Multi-target single-body conjugates

Abstract

The present invention relates generally to compounds, compositions and methods useful for modulating gene expression of multiple target nucleic acids with a single chemical entity. [Solution] A multitargeting molecule comprising a first double-stranded siRNA molecule and a second double-stranded siRNA molecule, wherein the first siRNA and the second siRNA are covalently linked to each other, and at least one ligand is conjugated to the multitargeting molecule.

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 / 194,003, filed July 17, 2015, the contents of which are incorporated herein by reference in their entirety. [Technical Field]

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to compounds, compositions and methods useful for modulating gene expression of multiple targets. [Background technology]

[0003] There is a need in the art for molecules capable of targeting more than one target, and the present disclosure provides some solutions to that need. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the present disclosure 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 target gene expression can be included in the multi-target molecule disclosed herein.

[0005] "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 those 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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).

[0010] The present inventors have found that multi-targeting molecules conjugated with a ligand are particularly effective in regulating gene expression. Accordingly, in some embodiments, at least one ligand is conjugated to the multi-targeting molecule. Accordingly, 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 at any position in the multi-targeting molecule. In some embodiments, at least two effector molecules in the multi-targeting molecule are conjugated with at least one ligand. Without intending to be bound by any particular theory, the ligand may enhance the delivery or pharmacokinetic profile of the conjugated multitargeted molecule.

[0011] At least two effector molecules in the multi-targeting molecule disclosed herein can be covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, as generally known in the art and as described herein. Thus, in some embodiments, the two effector molecules are linked to each other via a nucleotide-based linker. In some other embodiments, the two effector molecules are linked to each other via a non-nucleotide-based linker.

[0012] As disclosed herein, at least two effector molecules in the multi-targeting molecule described herein can be non-covalently linked to each other.Therefore, in some embodiments, multi-targeting molecule is assembled from two effector molecules, and each effector molecule is bound to at least one ligand.In some embodiments, multi-targeting molecule is assembled from two siRNAs, and each siRNA is conjugated to at least one ligand.

[0013] As disclosed herein, at least two effector molecules in the multi-targeting molecules described herein can be covalently linked to each other via a nucleotide-based linker. Without limitation, the nucleotide-based linker linking the effector molecules can be all DNA, all RNA, or a mixture of DNA and RNA. In some embodiments, the nucleotide-based linker linking the two effector molecules is all DNA. The RNA and DNA can be natural or modified. Furthermore, the nucleotide-based linker linking the two effector molecules can be unmodified or contain one or more nucleic acid modifications described in the present disclosure. Thus, in some embodiments, the nucleotide-based linker linking the effector molecules contains at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleic acid base, a modified sugar, and any combination thereof.

[0014] The nucleotide-based linker linking the effector molecules can comprise one or two nucleic acid strands, and can be single-stranded, double-stranded, or contain single-stranded and double-stranded regions. In some embodiments, the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands that do not form a double-stranded structure. In other words, the nucleotide-based linker comprises two strands that do not hybridize to each other. In some embodiments, the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands, one of which comprises all DNA and the other of which comprises a mixture of DNA and 2'-O alkyl modifications. In some embodiments, the linker linking the effector molecules comprises the nucleotide sequence uuu or (dT)n (where n is 1 to 20). In some embodiments, the linker linking the effector molecules comprises the nucleotide sequence -(CH2) 12 -(C12 linker or Q50), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker or Q51), Q151, Q173, -CH2CH2O-(CH2CH2) n —CH2CH2O—CH2CH2O— (where n is 0 or 1 to 20); —(CH2)9—(CH2) n -CH2- (where n is 0 or 1-20); mono-, di-, tri-, tetra-, penta-, or polyprolinol optionally conjugated to a ligand; or mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol optionally conjugated to a ligand. In some embodiments, the linker connecting the effector molecules comprises a molecule selected from those shown in Figures 16-23 and 26. In some embodiments, the linker comprises a monomer selected from the monomers described below in the section entitled "Exemplary Ligand Monomers," e.g., Monomers 1-30 described below in paragraphs

[0316] -

[0365] . Exemplary linkers are also described in the Examples section of this disclosure, e.g., Examples 1-23. [Brief explanation of the drawings]

[0015] [Figure 1]1 shows the design of a multi-target single entity conjugate according to some embodiments of the present invention. [Figure 2] FIG. 1 is a schematic diagram of an exemplary study design. [Figure 3A] Figure 1 shows the in vivo activity of a mixture of two siRNAs directed against two different targets. [Figure 3B] Figure 1 shows the in vivo activity of a mixture of two siRNAs directed against two different targets. [Figure 4] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 5] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 6] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 7] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 8] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 9] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 10] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 11] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 12] 1 shows the in vivo activity of exemplary embodiments of multitargeting molecules. [Figure 13] 1 shows the activity of exemplary embodiments of multitarget molecules as measured by relative protein concentration. [Figure 14-1] 1 shows duplex analysis and thermal melting profiles from one embodiment of a multi-target single entity conjugate. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15] FIG. 1 is a schematic diagram of three basic designs of some exemplary multitargeting molecules. [Figure 16]FIG. 1 is a schematic diagram of a multitargeting molecule-GalNAc conjugate (eg, bis(siRNA)-GalNAc conjugate) according to some embodiments of the present invention. [Figure 17-1] FIG. 1 is a schematic diagram of a multitargeting molecule-GalNAc conjugate (eg, bis(siRNA)-GalNAc conjugate) according to some embodiments of the present invention. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18] FIG. 1 shows a schematic diagram of some exemplary multi-target molecular designs according to some embodiments of the present invention. [Figure 19-1] Several triantennary monomers for multi-target molecule design are presented. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 19-3] This is a continuation of Figure 19-2. [Figure 20-1] 1 shows a schematic diagram of the synthesis reagents for exemplary GalNAc-conjugated multi-targeting molecules (eg, bis(siRNA)-GalNAc conjugates). [Figure 20-2] This is a continuation of Figure 20-1. [Figure 20-3] This is a continuation of Figure 20-2. [Figure 21] 1 shows some exemplary prolinol-based linker molecules. [Figure 22-1] An exemplary post-synthetic conjugation scheme is shown. [Figure 22-2] This is a continuation of Figure 22-1. [Figure 22-3] This is a continuation of Figure 22-2. [Figure 23] Examples of (1+1) or (1+1+1) type designs in bridges / linkers are shown. [Figure 24] FIG. 1 is a schematic diagram of a bis(siRNA)-GalNAc orientation according to an embodiment of the present invention. [Figure 25] FIG. 1 is a schematic diagram of a bis(siRNA)-GalNAc orientation according to an embodiment of the present invention. [Figure 26-1] Some exemplary linkers and monomers are shown. [Figure 26-2] This is a continuation of Figure 26-1. DETAILED DESCRIPTION OF THE INVENTION

[0016] 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.

[0017] 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.

