methods (2001), 23, 206-217; Gait et al., Applications of Chemically Synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36; Gallo et al., Tetrahedron (2001), 57, 5707-5713).

[0381] Nucleic acids, such as oligonucleotides, can be conveniently and routinely produced by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides such as phosphorothioates and alkylated derivatives. The present invention is not limited by the method of synthesis.

[0382] Methods for purifying and analyzing nucleic acids are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. These synthesis and analysis methods can be performed in multi-well plates. The method of the present invention is not limited by the oligomer purification method.

[0383] Nucleic acids such as oligonucleotides can also be prepared using liquid-phase or solid-phase organic synthesis, or enzymatically by methods known in the art. Organic synthesis offers the advantage of easily preparing oligonucleotide chains containing unnatural or modified nucleotides. Any other means for such synthesis known in the art can additionally or alternatively be used. It is also known to use similar techniques to prepare other nucleic acids, such as those containing phosphorothioate, phosphorodithioate, and alkylated derivatives of the intersugar linkage. Double-stranded nucleic acids can be prepared using a two-step method. First, the individual strands of the double-stranded molecule are prepared separately. Then, the constituent strands are annealed.

[0384] Regardless of the method of synthesis, the nucleic acid can be prepared in a solution (e.g., water and / or organic solution) appropriate for formulation. For example, the nucleic acid preparation can be precipitated, redissolved in pure double-distilled water, and then lyophilized. The dried nucleic acid can then be resuspended in a solution appropriate for the intended formulation method.

[0385] Teachings regarding the synthesis of particular modified nucleic acids can be found in the following U.S. patents or pending patent applications: U.S. Pat. Nos. 5,138,045 and 5,218,105 for polyamine-conjugated oligonucleotides; U.S. Pat. No. 5,212,295 for monomers for the preparation of oligonucleotides with chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307 for oligonucleotides with modified backbones; and U.S. Pat. Nos. 5,378,825 and 5,541,307 for backbone-modified oligonucleotides and their synthesis by reductive coupling. U.S. Patent No. 5,386,023 for the preparation of; U.S. Patent No. 5,457,191 for modified nucleobases based on 3-deazapurine ring systems and methods for their synthesis; U.S. Patent No. 5,459,255 for modified nucleobases based on N-2 substituted purines; U.S. Patent No. 5,521,302 for methods for preparing oligonucleotides with chiral phosphorus linkages; U.S. Patent No. 5,539,082 for peptide nucleic acids; U.S. Patent No. 5,554,746 for oligonucleotides with β-lactam backbones; U.S. Pat. No. 5,571,902 relates to methods and materials for the synthesis of nucleotides; U.S. Pat. No. 5,578,718 relates to nucleosides having alkylthio groups (such groups can be used as linkers to other moieties attached to any of the various positions of the nucleoside); U.S. Pat. Nos. 5,587,361 and 5,599,797 relate to oligonucleotides having phosphorothioate linkages of high chiral purity; 2'-O-alkylguanosine and related compounds (2,6-diaminopurine compounds); U.S. Patent No. 5,506,351 for methods of preparing oligonucleotides having N-2 substituted purines; U.S. Patent No. 5,587,469 for oligonucleotides having 3-deazapurines; U.S. Patent No. 5,223,168 and U.S. Patent No. 5,608,046, both for conjugated 4'-desmethyl nucleoside analogs; U.S. Patent Nos. 5,602,240 and 5,610,289 for backbone-modified oligonucleotide analogs;and U.S. Patent Nos. 6,262,241 and 5,459,255, which relate inter alia to methods for synthesizing 2'-fluoro-oligonucleotides.

[0386] Compositions and methods for formulating pharmaceutical compositions The effector molecule conjugated to the ligand or multitargeting molecule can be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of a pharmaceutical composition or formulation. The composition and method for the formulation of a pharmaceutical composition depend on several criteria, including but not limited to the route of administration, the extent of the disease, or the dose to be administered.

[0387] Effector molecules conjugated to ligands or multitargeting molecules can be used in pharmaceutical compositions by combining such oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in compositions to be delivered parenterally. Thus, in some embodiments, pharmaceutical compositions comprising an effector molecule conjugated to a ligand via a linker disclosed herein and a pharmaceutically acceptable diluent are used in the methods described herein. In some embodiments, pharmaceutical compositions comprising a multitargeting molecule and a pharmaceutically acceptable diluent are used in the methods described herein. In some embodiments, the pharmaceutically acceptable diluent is PBS.

[0388] Pharmaceutical compositions comprising multitargeting molecules include any pharmaceutically acceptable salts, esters, or salts of such esters. In some embodiments, pharmaceutical compositions comprising multitargeting molecules include one or more oligonucleotides that can provide (directly or indirectly) biologically active metabolites or residues thereof upon administration to an animal, such as a human. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0389] Prodrugs can involve the incorporation of additional nucleosides at one or both ends of the multitarget molecule that are cleaved by endogenous nucleases in the body to form the active molecule.

[0390] The pharmaceutical compositions of the present invention can be administered in various ways depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary, e.g., using a nebulizer, by inhalation or insufflation of powder or aerosol; intratracheal, intranasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, e.g., via an implantable device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular, administration. Multitargeting molecules can be delivered to target specific tissues, such as the liver (e.g., hepatocytes of the liver).

[0391] Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the subject multitargeting molecules of the present invention are mixed with topical delivery agents, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The multitargeting molecules that are the subject of the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the multitargeting molecules can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 These include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0392] There are many organized surfactant structures other than microemulsions that have been studied and used for drug formulation. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted great interest from the perspective of drug delivery due to the specificity and prolonged action they offer. As used in the present invention, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayer.

[0393] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse with the cell wall as efficiently, but are taken up by macrophages in vivo.

[0394] Further advantages of liposomes are as follows: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water- and lipid-soluble drugs; liposomes can protect the drugs encapsulated in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size and liposome water content.

[0395] Liposomes are useful for transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to fuse with cell membranes, and as the fusion of liposomes with cells progresses, the contents of the liposomes are transferred into the cells, where the active agent can act.

[0396] Liposomal formulations have been the subject of extensive research as a delivery method for many drugs. For topical administration, there is growing evidence that liposomes offer several advantages over 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 site, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0397] Several reports have detailed the ability of liposomes to deliver drugs, including high molecular weight DNA, into the skin. Compounds including analgesics, antibodies, hormones, and high molecular weight DNA have been delivered to the skin. In most cases, targeting of the upper epidermis was achieved by application.

[0398] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are internalized in endosomes. Due to the acidic pH inside the endosomes, the liposomes are ruptured, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0399] Liposomes that are pH-sensitive or negatively charged entrap DNA rather than complexing with it. Because both DNA and lipids are similarly charged, repulsion occurs rather than complexation. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected within the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0400] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0401] Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. When liposomes containing interferon were applied to the skin of guinea pigs, relief of cutaneous herpes was achieved, whereas delivery of interferon by other means (e.g., as a solution or emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, another study tested the efficacy of interferon administered as part of a liposomal formulation versus administration using an aqueous system, concluding that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0402] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. Results indicated that these nonionic liposomal systems were effective in promoting the deposition of cyclosporine A into various layers of the skin (Hu et al., STP Pharma. Sci., 1994, 4, 6, 466).

[0403] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, result in increased circulation life compared to liposomes without such specialized lipids. Examples of sterically stabilized liposomes include those in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1 or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without intending to be bound by any particular theory, it is believed in the art that the increased circulation lifetime of sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0404] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) report the use of monosialoganglioside G, which improves the blood half-life of liposomes. M1 (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88 / 04924 (both by Allen et al.) report the ability of (1) sphingomyelin and (2) ganglioside G M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0405] Many liposomes containing lipids derivatized with one or more hydrophilic polymers and methods for their preparation are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe a method for preparing liposomes containing a nonionic detergent, 2C, containing a PEG moiety. 1215G(FEBS Lett., 1984, 167, 79) described liposomes containing PEG-derivatized phosphatidylethanolamine (PE) or PEG stearate as liposomes containing PEG-derivatized phosphatidylethanolamine (PE). Illum et al. (FEBS Lett., 1984, 167, 79) described that hydrophilic coating of polystyrene particles with polymeric glycols significantly increased their blood half-life. Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899) described synthetic phospholipids modified by the attachment of carboxylic acid groups of polyalkylene glycols (e.g., PEG). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing PEG-derivatized phosphatidylethanolamine (PE) or PEG stearate have significantly increased blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently bound PEG moieties on their outer surface are described by Fisher in European Patent No. 0 445 131 B1 and International Publication No. WO 90 / 04384. Liposomal compositions containing 1 to 20 mole percent PE derivatized with PEG and methods of use thereof have been described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. 0 496 813 B1). Liposomes containing several other lipid-polymer conjugates are disclosed in WO 91 / 05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and WO 94 / 20073 (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in WO 96 / 10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0406] Some liposomes containing nucleic acids are known in the art. Thierry et al., International Publication No. 96 / 40062, discloses a method for encapsulating high molecular weight nucleic acids into liposomes. Tagawa et al., U.S. Patent No. 5,264,221, discloses protein-bound liposomes and claims that the contents of such liposomes can contain dsRNA. Rahman et al., U.S. Patent No. 5,665,710, describes a specific method for encapsulating oligodeoxynucleotides into liposomes. Love et al., International Publication No. 97 / 04787, discloses liposomes containing dsRNA targeted to the raf gene.