[0018] The various embodiments described herein are based on a multi-target molecule comprising at least two nucleic acid-based effector molecules, each capable of regulating gene expression of a target gene. Without limitation, each effector molecule in a multi-target molecule can be directed to the same target gene, different target genes, or different locations of the same target gene. Generally, a multi-target molecule comprises at least two effector molecules.

[0019] sticky end As disclosed herein, in some embodiments, at least two effector molecules in a multi-target molecule are non-covalently linked to each other by hybridization of nucleotides between the effector molecules, and each effector molecule is conjugated with at least one ligand. For example, a portion of the oligonucleotide chain of the first effector molecule hybridizes with a portion of the oligonucleotide chain of the second effector molecule.

[0020] In some embodiments, the multi-targeting molecule is assembled from two siRNAs, where the two siRNAs can be non-covalently linked to each other, and each siRNA is linked to at least one ligand.Therefore, in some embodiments, the multi-targeting molecule comprises a first siRNA and a second siRNA, where the first ligand is linked to the first siRNA, and the second ligand is linked to the second siRNA.Generally, a part of the first siRNA is hybridized to a part of the second siRNA molecule.Without limitation, either the 5' or 3' end part of the first siRNA can be hybridized to a part of the second siRNA depending on the nature of the single strand (sense strand or antisense strand).

[0021] In some embodiments, siRNA comprises a first strand and a second strand.Therefore, in some embodiments, a part of one of the strands in the first siRNA is hybridized with a part of one of the strands in the second siRNA.The strands of the first and second siRNA that hybridize can be both sense strands, both antisense strands, or one sense strand and the other antisense strand.

[0022] In some embodiments, the 3' end of the strand in the first siRNA is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the 3' end of the strand of the second siRNA molecule. In some embodiments, the 3' end of the sense strand of the first siRNA is completely complementary to the 3' end of the antisense strand of the second siRNA molecule. In some other embodiments, the 3' end of the antisense strand of the first siRNA is completely complementary to the 3' end of the sense strand of the second siRNA molecule. In some embodiments, the 3' end of the sense strand of the first siRNA is completely complementary to the 3' end of the sense strand of the second siRNA molecule. In some other embodiments, the 3' end of the antisense strand of the first siRNA is completely complementary to the 3' end of the antisense strand of the second siRNA molecule.

[0023] In some embodiments, the 3' end of the strand in the first siRNA is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the 5' end of the strand of the second siRNA molecule. In some embodiments, the 3' end of the sense strand of the first siRNA is completely complementary to the 5' end of the antisense strand of the second siRNA molecule. In some other embodiments, the 3' end of the antisense strand of the first siRNA is completely complementary to the 5' end of the sense strand of the second siRNA molecule. In some other embodiments, the 3' end of the sense strand of the first siRNA is completely complementary to the 5' end of the sense strand of the second siRNA molecule. In some other embodiments, the 3' end of the antisense strand of the first siRNA is completely complementary to the 5' end of the antisense strand of the second siRNA molecule. In some embodiments, the 3' end of the strand in the first siRNA is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95% or more) complementary to the 5' end of the strand of the second siRNA molecule. In some embodiments, the 5' end of the sense strand of the first siRNA is completely complementary to the 5' end of the antisense strand of the second siRNA molecule. In some other embodiments, the 5' end of the antisense strand of the first siRNA is completely complementary to the 5' end of the sense strand of the second siRNA molecule. In some embodiments, the 5' end of the sense strand of the first siRNA is completely complementary to the 5' end of the sense strand of the second siRNA molecule. In some other embodiments, the 5' end of the antisense strand of the first siRNA is completely complementary to the 5' end of the antisense strand of the second siRNA molecule.

[0024] Generally, the part of the strand in the first siRNA that is complementary to a part of the strand in the second siRNA can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.Similarly, the part of the strand in the second siRNA that is complementary to a part of the strand in the first siRNA can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.It should be noted that the two parts do not have to be the same length.Thus, one can be shorter than the other.

[0025] Without limitation, the length of complementarity between the strands of the first and second siRNA should be sufficient for hybridization under physiological conditions. Thus, the length of the complementary sequence can range from about 1 nucleotide to about 25 nucleotides. For example, the length of the complementary sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. As described above, complete complementarity between the strands of the first and second siRNA may not be required. Thus, the complementary portion may contain one or more (e.g., 1, 2, 3, 4, 5, or more) nucleotide mismatches, bulges, or loops.

[0026] The strand part in the first siRNA that has complementarity with the strand of the second siRNA can be all DNA, all RNA or a mixture of DNA and RNA.RNA and DNA can be natural and modified.Therefore, the strand part in the first siRNA that has complementarity with the strand of the second siRNA can be unmodified or comprise one or more nucleic acid modifications as described herein.

[0027] Similarly, the strand portion in the second siRNA that has complementarity with the strand of the first siRNA can be all DNA, all RNA or a mixture of DNA and RNA.RNA and DNA can be natural and modified.Therefore, the strand portion in the second siRNA that has complementarity with the strand of the first siRNA can be unmodified or comprise one or more nucleic acid modifications as described herein.

[0028] In some embodiments, the portion of the strand in the first siRNA that is complementary to a portion of the strand in the second siRNA is entirely RNA. In some embodiments, the portion of the strand in the first siRNA that is complementary to a portion of the strand in the second siRNA is entirely DNA. Furthermore, the complementary region can be unmodified or contain one or more nucleic acid modifications described in the present disclosure. Thus, in some embodiments, the complementary region contains at least one modification selected from the group consisting of modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof.

[0029] In some embodiments, the portion of the strand in the first siRNA that is complementary to the strand of the second siRNA is all RNA, and the portion of the strand in the second siRNA that is complementary to the strand of the first siRNA is all DNA.

[0030] In another embodiment, the portion of the strand in the first siRNA that is complementary to the strand of the second siRNA and the portion of the strand in the second siRNA that is complementary to the strand of the first siRNA are both DNA.

[0031] In some embodiments, the multi-targeting molecule is assembled from two separate siRNA molecules, wherein at least one ligand is bound to each siRNA, and wherein a portion of the sense strand of the first siRNA hybridizes to a portion of the antisense strand of the second siRNA molecule. In some other embodiments, the multi-targeting molecule is assembled from two separate siRNA molecules, wherein at least one ligand is bound to each siRNA, and wherein a portion of the antisense strand of the first siRNA hybridizes to a portion of the antisense strand of the second siRNA molecule. In still other embodiments, the multi-targeting molecule is assembled from two separate siRNA molecules, wherein at least one ligand is bound to each siRNA, and wherein a portion of the antisense strand of the first siRNA hybridizes to a portion of the antisense strand of the second siRNA molecule. In still other embodiments, the multi-targeting molecule is assembled from two separate siRNA molecules, wherein at least one ligand is bound to each siRNA, and wherein a portion of the sense strand of the first siRNA hybridizes to a portion of the sense strand of the second siRNA molecule.

[0032] In various embodiments of a multi-targeting molecule in which at least two siRNAs, each having at least one ligand, are non-covalently linked to each other, the sense strand of the siRNA in the multi-targeting molecule may include a single-stranded overhang at its 3' end, along with a ligand at the 3' end of the antisense strand. In the context of the present invention, a single-stranded overhang means that the 3' end of the sense strand extends beyond the 5' end of its complementary antisense sequence. Without limitation, the overhang may comprise from about 1 nucleotide to about 25 nucleotides. For example, the single-stranded overhang may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0033] In various embodiments of a multi-targeting molecule in which at least two siRNAs, each having at least one ligand, are non-covalently linked to each other, the antisense strands of the siRNAs in the multi-targeting molecule may contain a single-stranded overhang at their 3' ends, along with a ligand at the 3' end of the sense strand. In the context of the present invention, a single-stranded overhang means that the 3' end of the antisense strand extends beyond the 5' end of its complementary sense sequence. Without limitation, the overhang may contain from about 1 nucleotide to about 25 nucleotides. For example, the single-stranded overhang may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0034] Without limitation, the single-stranded overhangs in the sense strand and / or antisense strand can be all DNA, all RNA, or a mixture of DNA and RNA. In some embodiments, the single-stranded overhangs are all RNA. In some embodiments, the single-stranded overhangs are all DNA. Furthermore, the single-stranded overhangs can be unmodified or contain one or more nucleic acid modifications described in this disclosure. Thus, in some embodiments, the single-stranded overhangs contain at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof.

[0035] In some embodiments, the single-stranded overhang in the sense strand of the first siRNA is all RNA, and the complementary single strand in the antisense strand of the second siRNA is all DNA.In another embodiment, the single-stranded overhang in the antisense strand of the first siRNA is all RNA, and the complementary single strand in the antisense strand of the second siRNA is all DNA.In some embodiments, the single-stranded overhang in the single-stranded overhang in the first siRNA and the single-stranded overhang in the second siRNA are both DNA.

[0036] The single-stranded overhang in the first siRNA can be of the same length as the single-stranded overhang in its complementary strand of the second siRNA.In addition, there can be 0, 1, 2, 3, 4, 5 or more nucleobases at the 5' end of the single-stranded overhang that does not have complementary nucleobases in the single-stranded overhang of other sequences.Therefore, when the two siRNAs in a multi-targeting molecule are assembled together, there can be 0 (for example, a gap), 1, 2, 3, 4, 5 or more nucleobases between the 3' end of the single-stranded overhang of the sense strand of the first siRNA and the sense strand of the second siRNA.Similarly, when the two siRNAs in a multi-targeting molecule are assembled together, there can be 0 (for example, a gap), 1, 2, 3, 4, 5 or more nucleobases between the 3' end of the single-stranded overhang of the antisense strand of the first siRNA and the sense strand of the second siRNA.

[0037] In various embodiments of a multi-targeting molecule in which at least two siRNAs, each having at least one ligand, are non-covalently 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 to any position of each siRNA. For example, one ligand can be attached to the sense strand of the first siRNA, and the other ligand can be attached to the sense strand of the second siRNA; or one ligand can be attached to the sense strand of the first siRNA, and the other ligand can be attached to the antisense strand of the second siRNA; or one ligand can be attached to the antisense strand of the first siRNA, and the other ligand can be attached to the antisense strand of the second siRNA. Without limitation, the first ligand can be independently attached 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 attached to the 5'-end, 3'-end, or internal position of one strand (sense or antisense) of the second siRNA.

[0038] 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.

[0039] 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 at 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 at 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 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 of 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.

[0040] covalent bond In some embodiments, at least two nucleic acid-based effector molecules in a multi-targeting molecule can be covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, as generally known in the art and described herein. Thus, in some embodiments, at least two effector molecules in a multi-targeting molecule are linked via a nucleotide-based linker. In some other embodiments, at least two effector molecules are linked via a non-nucleotide-based linker.

[0041] It should be noted that a nucleotide-based linker may form part of one or both of the effector molecules that are linked together. This means that at least a portion of the nucleotide sequence of the linker is required for one function of the effector molecule. In a preferred embodiment, the nucleotide sequence of the linker does not form part of the effector molecule. In other words, neither of the effector molecules requires any portion of the nucleotide sequence of the linker to regulate gene expression. For example, if the linker sequence is removed from the effector molecule, the effector molecule can still regulate gene expression at a similar level (e.g., within 95%) compared to when the linker is present. If the effector molecule requires complementarity with a target gene for activity, the linker may or may not be part of the effector molecule that requires complementarity with the target sequence. In some embodiments, the linker does not have complementarity with the target sequence (e.g., less than 5% complementarity) or hybridizes to the target sequence.

[0042] In some embodiments, a first strand of a double-stranded nucleotide-based linker linking two effector molecules comprises a nucleotide sequence that is substantially complementary to a second strand of said double-stranded nucleotide-based linker. In some embodiments, the first strand of the linker comprises a nucleobase sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95% or more) complementary to the nucleobase sequence of the second strand of the linker. In some embodiments, the first strand of the linker comprises a nucleobase sequence that is completely complementary to the nucleobase sequence of the second strand of the linker linking two effector molecules.

[0043] Without limitation, the nucleotide-based linker linking the effector molecules can be all DNA, all RNA, or a mixture of DNA and RNA. In some embodiments, the nucleotide-based linker linking two effector molecules is all DNA. The RNA and DNA can be natural or modified. Thus, in some embodiments, the nucleotide-based linker linking the effector molecules comprises at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. Exemplary modifications of linkers include, but are not limited to, locked nucleic acids (e.g., LNA, ENA, and BNA), 2'-O-alkylnucleosides, 2'-halonucleosides (such as 2'-F nucleotides), 2'-aminonucleosides, 2'-S-alkylnucleosides, abasic nucleosides, 2'-cyanonucleosides, 2'-mercaptonucleosides; 2'-MOE nucleosides, acylnucleosides, (S)-cEt monomers, and modified internucleotide linkages (phosphodiester, phosphotriester, hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate, phosphorothioate, phosphorodithioate, methylenemethylimino, thiodiester, thionocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, boranophosphate ... phosphate), boranophosphate ester, amide, hydroxyamino, siloxane, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonic acid ester, thioformacetal, formacetal, oxime, methyleneimino, methylenecarbonylamino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether, thioether, and thioacetamide. Nucleic acid modifications are described in more detail below in this disclosure.

[0044] In some embodiments, at least one of the internucleoside linkages between the linker and the effector molecule that connects the effector molecules is a modified internucleoside linkage. In some embodiments, the internucleoside linkage connecting the 5' end of the linker to the 3' end of one of the effector molecules is a modified internucleoside linkage. In some embodiments, the internucleoside linkage connecting the 3' end of the linker to the 5' end of one of the effector molecules is a modified internucleoside linkage.