[0407] Transfersomes, another type of liposome, are highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets, which are highly deformable and therefore can easily penetrate pores smaller than these lipid droplets. Transfersomes can adapt to the environment in which they are used, for example, self-optimizing (adapting to the shape of skin pores), self-repairing, and often self-filling, reaching their target without fragmentation. To prepare transfersomes, a surface-active agent, usually a surfactant, can be added to a standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been found to be as effective as subcutaneous injection of a solution containing serum albumin.

[0408] Research Tools In some cases, oligonucleotides capable of modulating gene expression have been used as research tools. For example, researchers investigating the function of a particular gene product can design oligonucleotides that reduce the amount of that gene product present in a cell or animal and observe phenotypic changes in the cell or animal. In some embodiments, the invention provides methods for reducing the amount of two different targets in a cell or animal. In some embodiments, the two different targets can be two different genes or gene products. In some embodiments, the two different targets can be the same gene or gene product. In some embodiments, researchers can use such techniques to characterize proteins or untranslated nucleic acids. In some embodiments, such experiments are used to examine the kinetics and / or turnover of gene products and / or certain cellular functions. In some embodiments, such experiments are used to examine the relationship or interrelationship between different genes or gene products.

[0409] kit In some embodiments, the present invention provides kits comprising one or more multi-targeting molecules. In some embodiments, such kits are intended for therapeutic use. In some embodiments, such kits are intended for research use.

[0410] In some embodiments, the present invention provides a kit comprising at least one effector molecule conjugated to a ligand via a linker as described herein. In some embodiments, such kits are intended for therapeutic use. In some embodiments, such kits are intended for research use.

[0411] While the specific compounds, compositions, and methods described herein have been described in detail in accordance with several embodiments, the following examples are merely used to illustrate the compounds described herein and are not intended to limit the invention. All references, GenBank accession numbers, and the like cited in this application are hereby incorporated by reference in their entirety.

[0412] definition Unless specifically defined, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Some such techniques and procedures can be found, for example, in "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications," Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2002. nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. To the extent permitted, all patents, applications, published applications, and other publications and other materials referred to in this disclosure are incorporated herein by reference in their entirety.

[0413] Unless otherwise indicated, the following terms have the following meanings:

[0414] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be regulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target protein (including, but not limited to, pre-mRNA and mRNA or portions thereof), as well as cDNA and miRNA obtained from such RNA. For example, a target nucleic acid may be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a particular disorder or pathology. In some embodiments, a target nucleic acid may be a nucleic acid molecule derived from an infectious agent.

[0415] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in mRNA levels in a cell of a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between these. In one preferred embodiment, mRNA levels are reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between 5% and 100%.

[0416] As used herein, the term "modulate gene expression" means up-regulating or down-regulating the expression of a gene encoding one or more proteins or protein subunits or levels of an RNA molecule or equivalent RNA molecule such that expression, level, or activity is higher or lower than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," although use of the term "modulate" is not limited to this definition.

[0417] As used herein, gene expression modulation occurs when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The percentage and / or fold difference can be calculated, for example, relative to a control or non-control, as follows:

number

[0418] As used herein, the terms "inhibit," "down-regulate," or "reduce," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced by at least 10% relative to a corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% (i.e., no gene expression).

[0419] As used herein, the terms "increase" or "up-regulate," with respect to gene expression, means that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased by at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more, relative to a corresponding unmodulated control.

[0420] As used herein, the term "increased" or "increase" generally refers to an increase by a statistically significant amount; for the avoidance of doubt, "increased" means an increase of at least 10% compared to a base line level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% compared to a base line level, or any increase between 10 and 100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more increase compared to a base line level.

[0421] As used herein, the term "reduced" or "reducing" generally refers to a statistically significant decrease. However, for the avoidance of doubt, "reduced" refers to a decrease of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of less than 100% (i.e., a zero level compared to a reference sample), or any decrease between 10 and 100%.

[0422] "Specifically hybridizable" and "complementary" mean that a nucleic acid can form hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types. In the context of the nucleic acid molecules of the present invention, the binding free energy between a nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, such as RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, Am. Chem. Soc. 109: 3783-3785). Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to a situation in which some (but not all) nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, except for regions of the polynucleotide strands selected to be non-complementary (e.g., overhangs). Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0423] The term "off-target" and the phrase "off-target effect" refer to any instance in which an effector molecule for a given target causes an unintended effect by interacting, either directly or indirectly, with another target sequence, DNA sequence, or cellular protein or other moiety. For example, an "off-target effect" can occur when partial homology or complementarity between other transcripts and the sense and / or antisense strands of an siRNA results in the simultaneous degradation of other transcripts.

[0424] As used herein, the term "nucleoside" refers to a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with mimetic base and / or sugar groups.

[0425] As used herein, the term "nucleotide" refers to a glycosomine comprising a nucleobase and a sugar to which a phosphate group is covalently attached. Nucleotides can be modified with any of a variety of substituents.

[0426] As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase can include any atom or group of atoms that is capable of hydrogen bonding to a base of another nucleic acid.

[0427] As used herein, the term "heterocyclic base moiety" refers to a nucleobase that comprises a heterocycle.

[0428] As used herein, the term "oligomeric compound" refers to a polymeric structure comprising two or more substructures and capable of hybridizing to a region of a nucleic acid molecule. In some embodiments, the oligomeric compound is an oligonucleoside. In some embodiments, the oligomeric compound is an oligonucleotide. In some embodiments, the oligomeric compound is an antisense compound. In some embodiments, the oligomeric compound is an antidote compound. In some embodiments, the oligomeric compound comprises a conjugate group.

[0429] As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0430] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising multiple linked nucleosides. In certain embodiments, one or more nucleotides of an oligonucleotide are modified. In some embodiments, an oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, an oligonucleotide is composed of natural and / or non-natural nucleobases, sugars, and covalent internucleoside linkages, and may further comprise non-nucleic acid conjugates.

[0431] As used herein, "internucleoside linkage" refers to the covalent bond between adjacent nucleosides.

[0432] As used herein, "naturally occurring internucleoside linkage" refers to a 3'-5' phosphodiester linkage.

[0433] As used herein, the term "detecting siRNA activity" or "measuring siRNA activity" means that a test for detecting or measuring siRNA activity is carried out on a specific sample and compared with that of a control sample. Such detection and / or measurement may include a value of zero. Therefore, even if the test for detecting siRNA activity shows that there is no siRNA activity (zero siRNA activity), the step of "detecting siRNA activity" is still carried out.

[0434] As used herein, the term "control sample" refers to a sample that has not been contacted with a reporter oligomeric compound.

[0435] As used herein, the term "motif" refers to a pattern of unmodified and modified nucleotides in an oligomeric compound.

[0436] As used herein, the term "chimeric oligomer" refers to an oligomeric compound having at least one sugar, nucleobase, or internucleoside linkage that is modified in a different manner compared to at least one other sugar, nucleobase, or internucleoside linkage within the same oligomeric compound. The remainder of the sugars, nucleobases, and internucleoside linkages can be independently modified or unmodified, and can be the same or different.

[0437] As used herein, the term "chimeric oligonucleotide" refers to an oligonucleotide having at least one sugar, nucleobase, or internucleoside linkage that is modified in a different manner compared to at least one other sugar, nucleobase, or internucleoside linkage within the same oligonucleotide. The remainder of the sugars, nucleobases, and internucleoside linkages can be independently modified or unmodified, and can be the same or different.

[0438] As used herein, the term "mixed backbone oligomeric compound" refers to an oligomeric compound in which at least one internucleoside linkage of the oligomeric compound is different from at least one other internucleoside linkage of the oligomeric compound.

[0439] As used herein, the term "target protein" refers to a protein whose modulation is desired.

[0440] As used herein, the term "target gene" refers to a gene that encodes a target protein.