[0045] In some embodiments, the first (e.g., first, second, third, fourth, or fifth) internucleoside linkage at the 5' and / or 3' end of the linker connecting the effector molecules is a modified internucleoside linkage, hi some embodiments, one, two, three, four, five, or more internucleoside linkages from the 5' and / or 3' end of the linker are modified internucleoside linkages.

[0046] In some embodiments, the linker linking the effector molecules comprises at least one (eg, 1, 2, 3, 4, 5, 6 or more) modified internucleoside linkage at an internal position of the linker.

[0047] Without limitation, the nucleotide-based linker linking the effector molecules can be of any desired length. For example, the nucleotide-based linker linking the effector molecules can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length. In some embodiments, the nucleotide-based linker linking the effector molecules can range in length from 1 nucleotide to 5 nucleotides. In certain embodiments, the nucleotide-based linker linking two effector molecules is 4 nucleotides in length.

[0048] When a nucleotide-based linker linking effector molecules contains a nucleic acid modification, such modification can be located at any position in the linker. For example, the modification can be at the 5'-nucleotide, 3'-nucleotide, or internal nucleotide of the linker. In some embodiments, the first (e.g., the first, second, third, fourth, or fifth) nucleotide at the 5' and / or 3' end of the linker contains a nucleic acid modification. In some embodiments, one, two, three, four, five, or more nucleotides from the 5' and / or 3' end of the linker contain a nucleic acid modification. In some embodiments, one, two, three, four, five, or more internal nucleotides of the linker contain a nucleic acid modification. In some embodiments, the internal nucleotides of the linker contain all DNA in the sense strand. In another embodiment, the internal nucleotides of the linker contain a mixture of DNA and 2'-O alkyl modifications in the antisense strand.

[0049] The nucleotide-based linker that connects effector molecules can comprise one or two nucleic acid strands, and can be single-stranded, double-stranded, or comprise single-stranded and double-stranded regions.In some embodiments, the nucleotide-based linker that connects effector molecules comprises two nucleic acid strands that do not form a double-stranded structure.In other words, the nucleotide-based linker comprises two strands that do not hybridize with each other.

[0050] In some embodiments, a nucleotide-based linker linking effector molecules comprises two nucleic acid strands, wherein the nucleotide sequence of a first strand of the linker comprises at least one (e.g., one, two, three, four, five or more) nucleotide mismatch with the nucleotide sequence of a second strand of the linker. In some embodiments, at least one of the linker strands comprises a bulge or a loop. For example, at least one of the linker strands comprises at least one (e.g., one, two, three, four, five or more contiguous or non-contiguous) non-complementary nucleobase with the other linker strand.

[0051] Without limitation, the nucleotide-based linker linking the effector molecules may comprise one or more nucleic acid modifications disclosed herein. When the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands, each strand may independently be unmodified or comprise one or more nucleic acid modifications disclosed herein. Thus, in some embodiments, the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands, wherein each strand is unmodified. In some embodiments, the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands, wherein one strand is unmodified and the other strand comprises at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. In some embodiments, the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands, wherein both strands comprise at least one modification independently selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof.

[0052] In some embodiments, the nucleotide-based linker linking the effector molecules comprises two nucleic acid strands, one of which comprises all DNA and the other of which comprises a mixture of DNA and 2'-O alkyl modifications.

[0053] The nucleotide-based linker linking the effector molecules can be resistant to degradation or cleavage by single- or double-stranded nucleases. Alternatively, the nucleotide-based linker linking the effector molecules can be a cleavable linker. For example, the linker linking the effector molecules can be cleaved by single- or double-stranded nucleases.

[0054] As described herein, the linker that connects the effector molecules in the multi-targeting molecule can be a non-nucleotide-based linker. In some embodiments, the non-nucleotide-based linker that connects two oligonucleotides comprises a cleavable group.

[0055] In some embodiments, the non-nucleotide-based linker linking the two oligonucleotides comprises at least one disulfide group.

[0056] As disclosed herein, in some embodiments, at least two effector molecules in a multi-targeting molecule are covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, and the multi-targeting molecule is further conjugated with at least one ligand. Without limitation, the ligand may be present at any position in the multi-targeting molecule. For example, the ligand may be present at the end of one of the at least two effector molecules covalently linked by the linker, at an internal position in one of the at least two effector molecules covalently linked by the linker, or at a position in the linker.

[0057] In some embodiments, a multi-targeting molecule comprising at least two covalently linked effector molecules is conjugated with at least one ligand. Without limitation, the ligands can be the same or different. The two ligands can be independently conjugated at any position in the multi-targeting molecule. For example, the first ligand can be present in the first effector molecule, and the second ligand can be present in the linker that connects the first effector molecule to the second effector molecule, or the first ligand can be present in the first effector molecule, and the second ligand can be present in the second effector molecule that is covalently linked to the first effector molecule; or both ligands can be present in the same effector molecule; or both ligands can be present in the linker that connects the effector molecules.

[0058] In some embodiments, the linker linking the effector molecules comprises a monomer selected from the group consisting of Q151 (FIG. 26), Q173 (FIG. 26), the monomers shown in FIGS. 19-24, and the monomers described below in the section entitled "Exemplary Ligand Monomers." For example, the monomers described in paragraphs

[0316] -

[0365] . Without limitation, the ligand can be present at any position in the linker. For example, the ligand can be conjugated at a central position or within 1, 2, or 3 monomers or units of the center of the linker. In some embodiments, the ligand-bearing monomer serves as the linker.

[0059] In some embodiments, the multi-targeting molecule is assembled from two siRNAs, wherein the two siRNAs are covalently linked to each other via a nucleotide-based or non-nucleotide-based linker. In some embodiments, the linker linking the two siRNAs comprises the nucleotide sequence uuu or (dT)n, where n is 1 to 20. In some embodiments, the linker linking the effector molecules comprises the nucleotide sequence -(CH2) 12 -(C12 linker or Q50), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker or Q51), Q151, Q173, -CH2CH2O-(CH2CH2) n —CH2CH2O—CH2CH2O— (where n is 0 or 1 to 20); —(CH2)9—(CH2) n-CH2- (where n is 0 or 1-20); mono-, di-, tri-, tetra-, penta-, or polyprolinol optionally conjugated to a ligand; or mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol optionally conjugated to a ligand. In some embodiments, the linker connecting two siRNAs comprises a molecule selected from those shown in Figures 16-23 and 26. In some embodiments, the linker comprises a monomer selected from the monomers described below in the section entitled "Exemplary Ligand Monomers," e.g., monomers 1-30 described below in paragraphs

[0316] -

[0365] . Exemplary linkers are also described in the Examples section of this disclosure, e.g., Examples 1-23.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] In some embodiments, at least one effector molecule in the multi-target molecule is an siRNA. In some embodiments, the multi-target molecule comprises at least an siRNA. 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.

[0066] 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.

[0067] 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.