[0441] As used herein, the terms "targeting" or "targeted" refer to the association of the antisense strand of an siRNA with a specific target nucleic acid molecule or a specific region of nucleotides within a target nucleic acid molecule.

[0442] As used herein, the term "nucleobase complementarity" refers to a nucleobase that can base pair with another nucleobase.For example, in DNA, adenine (A) is complementary to thymine (T).For example, in RNA, adenine (A) is complementary to uracil (U).In some embodiments, complementary nucleobase refers to the nucleobase of an antisense compound that can base pair with the nucleobase of its target nucleic acid.For example, if the nucleobase at a specific position of an antisense compound can hydrogen bond with the nucleobase at a specific position of target nucleic acid, the hydrogen bond position between oligonucleotide and target nucleic acid is considered to be complementary at this nucleobase pair.

[0443] As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise support hybridization.

[0444] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize with another oligomeric compound or nucleic acid through nucleobase complementarity. In some embodiments, an oligomeric compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bind to each other, allowing stable association between the antisense compound and the target. Those skilled in the art will recognize that mismatches can be included without eliminating the ability of the oligomeric compound to maintain association. Thus, described herein are oligomeric compounds (e.g., siRNa, multi-target molecules, etc.) that can contain up to about 20% mismatched nucleotides (i.e., nucleobases that are not complementary to the corresponding nucleotides of the target). Preferably, oligomeric compounds such as siRNAs and multi-target molecules contain no more than about 15%, more preferably no more than about 10%, and most preferably no more than 5% mismatches. The remaining nucleotides are complementary nucleobases or do not disrupt hybridization (e.g., universal bases). Those skilled in the art will recognize that the compounds provided herein may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target nucleic acid.

[0445] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (for example, the antisense strand of siRNA and the antisense and sense strands of its target nucleic acid or siRNA).Not limited to a specific mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleoside or nucleotide bases (nucleobases), which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds.For example, the natural base adenine is a nucleobase that is complementary to the natural nucleobases thymidine and uracil, which pair through the formation of hydrogen bonds.The natural base guanine is a nucleobase that is complementary to the natural bases cytosine and 5-methylcytosine.Hybridization can occur under various circumstances.

[0446] As used herein, the term " specifically hybridize " refers to the ability of an oligomeric compound to hybridize with one nucleic acid site with higher affinity than to hybridize with other nucleic acid sites.In some embodiments, the antisense strand of siRNA specifically hybridizes with two or more target sites.

[0447] As used herein, "design" or "designed" refers to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

[0448] As used herein, the term "modulation" refers to the perturbation of function or activity compared to the level of function or activity prior to modulation. For example, modulation includes a change in gene expression, either an increase (stimulation or induction) or a decrease (inhibition or suppression). As another example, modulation of expression can include perturbation of splice site selection in pre-mRNA processing.

[0449] As used herein, the term "expression" refers to all the functions and steps by which a gene's coded information is converted into structures present and operating in a cell, including, but not limited to, the products of transcription and translation.

[0450] As used herein, "variant" refers to alternative RNA transcripts that can be produced from the same genomic region of DNA. Variants include, but are not limited to, "pre-mRNA variants," which are transcripts produced from the same genomic DNA that differ in either their start or end positions from other transcripts produced from the same genomic DNA, and include both intronic and exon sequences. Variants may also have, but are not limited to, alternative splice sites or alternative start and stop codons.

[0451] As used herein, a "high-affinity modified monomer" refers to a monomer that has at least one modified nucleobase, internucleoside linkage, or sugar moiety, compared to a naturally occurring monomer, such that this modification increases the affinity of the antisense compound comprising the high-affinity modified monomer for its target nucleic acid. High-affinity modifications include, but are not limited to, monomers (e.g., nucleosides and nucleotides) that contain 2'-modified sugars.

[0452] As used herein, the term "2'-modified" or "2'-substituted" refers to a sugar containing a substituent at the 2' position other than H or OH. 2'-modified monomers include, but are not limited to, 2'-substituents such as allyl, amino, azido, thio, O-allyl, O-C1-C2 10 Included are BNAs and monomers (e.g., nucleosides and nucleotides) having alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 In some embodiments, the oligomeric compound has the formula 2'-O(CH2) nIn some embodiments, the oligomeric compounds comprise 2' modified monomers that lack H (where n is 1-6). In some embodiments, the oligomeric compounds comprise 2' modified monomers that lack the formula 2'-OCH3. In some embodiments, the oligomeric compounds comprise 2' modified monomers that lack the formula above or, in another embodiment, 2'-O(CH2)2OCH3.

[0453] As used herein, the term "locked nucleic acid" or "LNA" or "locked nucleoside" or "locked nucleotide" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic system. Locked nucleic acids are also called bicyclic nucleic acids (BNAs).

[0454] As used herein, unless otherwise indicated, the term "methyleneoxyLNA" refers solely to β-D-methyleneoxyLNA.

[0455] As used herein, the term "MOE" refers to a 2'-O-methoxyethyl substituent.

[0456] As used herein, the term "gapmer" refers to a chimeric oligomeric compound comprising a central region (the "gap") and regions on either side of the central region (the "wings"), wherein the gap contains at least one modification that differs from each wing. Such modifications include nucleobase, monomer linkage, and sugar modifications, as well as the absence of a modification (unmodified). Thus, in some embodiments, the nucleotide linkage in each of the wings differs from the nucleotide linkage in the gap. In some embodiments, each wing contains nucleotides with a high-affinity modification, and the gap contains nucleotides that do not contain that modification. In some embodiments, the nucleotides in the gap and the nucleotides in the wings all contain a high-affinity modification, but the high-affinity modification in the gap differs from the high-affinity modification in the wings. In some embodiments, the modifications in the wings are the same as each other. In some embodiments, the modifications in the wings differ from each other. In some embodiments, the nucleotides in the gap are unmodified and the nucleotides in the wings are modified. In some embodiments, the modifications in each wing are the same. In some embodiments, the modification in one wing differs from the modification in the other wing. In some embodiments, the oligomeric compounds are gapmers having 2'-deoxynucleotides in the gap and nucleotides with high affinity modifications in the wings.

[0457] As used herein, the term "prodrug" refers to a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and / or conditions.

[0458] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of an active compound that retains the desired biological activity of the active compound and does not impart undesired toxicological effects.

[0459] As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification incorporated at either end of an antisense compound.

[0460] As used herein, the term "prevention" refers to delaying or forestalling the onset or occurrence of a condition or disease for a period of hours to days, preferably weeks to months.

[0461] As used herein, the term "amelioration" refers to a reduction in at least one activity or severity indicator of a condition or disease. The severity of the indicator can be determined by subjective or objective measures known to those skilled in the art.

[0462] As used herein, the term "treatment" refers to administering a composition of the present invention to effect an alteration or improvement of a disease or condition. Prevention, improvement, and / or treatment may require the administration of multiple doses at regular intervals or prior to the onset of the condition or disease to alter the course of the disease or condition. Furthermore, a single agent may be used sequentially or simultaneously in one individual for the prevention, improvement, and treatment of a condition or disease, respectively.

[0463] As used herein, the term "pharmaceutical agent" refers to a substance that provides a therapeutic benefit when administered to a subject. In some embodiments, the pharmaceutical agent is an active pharmaceutical agent. In some embodiments, the pharmaceutical agent is a prodrug.

[0464] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent that confers a therapeutic benefit on an animal.

[0465] As used herein, "administering" means providing a pharmaceutical agent to an animal and includes, but is not limited to, administration by a medical professional and self-administration.

[0466] As used herein, the term "co-administer" means providing two or more pharmaceutical agents to an animal. In some embodiments, the two or more pharmaceutical agents are administered together. In some embodiments, the two or more pharmaceutical agents are administered separately. In some embodiments, the two or more pharmaceutical agents are administered simultaneously. In some embodiments, the two or more pharmaceutical agents are administered at different times. In some embodiments, the two or more pharmaceutical agents are administered by the same route of administration. In some embodiments, the two or more pharmaceutical agents are administered by different routes of administration. In some embodiments, the two or more pharmaceutical agents are contained in the same pharmaceutical formulation. In some embodiments, the two or more pharmaceutical agents are in the form of separate formulations.

[0467] As used herein, the term "pharmaceutical composition" refers to a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution. In some embodiments, a pharmaceutical composition comprises a pharmaceutical agent and a diluent and / or carrier.

[0468] As used herein, the term "in vitro" refers to events that occur not within an organism (e.g., an animal or plant) but in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc. As used herein, the term "ex vivo" refers to cells removed from a living organism and cultured outside the organism (e.g., in a test tube). As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal, plant, and / or microorganism).

[0469] As used herein, the term "subject" or "patient" refers to any living organism to which a composition disclosed herein can be administered, for example, for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Typically, animals are vertebrates such as primates, rodents, livestock, or sport animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys (Rhesus). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and sport animals include cattle, horses, pigs, deer, bison, water buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, and ostriches, and fish, such as trout, catfish, and salmon. A patient or subject includes any subset of those listed above, e.g., all of the foregoing, but excluding one or more groups or species, such as humans, primates, or rodents. In some embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient" and "subject" are used interchangeably herein. A subject can be male or female.

[0470] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects to serve as animal models of human diseases and disorders. Furthermore, the compounds, compositions, and methods described herein can be used with livestock and / or pets.

[0471] In some embodiments, the subject is a human. In other embodiments, the subject is an experimental animal or surrogate animal for a disease model. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, as well as fetuses, whether male or female. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is also intended to include transgenic species. In some embodiments, the subject may be of European descent. In some embodiments, the subject may be African American. In some embodiments, the subject may be of Asian descent.

[0472] In jurisdictions that prohibit the patenting of methods performed on the human body, "administering" a composition to a human subject will be limited to prescribing a controlled substance for the human subject to self-administer by any method (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the statute or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods performed on the human body, "administering" a composition will encompass both methods performed on the human body and the aforementioned activities.

[0473] As used herein, the term "parenteral administration" refers to administration by injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

[0474] As used herein, the term "subcutaneous administration" refers to administration just below the skin. "Intravenous administration" means administration into a vein.

[0475] As used herein, the term "dose" refers to a specified amount of a pharmaceutical agent delivered in a single administration. In some embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dose requires an amount that cannot be easily delivered by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In some embodiments, a dose may be administered in two or more injections to minimize injection site reactions in individuals.

[0476] As used herein, the term "unit dosage form" refers to the form in which a pharmaceutical agent is provided. In some embodiments, the unit dosage form is a vial containing lyophilized antisense oligonucleotide. In some embodiments, the unit dosage form is a vial containing reconstituted antisense oligonucleotide.

[0477] As used herein, the term "active pharmaceutical ingredient" refers to the substance in a pharmaceutical composition that imparts a desired effect.

[0478] As used herein, the term "side effect" refers to a physiological response attributed to a treatment other than the desired effect. In some embodiments, side effects include, but are not limited to, injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. For example, elevated serum aminotransferase levels may indicate hepatotoxicity or liver function abnormalities. For example, elevated bilirubin may indicate hepatotoxicity or liver function abnormalities.

[0479] As used herein, the term "alkyl," as used herein, refers to a saturated straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. Alkyl groups typically contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms (C1-C12 alkyl), with 1 to about 6 carbon atoms being more preferred. As used herein, the term "lower alkyl" contains 1 to about 6 carbon atoms. As used herein, alkyl groups can optionally include one or more additional substituents.

[0480] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain radical containing up to 24 carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein can optionally contain one or more additional substituents.

[0481] As used herein, the term "alkynyl," as used herein, refers to a straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more additional substituents.

[0482] As used herein, the term "aminoalkyl," as used herein, refers to an amino-substituted alkyl radical. This term is meant to include C1-C12 alkyl groups having an amino substituent at any position, where the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and / or amino portions of the aminoalkyl group can be further substituted with substituents.

[0483] As used herein, the term "aliphatic" refers to a straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms, with the saturation between any two carbon atoms being a single, double, or triple bond. Aliphatic groups preferably contain from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms, with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of the aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Examples of such heteroatom-interrupted aliphatic groups include, but are not limited to, polyalkoxy groups, such as polyalkylene glycols, polyamines, and polyimines. The aliphatic groups used herein may optionally contain other substituents.

[0484] As used herein, the term "alicyclic" or "alicyclyl" refers to a ring system in which the ring is aliphatic. The ring system can include one or more rings in which at least one ring is aliphatic. Preferred alicyclic compounds include rings having from about 5 to about 9 carbon atoms in the ring. As used herein, alicyclics can optionally include other substituents. As used herein, the term "alkoxy," as used herein, refers to a radical formed between an alkyl group and an oxygen atom, where the oxygen atom is used to attach the alkoxy group to the parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. As used herein, alkoxy groups can optionally include other substituents. As used herein, the terms "halo" and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0485] As used herein, the terms "aryl" and "aromatic," as used herein, refer to a monocyclic or polycyclic carbocyclic ring system radical having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in the ring or rings. Aryl groups as used herein can optionally include other substituents.

[0486] As used herein, the terms "aralkyl" and "arylalkyl," as used herein, refer to a radical formed between an alkyl group and an aryl group, where the alkyl group is used to attach the aralkyl group to the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, and the like. Aralkyl groups as used herein can optionally include additional substituents attached to the alkyl, aryl, or both groups that form the radical group.

[0487] As used herein, the term "heterocyclic radical" refers to a radical monocyclic or polycyclic ring system that contains at least one heteroatom and is unsaturated, partially saturated, or fully saturated, and thus includes heteroaryl groups. Heterocyclic is also meant to include fused ring systems, where one or more of the fused rings contain at least one heteroatom, and the other rings may contain one or more heteroatoms, or optionally no heteroatoms. Heterocyclic groups typically contain at least one atom selected from sulfur, nitrogen, or oxygen. Examples of heterocyclic groups include [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and the like. As used herein, heterocyclic groups may optionally contain other substituents. As used herein, the terms "heteroaryl" and "heteroaromatic" refer to a radical containing a monocyclic or polycyclic aromatic ring, ring system, or fused ring system in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl is also meant to include fused ring systems, including systems in which one or more fused rings do not contain heteroatoms. Heteroaryl groups typically contain one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. A heteroaryl radical can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. Heteroaryl groups as used herein can optionally include other substituents.

[0488] As used herein, the term "heteroarylalkyl" refers to a heteroaryl group, as previously defined, having an alkyl radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, naphthyridinylpropyl, and the like. Heteroarylalkyl groups as used herein can optionally include other substituents on either or both of the heteroaryl or alkyl portions.

[0489] As used herein, the term "monocyclic or polycyclic structure" encompasses all ring systems, whether monocyclic or polycyclic with fused or linked rings, including monocyclic or mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, and heteroarylalkyl. Such monocyclic and polycyclic structures can be homogeneous or contain rings with varying degrees of saturation, such as fully saturated, partially saturated, or fully unsaturated. Each ring contains ring atoms selected from C, N, O, and S, thereby forming heterocyclic and mixed motifs, such as benzimidazole, where one ring contains only carbon ring atoms and the fused ring contains two nitrogen atoms. The monocyclic or polycyclic structure can be further substituted with substituents, such as phthalimide, which has two =O groups attached to one of the rings. In another embodiment, the monocyclic or polycyclic structure can be attached to the parent molecule directly through a ring atom, through a substituent, or through a difunctional linking moiety.

[0490] As used herein, the term "acyl," as used herein, refers to a radical formed by removal of a hydroxyl group from an organic acid and has the general formula -C(O)-X, where X is typically aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates, and the like. As used herein, acyl groups can optionally include other substituents.

[0491] As used herein, the term "hydrocarbyl" includes groups containing C, O, and H. Included are straight-chain, branched, and cyclic groups of any degree of saturation. Such hydrocarbyl groups may contain one or more heteroatoms selected from N, O, and S, and may be further mono- or polysubstituted with one or more substituents.

[0492] As used herein, the terms "substituent" and "substituent group," as used herein, include groups that are typically added to other groups or parent compounds to enhance a desired property or impart a desired effect. Substituents can be either protected or unprotected and can be added to one available site or multiple available sites within the parent compound. Substituents can also be further substituted with other substituents and can be attached to the parent compound directly or through a linking group such as an alkyl or hydrocarbyl group. Such groups include, but are not limited to, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxo (—O-Raa), aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino (—NRbbRcc), imino (═NRbb), amido (—C(O)N-RbbRcc or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)NRbbR cc or -N(Rbb)C(O)ORaa), ureido (-N(Rbb)C(O)NRbbRcc), thioureido (-N(Rbb)C(S)NRbbRcc), guanidinyl (-N(Rbb)C(=NRbb)NRbbRcc), amidinyl (-C(=NRbb)-NRbbRcc or -N(Rbb)C(NRbb)Raa), thiol (-SRbb), sulfinyl (-S(O)Rbb), sulfonyl (-S(O)Rbb), sulfonamidyl (-S(O)NRbbRcc or -N(Rbb)S(O)Rbb), and conjugate groups. Each Ra, Rbb, and Rcc is independently H, an optionally linked chemical functionality, or another substituent, with a preferred list including, but not limited to, H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic, and heteroarylalkyl.