[0068] As used herein, the term "antisense strand" refers to an oligomeric compound that is substantially complementary or 100% complementary to a target sequence of interest. 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.

[0069] 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.

[0070] In some embodiments, the double-stranded region of the double-stranded oligonucleotide is equal to or at least as long as 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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."

[0076] 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 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.

[0077] 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.

[0078] 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 of mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, for example, by determining the 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).

[0079] 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. The dsRNA agent includes a sense strand and an antisense strand, each having 14 to 40 nucleotides. The dsRNA agent has the formula (I): [ka] It is expressed by:

[0080] In Formula (I), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide comprising 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 comprise a 2'-OMe modification.

[0081] C1 is a thermolabile nucleotide located at the site 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: 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.

[0082] 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 may be at the 2' position of the ribose sugar of the nucleotide, or may 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.

[0083] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0084] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0085] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length.

[0086] q 5 are independently 0 to 10 nucleotides in length.

[0087] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0088] Or, n 4 is 0 to 3 nucleotides in length.

[0089] In some embodiments, n 4may 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).

[0090] In some embodiments, n 4 , q 2 , and q 6 are each 1.

[0091] In some embodiments, n 2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] In some embodiments, T1' and T3' are separated by a length of 11 nucleotides (i.e., the T1' and T3' nucleotides are not counted.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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).

[0102] 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.

[0103] 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).

[0104] 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.

[0105] 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).

[0106] 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.

[0107] 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).

[0108] 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.

[0109] 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.

[0110] In some embodiments, the sense and antisense strands of a dsRNA agent each contain at least two different modifications.

[0111] 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.

[0112] In some embodiments, a dsRNA agent of the invention does not include any 2'-F modifications.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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, wherein 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 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.

[0119] In some embodiments, the sense strand sequence of the dsRNA agent has the formula (Is): [ka] is represented by During the ceremony, B1, B2, and B3 each independently represent 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; C1 is a heat-labile nucleotide (e.g., a UNA or GNA, a mismatch, an abasic, or an acyclic nucleotide such as DNA) located at a site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand); T1 represents a non-ribose, acyclic, or nucleotide containing a chemical modification at the 2'-position or equivalent position of the backbone, which modification confers less steric bulk to the nucleotide than a 2'-OMe modification; for example, T1 is selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 or n 3 are independently 4 to 15 nucleotides in length; n 5 is 1 to 6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; n 2 is 0 to 3 nucleotides in length.

[0120] In some embodiments, the 19, 20, 21, or 22 nucleotide long sense strand sequence of the dsRNA agent has the formula (Is): [ka] is represented by During the ceremony, B1, B2, and B3 each independently represent 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; C1 is a heat-labile nucleotide (e.g., a UNA or GNA, a mismatch, an abasic, or an acyclic nucleotide such as DNA) located at a site opposite the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand); T1 represents a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 or n 3are independently 4 to 15 nucleotides in length; n 5 is 1 to 6 nucleotides in length; n 4 is 1 to 3 nucleotides in length; n 2 is 0 to 3 nucleotides in length.

[0121] In some embodiments, the dsRNA agent of Formula (Is) further comprises a 3' and / or 5' overhang from 1 to 10 nucleotides in length. In one example, the dsRNA agent of Formula (Is) comprises a 5' overhang.

[0122] In some embodiments, C1 comprises one heat-destabilizing nucleotide at position 14, 15, 16, or 17 from the 5' end of the sense strand. For example, C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic, or DNA. In one specific example, C1 is GNA.

[0123] In some embodiments, T1 comprises DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 from the 5' end of the sense strand.

[0124] In some embodiments, a dsRNA agent of the invention comprises a sense strand (Is), where C1 is an acyclic nucleotide (e.g., UNA or GNA), a mismatch, an abasic, or DNA; and T1 comprises DNA, RNA, LNA, 2'-F, or 2'-F-5'-methyl at position 11 from the 5' end of the sense strand.

[0125] In some embodiments, the antisense strand of the dsRNA agent has the formula (Ia): [ka] is represented by During the ceremony, B1', B2', B3', and B4' each independently represent 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; T1', T2', and T3' each independently represent a nucleotide containing a non-ribose, acyclic, or backbone chemical modification at the 2'-position or equivalent position, which modification confers less steric bulk to the nucleotide than a 2'-OMe modification; for example, T1', T2', and T3' each independently are selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; q 1 are independently 4 to 15 nucleotides in length; q 3 or q 7 are independently 1 to 6 nucleotides in length; q 2 or q 6 are independently 1 to 3 nucleotides in length; q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length.

[0126] In some embodiments, the 19, 20, 21, 22, 23, 24, or 25 nucleotide long antisense strand sequence of the dsRNA agent has Formula (Ia): [ka] is represented by During the ceremony, B1', B2', B3', and B4' each independently represent 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; T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; q 1 are independently 4 to 15 nucleotides in length; q 3 or q 7 are independently 1 to 6 nucleotides in length; q 2 or q 6are independently 1 to 3 nucleotides in length; q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length.

[0127] In some embodiments, the dsRNA of Formula (Ia) further comprises a 3' and / or 5' overhang between 1 and 10 nucleotides in length. In one example, the dsRNA agent of Formula (Ia) has a 3' overhang.

[0128] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 or q 4are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent has 3' and / or 5' overhangs of 1 to 10 nucleotides in length on the antisense and / or sense strands.

[0129] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense and an antisense strand, each strand having 14-40 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 or q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent includes a 2-nucleotide long 3' overhang at the 3' end of the antisense strand.

[0130] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 15-30 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification; C1 is the acyclic nucleotide GNA; T1, T1', T2', and T3' are each independently DNA or RNA; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 or q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent has a 3' overhang of 1 to 6 nucleotides in length at the 3' end of the antisense strand.

[0131] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 19-23 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification; C1 is the acyclic nucleotide GNA; T1, T1', T2', and T3' are each independently DNA or RNA; n 1 , n 3 , q 1 , or q 3 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 , q 4 or q 5 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent has a 2-nucleotide long 3' overhang at the 3' end of the antisense strand.

[0132] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a 2'-OMe modification; C1 is the acyclic nucleotide GNA; T1, T1', T2', and T3' are each independently DNA or RNA; n1 , n 3 , q 1 , or q 3 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 , q 4 or q 5 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent has a 2-nucleotide long 3' overhang at the 3' end of the antisense strand.

[0133] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q 6 are independently 1 to 3 nucleotides in length; n 2 or q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent has a 5' overhang of 1 to 10 nucleotides in length at the 5' end of the sense strand.