[0493] The terms "bis(siRNA)" and "bis-siRNA" are used interchangeably herein and refer to two siRNAs that are covalently or non-covalently linked to form a single chemical entity that can cause RNAi activity in vitro and / or in vivo.Two linked siRNAs can target the same target gene at different sites, or two linked siRNAs can target the same site in target gene to cause RNAi-mediated gene silencing.In another embodiment of the present invention, two linked siRNAs can target two different genes.

[0494] For clarity, one of the siRNAs in the bis(siRNA) can regulate the gene expression of a first target nucleic acid, and the other siRNA in the bis(siRNA) can regulate the gene expression of a second target nucleic acid. In some embodiments, the first and second target nucleic acids are the same. In some other embodiments, the two siRNAs target the same nucleic acid sequence in the target nucleic acid.

[0495] In some other embodiments, one of the siRNAs in the bis(siRNA) is capable of regulating gene expression of a first target nucleic acid, and the other siRNA in the bis(siRNA) is capable of regulating gene expression of a second target nucleic acid, wherein the first and second target nucleic acids are different genes.

[0496] As used herein, one or more "linkers" include nucleotide and non-nucleotide linkers, or combinations thereof, that link two parts of a molecule, such as one or both strands of two individual siRNA molecules to generate a bis(siRNA). In some embodiments, a simple electrostatic or stacking interaction between two individual siRNAs can represent a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, aliphatic, alicyclic, heterocyclic compounds, and combinations thereof.

[0497] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradative molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degradative agents include redox agents that are selective for specific substrates or redox agents without substrate specificity, such as oxidizing or reducing enzymes or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linkers by reduction; esterases; amidases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and proteases, as well as phosphatases. For example, linkers can be cleaved by metabolic enzymes in vitro and / or in vivo. Exemplary metabolic enzymes include, but are not limited to, nucleases, proteases, peptidases, glycosylases, glycosidases, hydrolases, oxidase classes, and the like. [Example]

[0498] Example 1: Synthesis of bis(siRNA) with a cleavable linker bearing a targeting ligand [ka] As shown in Scheme 1, bis(siRNA)s are synthesized post-synthesis on a solid support or with a cleavable linker, followed by hybridization with a complementary strand.

[0499] Example 2: siRNA or Endosomotropic Agents with Cleavable Linkers Bearing Targeting Ligands [ka]

[0500] Example 3. Synthesis of siRNA-ASO with a cleavable linker bearing a targeting ligand [ka] As shown in Scheme 3, siRNA-ASO or ASO-siRNA is synthesized on a solid support or post-synthesis with a cleavable linker, followed by hybridization with a complementary strand.

[0501] Example 4. Synthesis of siRNA-anti-miR with a cleavable linker bearing a targeting ligand [ka] As shown in Scheme 4, siRNA-anti-miR or anti-miR-siRNA is synthesized on a solid support or post-synthesis with a cleavable linker, followed by hybridization with a complementary strand.

[0502] Example 5. Synthesis of bis(ASO) with a cleavable linker bearing a targeting ligand [ka] As shown in Scheme 5, bis(ASO)s are synthesized post-synthesis on a solid support or with a cleavable linker, followed by hybridization with a complementary strand.

[0503] Example 6. Synthesis of bis(anti-miR) with a cleavable linker bearing a targeting ligand [ka] As shown in Scheme 6, bis(anti-miRs) are synthesized on a solid support or post-synthesis with a cleavable linker, followed by hybridization with a complementary strand.

[0504] Example 7. Functionalized cleavable linkers and phosphoramidites [ka] As shown in Example 1 (Scheme 1), bis(siRNA)s are synthesized on a solid support by the sequential addition of one or more of these cleavable linkers, followed by hybridization with a complementary strand.

[0505] Example 8. Functionalized cleavable linkers and phosphoramidites [ka] As shown in Example 1 (Scheme 1), bis(siRNA)s are synthesized on a solid support by the sequential addition of one or more of these cleavable linkers, followed by hybridization with a complementary strand.

[0506] Example 9. Functionalized cleavable linkers and phosphoramidites [ka] As shown in Example 1 (Scheme 1), bis(siRNA)s are synthesized on a solid support by the sequential addition of one or more of these cleavable linkers, followed by hybridization with a complementary strand.

[0507] Example 10. Functionalized cleavable linkers and phosphoramidites [ka] As shown in Example 1 (Scheme 1), bis(siRNA)s are synthesized on a solid support by the sequential addition of one or more of these cleavable linkers, followed by hybridization with a complementary strand.

[0508] Example 11. Functionalized protease-cleavable linkers and phosphoramidites [ka] As shown in Example 1 (Scheme 1), bis(siRNA)s are synthesized on a solid support by the sequential addition of one or more of these cleavable linkers, followed by hybridization with a complementary strand.

[0509] Example 12. Synthesis of phosphoramidite 106 [ka] Compound (101) - Compound 100 (20 g, 51.4 mmol) was suspended in anhydrous dichloroethane (DCE) (200 mL). The reaction flask was evacuated and purged with argon. Trimethylsilyl trifluoromethanesulfonate (11.16 mL, 61.7 mmol) was added dropwise via syringe. The reaction was heated to 45 °C using a water bath and stirred overnight to obtain a clear solution. The reaction was monitored by TLC (5% MeOH / DCM) and developed using Hanessian stain. Upon completion, the reaction was cooled in an ice bath. Sodium bicarbonate (12.95 g, 154.2 mL) was taken and dissolved in 100 mL of water. The sodium bicarbonate solution was slowly added to the reaction mixture, causing effervescence. The reaction was allowed to stir for 20 minutes to fully neutralize. The mixture was added to a separatory funnel, the organic layer was separated, and the aqueous layer was washed with dichloromethane. The organic layers were combined and washed with brine. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated and placed under high vacuum to give (16.64 g) of 101. 1 H NMR (400MHz, DMSO-d6)δ 6.04(d,J=7.0Hz, 1H), 5.23(dd,J=3.9, 2.8Hz, 1H), 4.87(dd,J=6.9, 3.9Hz, 1H), 4.25(ddd,J=7.5, 5.0, 2.8Hz, 1H), 4.10(dd,J=11.6, 7.2Hz, 1H), 4.02(dd,J=11.5 , 5.0 Hz, 1H), 3.94 (tq, J = 6.9, 1.4 Hz, 1H), 3.89 (s, 1H), 2.06 (s, 3H), 2.00 (d, J = 3.6 Hz, 6H), 1.94 (d, J = 1.4 Hz, 3H). Calculated mass for CHNO: 329.31, found: 330.1 (M+H).

[0510] Compound (102)—Compound 101 (5 g, 15.19 mmol) and 5-benzyloxy-1-pentanol (3.21 mL, 16.71 mmol) were dissolved in dry dichloroethane (DCE) (60 mL). The reaction flask was evacuated, purged with argon, and cooled in an ice bath. Trimethylsilyl trifluoromethanesulfonate (0.550 mL, 3.04 mmol) was added via syringe. The reaction was monitored by TLC (5% MeOH / DCM) after 3.5 hours and developed using Hanessian stain. The reaction was complete. Sodium bicarbonate (383 mg, 4.56 mmol) was dissolved in 100 mL and cooled in an ice bath. The reaction mixture was added dropwise to the stirred sodium bicarbonate solution. The reaction was allowed to stir for 20 minutes to allow complete neutralization. The mixture was added to a separatory funnel, the organic layer was separated, and the aqueous layer was washed with dichloromethane. The organic layers were combined and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off, and the mother liquor was concentrated and placed under high vacuum to give (8.88 g) of 102. 1 H NMR (400MHz, DMSO-d6)δ 7.81(d,J=9.2Hz, 1H), 7.38-7.21(m,6H), 5.20(d,J=3.4Hz, 1H), 4.94(dd,J =11.3, 3.4Hz, 1H), 4.47(d,J=8.5Hz, 1H), 4.08-3.93(m,3H), 3.89(s,2H), 3 .89-3.80(m,1H), 3.69(dt,J=9.9, 6.1Hz, 1H), 3.45-3.34(m,5H), 3.33(s,3 H), 2.09(s,3H), 1.98(s,3H), 1.88(s,3H), 1.73(s,2H), 1.58-1.24(m,8H).