[0134] In some embodiments, the present invention relates to double-stranded RNA (dsRNA) agents for inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-40 nucleotides: [ka] Including, During the ceremony, B1, B2, B3, B1', B2', B3', and B4' each independently represent 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; C1 is an acyclic nucleotide (e.g., UNA or GNA); T1, T1', T2', and T3' each independently represent a nucleotide containing a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl; n 1 , n 3 , or q 1 are independently 4 to 15 nucleotides in length; n 5 , q 3 or q 7 are independently 1 to 6 nucleotides in length; n 4 , q 2 or q6 are independently 1 to 3 nucleotides in length; n 2 or q 4 are independently 0 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; Here, the dsRNA agent has a 5' overhang of 1 to 10 nucleotides in length at the 5' end of the sense strand and a 3' overhang of 1 to 10 nucleotides at the 5' end of the antisense strand.

[0135] In some embodiments, at least one of the effector molecules in the multitargeting molecules disclosed herein is a microRNA. In some embodiments, the multitargeting molecule includes at least microRNAs that are covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in this disclosure. 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 region 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.

[0136] MicroRNAs are also involved in regulating pathogens in hosts. See, for example, Jopling, CL, et al., Science (2005) vol. 309, pp. 1577-1581. Without intending to be bound by any particular theory, administration of microRNAs, microRNA mimics, and / or anti-microRNA oligonucleotides results in modulation of pathogen survival, growth, development, and / or replication. In some embodiments, the oligonucleotide is a microRNA, microRNA mimic, and / or anti-microRNA, wherein the microRNA is a host microRNA.

[0137] The number of miRNA sequences identified to date is large and continues to grow, illustrative examples of which can be found, for example, in "miRBase: microRNA sequences, targets and gene nomenclature" Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. NAR, 2006, 34, Database Issue, D140-D144; "The microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111; and further on the World Wide Web at http: / / microrna.dot.sanger.dot.ac.dot.uk / sequences / .

[0138] Mature miRNAs are generally characterized by a "seed region" comprising bases 2-7 at the 5' end. The seed region is thought to primarily define the specificity of the miRNA for the 3'UTR of its target mRNA and is used for computational target prediction. Although several hundred target mRNAs are predicted for each miRNA, relatively few targets have been experimentally verified to date. Recent deep sequencing techniques have led to changes in the current miRNA database, and the number of miRNAs has increased. *Furthermore, in some miRNA stem-loops, such as mir-302b, both the 5' and 3' stem-loop sequences were annotated as mature miRNAs, suggesting that both miRNA strands may have functional properties.

[0139] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is a ribozyme. In some embodiments, the multi-targeting molecule comprises at least two ribozymes that are covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in this disclosure.

[0140] Ribozymes are oligonucleotides containing specific catalytic domains with 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, which is 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 it 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 has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0141] 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 therein.

[0142] In some embodiments, at least one of the effector molecules in the multi-targeting molecule disclosed herein is an aptamer. In some embodiments, the multi-targeting molecule comprises at least two aptamers that are covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure. 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. Additionally, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers" that contain consensus sequences obtained from comparing two or more known aptamers to a given target.

[0143] Because transcription factors recognize their relatively short binding sequences even in the absence of surrounding genomic DNA, short oligonucleotides containing consensus binding sequences for specific transcription factors can be used as tools to manipulate gene expression in living cells. This approach involves intracellular delivery of such "decoy oligonucleotides," which are then recognized and bound by target factors. Thus, in some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is a decoy oligonucleotide. In some embodiments, the multi-targeting molecule comprises at least two decoy oligonucleotides covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure.

[0144] 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.

[0145] In some embodiments, at least one of the effector molecules in the multitargeting molecules disclosed herein is an miRNA mimic. In some embodiments, the multitargeting molecule comprises at least two miRNA mimics, either covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as those described herein. 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 synthetic non-coding RNAs (i.e., miRNAs 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) and contain one or more sequences that share identity with the mature strand of a given miRNA. The double-stranded miRNA mimics have a similar design as described above for the double-stranded oligonucleotides.

[0146] 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.

[0147] In some embodiments, at least one of the effector molecules in the multitargeting molecule disclosed herein is a supermir. In some embodiments, the multitargeting molecule comprises at least two supermirs, covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as those described in this disclosure, or non-covalently linked to each other. 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 functions 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. While the supermirs that are the subject of the present invention may have secondary structure, they are 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 at a non-end or in the middle of the supermir.

[0148] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an anti-mir. In some embodiments, the multi-targeting molecule comprises at least two anti-mirs, covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as those described in this disclosure. The terms "anti-mir," "microRNA inhibitor," or "miR inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the activity of specific miRNAs. Inhibitors can take various 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. Additionally, miRNA inhibitors may also contain additional sequences located 5' and 3' of the sequence reverse-complementary to the mature miRNA. The additional sequence may be reverse complementary to a sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequence may be any sequence (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences is any sequence capable of forming a hairpin. Thus, in some embodiments, the sequence reverse complementary to the miRNA has a hairpin structure located on the 5' and 3' sides. When the microRNA inhibitor is double-stranded, it may contain mismatches between nucleotides in the opposite strands. Additionally, the microRNA inhibitor may be attached to a conjugate moiety to facilitate uptake of the inhibitor into cells.

[0149] 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.

[0150] In some embodiments, at least one of the effector molecules in the multitargeting molecule disclosed herein is an antagomir. In some embodiments, the multitargeting molecule comprises at least two antagomirs, covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in this disclosure. Antagomirs are RNA-like oligonucleotides with various modifications for pharmacological properties such as RNAse protection and improved tissue and cellular uptake. They differ from conventional RNA, for example, by complete 2'-O-methylation of the sugars, a phosphorothioate intersugar linkage, and a cholesterol moiety, for example, at the 3' end. In a preferred embodiment, the antagomir comprises 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. Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes 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.

[0151] 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, covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in this disclosure. The U1 adaptor is a bifunctional oligonucleotide that interrupts the polyA site and has a targeting domain complementary to a site in the terminal exon of the target gene and an "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 early 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).

[0152] 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.

[0153] 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.

[0154] The effector domain can be about 8 nucleotides to about 30 nucleotides, about 10 nucleotides 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 2-11. In another embodiment, the U1 domain is fully complementary to nucleotides 2-11 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.

[0155] 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.

[0156] In some embodiments, the linker between the annealing domain and the effector domain is multivalent, e.g., 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.

[0157] 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.

[0158] 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 induce RNAa have also been found in humans. See E. Nature (2007). 448(7156):855-858.

[0159] Another surprising observation is that gene activation by RNAa is long-lasting. It has been found that the induction of gene expression lasts 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.

[0160] Thus, 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 molecules comprise at least two activator-RNAs that are covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in this disclosure.

[0161] Thus, in some embodiments, at least one of the effector molecules in the multi-targeting molecule disclosed herein is a triplex-forming oligonucleotide (TFO). In some embodiments, the multi-targeting molecule comprises at least two TFOs that are covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in the present disclosure. 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

[0162] 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.

[0163] Thus, for any given sequence, triplex-forming sequences can be devised. Triplex-forming oligonucleotides are preferably at least 15, more preferably 25, and even more preferably 30 or more nucleotides in length, up to 50 or 100 nucleotides.