[0511] Compound (102a)—Compound 102 (7.95 g, 15.19 mmol) was dissolved in 150 mL of anhydrous methanol. The reaction flask was evacuated and purged with argon. Sodium methoxide in methanol (0.5 M, 3.04 mL, 6.07 mmol) was added via syringe. The reaction was allowed to stir overnight at room temperature. The reaction was monitored by TLC (10% MeOH / DCM) and developed using Hanessian stain. Glacial acetic acid was added to the reaction to lower the pH to 7, and the reaction mixture was concentrated under reduced pressure. The residue was suspended in 100 mL of dichloromethane with minimal methanol to dissolve the crude product. The product was precipitated by adding the crude solution dropwise to a 50 / 50 ether / hexane (500 mL) solution. A precipitate formed, which was stirred for an additional 10 minutes. The product was filtered off and dried under high vacuum to yield (5.26 g) of 102a. 1 H NMR (400MHz, DMSO-d6)δ 7.62(d,J=9.0Hz, 1H), 7.38-7.21(m,5H), 4.62(s,3H), 4.42(s,2H), 4.20(d,J= 8.4Hz, 1H), 3.75-3.60(m,3H), 3.57-3.43(m,3H), 3.39(q,J=6.5, 5.3Hz, 4H), 3. 31 (d, J = 6.5 Hz, 8H), 3.26 (t, J = 6.2 Hz, 1H), 1.75 (s, 3H), 1.47 (dq, J = 28.9, 6.9, 6.5 Hz, 4H), 1.37-1.26 (m, 2H). Calculated mass for CHNO: 397.47; found: 420.2 (M + Na).

[0512] Compound (103) - Compound 102a (5.20 g, 13.09 mmol) and 50 mL of anhydrous pyridine were added to a reaction flask. The pyridine was removed under reduced pressure. This was repeated three times and the reaction was dried under high vacuum overnight. The next day, 4-(dimethylamino)pyridine (0.160 g, 1.31 mmol), triethylamine (1.78 mL, 13.09 mmol), and anhydrous pyridine were added to the reaction flask. The reaction was cooled to 0 °C using an ice bath. The reaction flask was evacuated and purged with argon. 4,4'-Dimethoxytrityl chloride (4.92 g, 14.53 mmol) was dissolved in anhydrous pyridine, and the resulting solution was added to the reaction flask via syringe. The reaction was allowed to reach room temperature and stirred overnight. The reaction was monitored by TLC (100% EtOAc) and developed using Hanessian stain. The reaction was quenched by the addition of methanol and concentrated under reduced pressure. The residue was dissolved in dichloromethane and added to a separatory funnel, and the organic layer was washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off, and the mother liquor was concentrated and placed under high vacuum to give (8.70 g) of 103. Compound 103 1 H NMR (400MHz, DMSO-d6)δ 7.63(d,J=9.0Hz, 1H), 7.44-7.37(m,3H), 7.36-7.15(m,17H), 7.09-7.01(m,1H), 6.92-6.78(m,7H), 4.62(d,J=6.0Hz, 1H), 4.52(d ,J=4.3Hz, 1H), 4.39(s,2H), 4.27(d,J=8.4Hz, 1H), 3.69(d,J=7.1Hz, 11H), 3.67-3.62(m,1H), 3.59(t,J=3.8Hz, 1H), 3.52(t,J=5.8 Hz, 1H), 3.43 (ddd, J = 9.9, 6.1, 3.0 Hz, 2H), 3.37 (t, J = 6.5 Hz, 3H), 3.33 (s, 5H), 3.18 (dd, J = 9.3, 7.0 Hz, 1H), 3.00 (dd, J = 9.3, 4.8 Hz, 1H), 2.92 (d, J = 12.9 Hz, 2H), 1.77 (s, 3H), 1.54-1.45 (m, 4H), 1.39-1.28 (m, 2H). Calculated mass for CHNO: 699.84; found: 722.3 (M + Na).

[0513] Compound (104)—Compound 103 (8.70 g, 12.44 mmol) and 4-(dimethylamino)pyridine (1.52 g, 12.44 mmol) were added to a reaction flask. The reaction flask was evacuated and purged with argon. Anhydrous pyridine (230 mL) was added via syringe. A solution of benzoic anhydride (7.03 g, 31.1 mmol) in pyridine was added to the reaction mixture via syringe, and the reaction was stirred overnight at room temperature. The reaction was monitored by TLC (30% EtOAc / hexane) and developed using Hanessian stain. After completion of the reaction, water was added to quench the reaction and stirred for 10 minutes. The solvent was removed under reduced pressure. EtOAc and water were added and placed in a separatory funnel. The organic layer was separated and washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated and placed under high vacuum to give (8.35 g) of 104. 1 H NMR (400 MHz, DMSO-d) δ 8.01-7.88(m,2H), 7.73(ddd,J=8.2, 3.9, 1.4Hz, 4H), 7.71-7.55(m,3H), 7.45(dt,J=39.3, 7.8Hz, 5H), 7.37-7.20(m,8H), 7.20-7.04(m,8H), 6.76- 6.58(m,5H), 5.84(d,J=3.3Hz, 1H), 5.33(dd,J=11.1, 3.4Hz, 1H), 4.64(d, J=8.5Hz, 1H), 4.41(s,2H), 4.31(dd,J=8.6, 5.9Hz, 1H), 4.16(dt,J=11.0, 8.9 Hz, 1H), 3.79-3.66 (m, 2H), 3.64 (s, 7H), 3.44 (dt, J = 9.8, 6.5 Hz, 1H), 3.37 (t, J = 6.4 Hz, 2H), 3.33 (s, 1H), 3.16 (dd, J = 8.5, 5.4 Hz, 1H), 2.95 (t, J = 8.6 Hz, 1H), 1.65 (s, 3H), 1.50 (ddt, J = 14.9, 10.6, 6.5 Hz, 4H), 1.32 (qd, J = 10.5, 9.3, 6.3 Hz, 2H). Calculated mass for CHNO: 908.06; found: 930.3 (M + Na).

[0514] Compound (105)—Compound 104 (3.74 g, 4.12 mmol) was added to the reaction flask. The reaction flask was evacuated and purged with argon three times. The starting material was dissolved by adding anhydrous tetrahydrofuran via syringe. Next, 10% palladium on carbon, Degussa type (374 mg, 10 wt%) was added. The reaction flask was evacuated and purged with argon three times. The reaction flask was then evacuated and purged twice with hydrogen from a balloon. The reaction was allowed to stir overnight at room temperature. The reaction was monitored by TLC (50 / 50 EtOAc / hexane) and developed using phosphomolybdic acid. After completion of the reaction, the flask was evacuated and purged with argon three times. The reaction mixture was filtered through Celite to remove the palladium on carbon and washed with methanol. The mother liquor was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (10% to 100% EtOAc / hexanes) and the product fractions were combined and concentrated under reduced pressure to give 105 (2.00 g 59%). 1 H NMR (400MHz, DMSO-d6)δ 8.04-7.85(m,1H), 7.79-7.64(m,4H), 7.64-7.54(m,1H), 7.51(t,J=7.7Hz, 2H), 7.40(t,J=7.7Hz, 2H), 7.34-7.21(m,2H), 7.22-7.01(m ,6H), 6.75-6.59(m,4H), 5.84(d,J=3.2Hz, 1H), 5.33(dd,J=11.1, 3.3Hz, 1H), 4.64(d,J=8.5Hz, 1H), 4.32(dt,J=9.7, 5.6Hz, 2H), 3.71(d t,J=9.7, 6.2Hz, 1H), 3.61(s,5H), 3.43(dt,J=10.0, 6.6Hz, 1H), 3.39-3.29(m,2H), 3.16(dd,J=8.6, 5.4Hz, 1H), 2.94(t,J=8.6Hz, 1H), 1.67(s,3H), 1.55-1.43(m,2H), 1.38(dt,J=13.0, 5.8Hz, 2H), 1.28(q,J=7.6Hz, 2H).Calculated mass value for C48H51NO11: 817.93, actual value: 840.3(M+Na)

[0515] Compound (106) - Compound 105 is added to a reaction flask, which is evacuated and purged with argon. The starting material is dissolved in dichloromethane, diisopropylamine is added, and the reaction is stirred at room temperature for 1-2 hours. After checking for completeness by TLC, the reaction is worked up using standard extraction conditions. The crude residue is purified on silica gel to give compound 106.