[0164] 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 cells treated with TFOs 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, which is 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).

[0165] 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.

[0166] Nucleic acid modification In some embodiments, the multi-targeting molecule comprises 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 multi-targeting molecule. For example, the modification may be present in one of the effector molecules or the linker connecting two effector molecules.

[0167] Nucleic acid modifications (nucleobases) 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.

[0168] 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 mimetics 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 comprise a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases containing conjugate moieties, such as ligands, described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl, or amide bonds.

[0169] The oligomeric compounds described herein can also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. 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 Pseudouracil, 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 Douracil, 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-3 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-listed bases and "universal bases" can also be used.

[0170] 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 the oligonucleoside duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazol, 4-methylbenzimidazol, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenathracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0171] Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application PCT / US Patent Application No. 09 / 038425, filed March 26, 2009; those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-85, 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.

[0172] 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 skilled in the art.

[0173] Nucleic acid modification (sugar) The multitargeting molecules provided herein 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.

[0174] 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)-; During the ceremony, 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.

[0175] 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.

[0176] 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.

[0177] 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, 8, 1-7; 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).

[0178] 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).

[0179] 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.

[0180] 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.

[0181] 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; Nos. 5,591,722; 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 WO 2005 / 121371.

[0182] 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 AMINE (n=1-10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0183] "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 Included are 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 functionality.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] The multitargeting molecules disclosed herein 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, e.g., 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 contain modifications to one or more of the constituent sugar atoms. The 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.

[0188] Sugar modifications may also include acyclic nucleotides in which a 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, an 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.

[0189] 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.

[0190] 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, e.g., 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.

[0191] The hydrogen attached to C4' and / or C1' can be replaced by a straight 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] NR 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, for each occurrence, independently 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 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is, for each occurrence, independently 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.

[0192] 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, for each occurrence, is independently 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.

[0193] In some embodiments, the LNA has the formula: [ka] and a bicyclic nucleoside having the formula: During the ceremony, 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-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0194] 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).

[0195] 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).

[0196] 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.

[0197] 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.

[0198] In some such embodiments, the Z group is in the (R)-configuration: [ka] is.

[0199] In some such embodiments, the Z group is in the (S)-configuration: [ka] is.

[0200] 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.

[0201] 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.

[0202] In some embodiments, the multi-targeting molecule has the formula: [ka] or the expression: [ka] or the expression: [ka] and at least one monomer of During the ceremony, 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-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0203] 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).

[0204] 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).

[0205] 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.

[0206] 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).

[0207] 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-).

[0208] 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-).

[0209] 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-.

[0210] 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-.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] In some embodiments, at least one Z is CH3-. In other embodiments, each Z is CH3-.

[0216] 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

[0217] In some embodiments, the Z group of each monomer of the above formula is in the (R)-configuration.

[0218] 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

[0219] In some embodiments, the Z group of each monomer of the above formula is in the (S)-configuration.

[0220] 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.

[0221] In some embodiments, the multi-targeting molecule has the formula: [ka] or the expression: [ka] or the expression: [ka] and at least one region of at least two consecutive monomers of

[0222] 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.

[0223] In some embodiments, a multitargeting molecule comprises 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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).

[0228] 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 any 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 O, S, Se, B, C, H, N, or OR (where R is alkyl or aryl).

[0229] 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.

[0230] 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."

[0231] 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 reactive 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.

[0232] 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')), hydroxyamino, 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, methyl Nonionic bonds containing mixed N, O, S, and CH moieties, such as phenylimino, methylenecarbonylamino, methylenemethylimino (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.

[0233] 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 in many cases, modification of the non-bridging oxygen may require modification of the 2'-OH, e.g., a modification that does not participate in cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0234] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates containing at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Sp isomer, phosphorothioates containing at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0235] In some embodiments, the multi-targeting molecule comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, including and including each) modified or non-phosphodiester linkage. In some embodiments, the multi-targeting molecule comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, including and including each) phosphorothioate linkage.

[0236] 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 theory, it is believed that the absence of a repeatedly charged backbone weakens binding to proteins that recognize polyanions (e.g., nucleases). Also without intending to be bound by 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.

[0237] The multitargeting molecules described herein contain one or more asymmetric centers and may therefore 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 optionally pure forms.

[0238] Nucleic acid modification (end modification) The termini of the multitargeting molecule or the effector molecule contained therein 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 to a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entity, such as a protecting group (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).

[0239] 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.

[0240] 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 selected from 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 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. The heterocyclic is preferably substituted with an aryl or heteroaryl. In some embodiments, one or both hydrogens on the C5' of the 5'-terminal nucleotide are substituted with a halogen, e.g., F.

[0241] 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.

[0242] 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.

[0243] Thermal destabilization modification 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 thermolabile nucleotide 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.

[0244] 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, e.g., unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA).

[0245] Exemplary abasic modifications are as follows: [ka]

[0246] Exemplary sugar modifications are as follows: [ka]

[0247] 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' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) 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.

[0248] 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

[0249] The thermodestabilizing modification may 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.

[0250] Further examples of abasic nucleotides, acyclic nucleotide modifications (such as UNA and GNA), and mismatch modifications are described in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0251] 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.

[0252] 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.

[0253] Exemplary phosphate modifications that have been shown to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are: [ka] is.

[0254] In some embodiments, the effector molecules in the multitargeting molecule comprise a 2'-5' linkage (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 inhibit binding of the sense and antisense strands, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0255] In another embodiment, the effector molecule in the multitargeting molecule comprises 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.

[0256] In one embodiment, a dsRNA agent of the invention is conjugated to a ligand via a carrier, which may 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.

[0257] 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)(HS )(S)PO-5'), 5'-phosphorothiolates ((HO)2(O)PS-5'); 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'-alkylphosphonates (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'-).

[0258] Target gene Without limitation, target genes for siRNA 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, allele genes found in cells characterized by loss of heterozygosity, or allele genes of polymorphic genes.

[0259] Specific exemplary target genes for siRNA 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; and BCL-2. Genes; 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; topoisomerase IIα gene; p73 gene; p21(WAF1 / CIP1) gene, p27(KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor gene; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, e.g., 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; αv-integrin gene; Flt-1 receptor gene; tubulin gene; human papilloma virus genes, genes required for human papilloma virus replication; human immunodeficiency virus genes, genes required for human immunodeficiency virus replication; hepatitis A virus genes, hepatitis A virus genes Genes required for Hepatitis B 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 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 genes, Mycobacterium leprae genes, genes required for Mycobacterium leprae replication, Staphylococcus aureus genes, genes required for Staphylococcus 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-required 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, CMBKR3 gene, C MBKR5v, 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 genes, DRPLA genes, SCA1 genes, SCA2 genes, MJD1 genes, CACNL1A4 genes, SCA7 genes, SCA8 genes, alleles found in loss of heterozygosity (LOH) cells, one allele of a polymorphic gene, and combinations thereof.