[0516] Example 13. Synthesis of phosphoramidite 113 [ka] Compound (107): Compound 101 (5.0 g, 15.19 mmol) and 2-(benzyloxy)ethanol (2.80 mL, 19.75 mmol) were dissolved in dry dichloroethane (DCE) (60 mL). The reaction flask was evacuated, purged with argon, and cooled in an ice bath. Trimethylsilyl trifluoromethanesulfonate (0.550 mL, 3.04 mmol) was added via syringe. The reaction was monitored by TLC (5% MeOH / DCM) after 3.5 hours and developed using Hanessian stain. The reaction was complete. Sodium bicarbonate (383 mg, 4.56 mmol) was dissolved in 100 mL and cooled in an ice bath. The reaction mixture was added dropwise to the stirred sodium bicarbonate solution. The reaction was allowed to stir for 20 minutes to fully neutralize. The mixture was added to a separatory funnel, the organic layer was separated, and the aqueous layer was washed with dichloromethane. The organic layers were combined and washed with brine. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated and placed under high vacuum to give (8.64 g) of 107. 1H NMR (400MHz, DMSO-d6)δ 7.38-7.22(m,4H), 5.21(d,J=3.4Hz, 1H), 4.97(dd,J=11.2, 3.4Hz, 1H), 4.56(d,J=8 .5Hz, 1H), 4.47(d,J=1.4Hz, 2H), 4.09-3.95(m,2H), 3.89(s,1H), 3.84(ddd,J=14.7 , 7.5, 3.4Hz, 1H), 3.63(ddd,J=10.8, 6.4, 3.6Hz, 1H), 3.54(dq,J=10.7, 5.2Hz, 2H), 3.44(t,J=5.1Hz, 1H), 2.09(d,J=4.4Hz, 2H), 1.98(s,2H), 1.88(s,2H), 1.73(s,1H).

[0517] Compound (107a)—Compound 107 (7.31 g, 15.19 mmol) was dissolved in 150 mL of anhydrous methanol. The reaction flask was evacuated and purged with argon. Sodium methoxide in methanol (0.5 M, 2.275 mL, 4.55 mmol) was added via syringe. The reaction was allowed to stir overnight at room temperature. The reaction was monitored by TLC (10% MeOH / DCM) and developed using Hanessian stain. After completion of the reaction, glacial acetic acid was added to the reaction to lower the pH to 7, and the reaction mixture was concentrated under reduced pressure. The residue was suspended in 100 mL of dichloromethane with minimal methanol to dissolve the crude product. The product was precipitated by adding the crude solution dropwise to a 50 / 50 ether / hexane (500 mL) solution. A precipitate formed which was stirred for an additional 10 min and the product was filtered off and dried under high vacuum to give (5.05 g) of 107a. 1H NMR (400MHz, DMSO-d6)δ 7.65(d,J=8.9Hz, 1H), 7.38-7.21(m,5H), 4.63(d,J=11.5Hz, 2H), 4.47(s,2H), 4.28(d,J=8.4Hz, 1H), 3.83(dt,J=9.1, 3.4Hz, 1H), 3.7 2(dt,J=10.7, 8.7Hz, 1H), 3.64(s,1H), 3.62-3.45(m,6H), 3.41(d,J=10.5Hz, 2H), 3.36-3.25(m,7H), 1.90-1.78(m,1H), 1.75(s,2H).

[0518] Compound (108): Compound 107a (5.0 g, 14.08 mmol) and imidazole (2.88 g, 42.24 mmol) were added to a reaction flask. The reaction flask was evacuated and purged with argon. Anhydrous pyridine was added via syringe to dissolve the starting material. The reaction was stirred at room temperature for 10 minutes, and then a solution of tert-butyldimethylsilyl chloride (3.18 g, 21.12 mmol) in pyridine was added via syringe. The reaction was stirred overnight at room temperature. The reaction was monitored by TLC (100% EtOAc) and developed using Hanessian stain. After completion of the reaction, methanol was added to quench the reaction and stirred for 10 minutes. The reaction mixture was concentrated under reduced pressure, and dichloromethane and water were added. The mixture was added to a separatory funnel, the organic layer was separated, and the aqueous layer was washed with dichloromethane. The organic layers were combined and washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated to give (8.80 g) of 108, which was used crude in the next reaction.

[0519] Compound (109): Compound 108 (6.61 g, 14.09 mmol) and 4-(dimethylamino)pyridine (1.72 g, 14.09 mmol) were added to a reaction flask. The reaction flask was evacuated and purged with argon. Anhydrous pyridine (200 ml) was added via syringe. A solution of benzoic anhydride (11.15 g, 49.31 mmol) in pyridine was added to the reaction mixture via syringe, and the reaction was stirred overnight at room temperature. The reaction was monitored by TLC (50% EtOAc / hexane) and developed using Hanessian stain. After completion of the reaction, water was added to quench the reaction and stirred for 10 minutes. The solvent was removed under reduced pressure. EtOAc and water were added and placed in a separatory funnel. The organic layer was separated and washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated. The residue was purified by flash chromatography on silica gel (0% to 100% EtOAc / hexanes) and the product fractions were combined and concentrated under reduced pressure to give 109 (4.22 g 44%). 1 H NMR (400MHz, DMSO-d6)δ 7.97(d,J=9.2Hz, 1H), 7.94-7.86(m,2H), 7.75-7.63(m,3H), 7.56(dt,J=15.1, 7.5Hz, 4H), 7.44-7.35(m,2H), 7.33(d,J=3. 5Hz, 4H), 7.30-7.23(m,1H), 5.67(d,J=3.3Hz, 1H), 5.27(dd,J=11.1, 3.3Hz, 1H), 4.73(d,J=8.5Hz, 1H), 4.51(s,2H), 4.35- 4.16 (m, 1H), 4.08 (t, J = 7.3 Hz, 1H), 3.97-3.85 (m, 1H), 3.76-3.67 (m, 2H), 3.67-3.53 (m, 4H), 1.65 (s, 3H), 0.89-0.77 (m, 2H), 0.76 (s, 8H), 0.64 (s, 1H), -0.06 (s, 3H), -0.14 (s, 3H). Calculated mass for C37H47NO9Si: 677.87; found: 678.3 (M+H), 700.3 (M+Na).

[0520] Compound (110): Compound 109 (3.0 g, 4.43 mmol) was added to a reaction flask. The reaction flask was evacuated and purged with argon three times. The starting material was dissolved by adding anhydrous methanol via syringe. Next, 10% palladium on carbon, Degussa type (300 mg, 10 wt%) was added. The reaction flask was evacuated and purged with argon three times. Next, the reaction flask was evacuated and purged with hydrogen from a balloon twice. The reaction was allowed to stir overnight at room temperature. The reaction was monitored by TLC (60% EtOAc / hexane) and developed using Hanessian stain. After completion of the reaction, the flask was evacuated and purged with argon three times. The reaction mixture was filtered through Celite to remove the palladium on carbon and washed with methanol. The mother liquor was concentrated under reduced pressure to give 110 (2.75 g). Calculated mass for C30H41NO9Si: 587.74, Found: 588.3 (M+H), 610.3 (M+Na)

[0521] Compound (111): Compound 110 (2.60 g, 4.43 mmol) and 50 mL of anhydrous pyridine were added to a reaction flask. The pyridine was removed under reduced pressure. The residue was coevaporated with pyridine three times and dried under high vacuum overnight. The next day, 4-(dimethylamino)pyridine (0.054 g, 0.443 mmol), triethylamine (0.604 mL, 4.43 mmol), and anhydrous pyridine were added to the reaction flask. The reaction was cooled to 0 °C using an ice bath. The reaction flask was evacuated and purged with argon. 4,4'-Dimethoxytrityl chloride (1.67 g, 4.92 mmol) was dissolved in anhydrous pyridine, and the resulting solution was added to the reaction flask via syringe. The reaction was allowed to reach room temperature and stirred overnight. The reaction was monitored by TLC (60% EtOAc / hexane) and developed using Hanessian stain. After the reaction was complete, methanol was added to quench the reaction and concentrated under reduced pressure. The residue was dissolved in dichloromethane and added to a separatory funnel, and the organic layer was washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off, and the mother liquor was concentrated. The residue was purified by flash chromatography on silica gel (0% to 50% EtOAc / hexanes), and the product fractions were combined and concentrated under reduced pressure to give 111 (1.96 g, 49.7%). 1H NMR(400MHz, DMSO-d6)δ 8.04(d,J=9.4Hz,1H),7.95-7.85(m,2H),7.76-7.71(m,2H),7.71-7.63(m,1H),7.63-7.56(m,1H),7.53(t,J=7.7Hz,2H),7.45-7.36(m ,4H), 7.34-7.23(m,6H), 7.23-7.15(m,1H), 6.91-6.83(m,4H), 5.70(d,J=4.3Hz, 1H), 5.30(dd,J=11.1, 3.4Hz, 1H), 4.75(d,J=8.5Hz, 1H ), 4.41-4.28(m,1H), 4.11(s,1H), 3.77-3.67(m,7H), 3.67-3.56(m,2H), 3.33(s,1H), 3.20(s,1H), 2.98-2.91(m,1H), 1.60(s,3H), 0.8 4(d,J=14.5Hz,1H),0.76(s,8H),0.73-0.64(m,1H),-0.07(s,3H),- 0.15 (s, 3H). Calculated mass value of C51H59NO11Siについての: 890.11, measured value: 912.4 (M+Na).

[0522] Compound (112): Compound 111 (1.86 g, 2.09 mmol) was added to a plastic reaction vessel. Dichloromethane (18.3 mL) was added to dissolve the starting material. Acetonitrile (55.8 mL), pyridine (37.2 mL), and trimethylamine (9.3 mL) were added. The reaction vessel was purged and cooled in an ice bath. The reaction was stirred, and then hydrogen fluoride pyridine complex (9.3 mL) was carefully added. The reaction was stirred and allowed to warm to room temperature over 5 hours. The reaction was monitored by TLC (35% EtOAc / hexane) and developed with phosphomolybdic acid. The reaction was 90% complete. 200 mL of saturated sodium bicarbonate was cooled in an ice bath and stirred. The reaction mixture was quenched by slowly adding the reaction mixture to the cooled bicarbonate solution. There was some effervescence, and the mixture was stirred for an additional 30 minutes. Dichloromethane was added, and the mixture was transferred to a separatory funnel. The organic layer was separated and washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated. The residue was purified by flash chromatography on silica gel (0% to 100% EtOAc / hexanes) and the product fractions were combined and concentrated under reduced pressure to give 112 (1.13 g, 69%). 1H NMR (400MHz, DMSO-d6)δ 8.01(d,J=9.4Hz, 1H), 7.95-7.83(m,2H), 7.79-7.72(m,1H), 7.72-7.55(m,3H), 7.52(d,J=7.7Hz, 2H), 7.47-7.35(m,4H), 7.35-7.24(m,6) H), 7.24-7.14(m,1H), 6.96-6.79(m,4H), 5.70(d,J=3.6Hz, 1H), 5.27(dd,J=11.1, 3.3Hz, 1H), 4.93(dd,J=6.4, 4.3Hz, 1H), 4.74(d,J=8.5H) z, 1H), 4.34(dt,J=11.1, 8.9Hz, 1H), 3.99(dq,J=20.8, 6.8Hz, 3H), 3.72(d,J=1.1Hz, 6H), 3.59-3.37(m,2H), 3.20(ddd,J=11.0, 7.8, 3.6H z, 1H), 2.95(dt,J=10.2, 4.1Hz, 1H), 1.98(s,2H), 1.60(s,3H), 1.16(t,J=7.1Hz, 2H).Calculated mass value for C45H45NO11: 775.85, actual value: 798.3(M+Na).

[0523] Compound (113) - Compound 112 is added to a reaction flask, which is evacuated and purged with argon. The starting material is dissolved in dichloromethane, and diisopropylamine is added via syringe. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite is added, and the reaction is stirred at room temperature for 1-2 hours. After checking for completeness by TLC, the reaction is worked up using standard extraction conditions. The crude residue is purified on silica gel to give compound 113.

[0524] Example 14. Synthesis of phosphoramidite 508 [ka] Compound (501): Compound 500 (6 g, 7.70 mmol) and 2-(benzyloxy)ethanol (1.20 mL, 8.117 mmol) were added to a reaction flask and dissolved in anhydrous toluene. The solvent was removed under reduced pressure. This was repeated three times and the mixture was placed under high vacuum and dried overnight. The next day, a stir bar and molecular sieves were added, evacuated, and purged with argon three times. Anhydrous ether was added via syringe, and the reaction was cooled to 0 °C using an ice bath. It was stirred for 10 minutes, and then trimethylsilyl trifluoromethanesulfonate (0.139 mL, 0.77 mmol) was added via syringe. The reaction was allowed to stir for 3 hours and then monitored by TLC (20% EtOAc / hexane) and developed using Hanessian stain. The reaction was complete and quenched with trimethylamine. The solution was diluted with ethyl acetate, and the molecular sieves were removed by filtration. The solution was added to a separatory funnel and the organic layer was washed with saturated sodium bicarbonate. The organic layer was separated and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off and the mother liquor was concentrated. The residue was purified by flash chromatography on silica gel (0% to 50% EtOAc / hexanes) and the product fractions were combined and concentrated under reduced pressure to give (5.87 g) of 501. Mass calculated for C43H38O11: 730.77, found: 753.2 (M+Na).

[0525] Compound (502): Compound 501 (5.62 g, 7.70 mmol) was dissolved in 150 ml of anhydrous methanol. The reaction flask was evacuated and purged with argon. Sodium methoxide in methanol (0.5 M, 6.16 ml, 3.08 mmol) was added via syringe. The reaction was allowed to stir at room temperature overnight. The reaction was monitored by TLC (5% MeOH / DCM) and developed using Hanessian stain. After the reaction was complete, it was neutralized to pH 7 by adding 20 drops of glacial acetic acid. The solvent was removed under reduced pressure to give (3.63 g) of 502. 15 H 22 Calculated mass for O7: 314.33, found: 337.1 (M+Na).

[0526] Compound (503): Compound 502 (2.42 g, 7.70 mmol) and imidazole (1.57 g, 23.1 mmol) were added to a reaction flask. The reaction flask was evacuated and purged with argon. Anhydrous pyridine was added via syringe to dissolve the starting material. The reaction was stirred at room temperature for 10 minutes, and then a solution of tert-butyldimethylsilyl chloride (1.74 g, 11.55 mmol) in pyridine was added via syringe. The reaction was stirred at room temperature overnight. The reaction was monitored by TLC (100% EtOAc) and developed using Hanessian stain. After completion of the reaction, methanol was added to quench the reaction and stirred for 10 minutes. The reaction mixture was concentrated under reduced pressure, and dichloromethane and water were added. The mixture was added to a separatory funnel, the organic layer was separated, and the aqueous layer was washed with dichloromethane. The organic layers were combined and washed with saturated sodium bicarbonate. The organic layers were separated and washed with brine solution. The organic layer was separated and dried over sodium sulfate. The solids were filtered off, and the mother liquor was concentrated. The residue was purified by flash chromatography on silica gel (0% to 100% EtOAc / hexanes), and the product fractions were combined and concentrated under reduced pressure to give 503 (2.57 g, 78.1%). 1 H NMR (400MHz, DMSO-d6)δ 7.40-7.16(m,2H), 4.74(dd,J=5.8, 4.8Hz, 1H), 4.67-4.54(m,1H), 4.48(s,1H), 3.78-3.67(m,1H), 3.65-3.48(m,2H), 3.45(ddd,J=9.3, 6.1, 3.4Hz, 1H), 3.42-3.23(m,1H), 0.84(s,4H), 0.02(s,3H).C 21 H 36 Calculated mass for O7Si: 428.60, found: 451.2 (M+Na).

[0527] Compound (504): Compound 503 (2.50 g, 5.84...

Claims

【Request Item 1】 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 wherein m and n are independently an integer selected from 1 to 12. A compound selected from the group consisting of:

2. The compound of claim 1, wherein the stereochemistry of the carbohydrate ring is the same as that of galactose.

3. The compound of claim 1, wherein the stereochemistry of the carbohydrate ring is the same as that of glucose.

4. The compound of claim 1, wherein the stereochemistry of the carbohydrate ring is the same as that of mannose. 【Request Item 5】 【Chemistry 4】 3. The compound of claim 2, wherein: 【Request Item 6】 【Chemistry 5】 3. The compound of claim 2, wherein: 【Request Item 7】 【Transformation 6】 4. The compound of claim 3, wherein: 【Request Item 8】 【Chemistry 7】 4. The compound of claim 3, wherein: 【Request Item 9】 【Transformation 8】 5. The compound of claim 4, wherein: 【Request Item 10】 【Chemistry 9】 wherein m and n are independently an integer selected from 1 to 12.

2. The compound of claim 1 selected from the group consisting of:

11. The compound of claim 10, wherein the stereochemistry of each carbohydrate ring is the same as that of galactose.

12. 11. The compound of claim 10, wherein the stereochemistry of the carbohydrate ring is the same as glucose.

13. 11. The compound of claim 10, wherein the stereochemistry of the carbohydrate ring is the same as that of mannose. 【Request Item 14】 【Chemistry 10】 wherein m and n are independently an integer selected from 1 to 12.

2. The compound of claim 1 selected from the group consisting of:

15. 15. The compound of claim 14, wherein the stereochemistry of each carbohydrate ring is the same as that of galactose.

16. 15. The compound of claim 14, wherein the stereochemistry of the carbohydrate ring is the same as that of glucose.

17. 15. The compound of claim 14, wherein the stereochemistry of the carbohydrate ring is the same as that of mannose. 【Request Item 18】 【Chemistry 11】 wherein m and n are independently an integer selected from 1 to 12.

2. The compound of claim 1 selected from the group consisting of:

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