[0260] Loss of heterozygosity (LOH) can result in hemizygous 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 genes whose loss promotes unwanted proliferation, e.g., tumor suppressor genes, as well as other sequences, including 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.

[0261] In some embodiments, the present invention provides multi-target molecules that regulate microRNAs.

[0262] Ligand In some embodiments, the multitargeting molecule is modified by the covalent attachment of one or more conjugate groups. Generally, the conjugate group modifies one or more properties of the attached multitargeting molecule, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are commonly used in the chemical arts and are attached to parent compounds, such as oligomeric compounds, directly or via an optional linking moiety or linking group. A preferred list of conjugate groups 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.

[0263] Preferred conjugate groups 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, e.g., 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, e.g., 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); adamantane acetic 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).

[0264] Generally, a wide variety of entities, e.g., ligands, can be attached to the oligomeric compounds described herein. 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, mycobacterium globulin), tomycin 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).

[0265] Peptide and peptidomimetic ligands include natural or modified peptides, e.g., D- or L-peptides; α-, β-, or γ-peptides; N-methylpeptides; azapeptides; peptides with one or more amides, i.e., peptides with 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.

[0266] 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.

[0267] 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 (PEIs), 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.

[0268] 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).

[0269] 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).

[0270] 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.

[0271] 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).

[0272] 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 = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CHCHNH) n Included are protonated amino groups derived from CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0273] As used herein, the term "targeting ligand" refers to any molecule that confers increased affinity to a selected target, e.g., 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.

[0274] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactose (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-galactosamine, 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.

[0275] 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.

[0276] 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 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.

[0277] When there are two or more ligands, the ligands may all have the same properties, or all have different properties, or some ligands may have the same properties and the rest have different properties.For example, the ligands may have targeting properties, endosomolytic activity, or PK regulation properties.In a preferred embodiment, the ligands all have different properties.

[0278] The ligand or tethered ligand may be present on the monomer when the monomer is incorporated into a component of a multitargeting molecule (e.g., an effector molecule or linker). In some embodiments, the ligand can be incorporated into a "precursor" monomer by coupling after the monomer is incorporated into a component of a multitargeting molecule (e.g., an effector molecule or linker). For example, a monomer having an amino-terminated tether (i.e., no ligand attached), e.g., monomer-linker-NH, can be incorporated into a component of a multitargeting molecule (e.g., an effector molecule or linker). Subsequent to incorporation of the precursor monomer into a component of a multitargeting molecule (e.g., an effector molecule or linker), a ligand having an electrophilic group, e.g., 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.

[0279] In another example, one can incorporate a monomer bearing a chemical group suitable for participating in a click chemistry reaction, such as an azide or alkyne terminal tether / linker. Subsequent manipulations, 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.

[0280] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside bond of the multi-targeting molecule. Conjugation to a purine nucleobase or its derivative can be performed at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is linked to a conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can also be performed at any position. In some embodiments, the 2-, 5-, and 6-position of the pyrimidine nucleobase can be substituted with a conjugate moiety. When conjugating a ligand to a 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.

[0281] Conjugation of a nucleoside to a sugar moiety may occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as an abasic residue. An 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.

[0282] 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.

[0283] 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 conjugating nucleic acids and related oligomeric compounds, with or without linking groups, are well described in the literature, 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.

[0284] Ligands can be attached to multitarget molecules via carrier monomers, e.g., ligand carriers. The carrier comprises (i) at least one "backbone attachment point," preferably two, and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally 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, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of a carrier monomer to which a selected moiety is attached. The selected moiety may 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.

[0285] 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; No. 4,824,941 Specification; Specification No. 4,835,263; Specification No. 4,876,335; Specification No. 4,904,582; Specification No. 4,958,013 No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,8 Specification No. 30; Specification No. 5,112,963; Specification No. 5,149,782; Specification No. 5,214,136; Specification No. 5,245, Specification No. 022; Specification No. 5,254,469; Specification No. 5,258,506; Specification No. 5,262,536; Specification No. 5,27 Specification No. 2,250; Specification No. 5,292,873; Specification No. 5,317,098; Specification No. 5,371,241; Specification No. 5,3 Specification No. 91,723; Specification No. 5,416,203, Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5 , 512,667; 5,514,785; 5,565,552; 5,567,810; Specification No. 5,574,142; Specification No. 5,585,481; Specification No. 5,587,371; Specification No. 5,595,726; 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.

[0286] In some embodiments, the multi-targeting molecule is [ka] and a ligand having the structure shown in During the ceremony, 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.

[0287] In some embodiments, the multi-targeting molecule has formula (II), (III), (IV), or (V): [ka] and a ligand of During the ceremony, 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 repeat units may be the same or different; Q and Q' are, for each occurrence, independently absent, -(P 7 -Q 7 -R 7 )p -T 7 -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, independently for each occurrence, 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; L1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is, each 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.

[0288] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0289] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0290] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0291] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0292] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0293] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0294] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0295] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0296] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0297] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0298] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0299] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0300] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0301] Exemplary Ligand Monomers In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0302] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0303] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0304] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0305] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0306] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0307] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0308] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0309] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0310] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0311] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0312] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0313] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0314] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0315] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0316] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0317] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0318] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0319] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0320] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0321] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0322] In some embodiments, L 2A and L 2B are all different.

[0323] In some preferred embodiments, L 3A and L 3B are both the same.

[0324] In some embodiments, L 3A and L 3B are all different.

[0325] In some preferred embodiments, L 4A and L 4B are both the same.

[0326] In some embodiments, L 4A and L 4B are all different.

[0327] In some preferred embodiments, L 5A , L 5B and L 5C are all the same.

[0328] In some embodiments, L 5A , L 5B and L 5C Two of them are the same.

[0329] In some embodiments, L 5A and L 5B is the same.

[0330] In some embodiments, L 5A and L 5C is the same.

[0331] In some embodiments, L 5B and L 5C is the same.

[0332] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0333] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0334] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0335] In some embodiments, the multitargeting molecule has the structure: [ka] where Y is O or S and n is 1-6.

[0336] In some embodiments, the 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.

[0337] In some embodiments, the multitargeting molecule has the structure: [ka] where Y is O or S and n is 1-6.

[0338] In some embodiments, the multitargeting molecule has the structure: [ka] The compound contains at least one, two, three or four monomers of the formula:

[0339] In some embodiments, the multitargeting molecule has the structure: [ka] where X is O or S.

[0340] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0341] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0342] In some embodiments, the multitargeting molecule has the structure: [ka] where R is O or S.

[0343] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0344] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0345] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0346] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0347] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0348] In some embodiments, the multitargeting molecule has the structure: [ka] where R is OH or NHCOOH.

[0349] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0350] 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'', for each occurrence, are each independently O or S.

[0351] In some embodiments, the multitargeting molecule has the structure: [ka] is conjugated with a ligand of

[0352] In some embodiments, the multitargeting molecule has the structure: [ka] The ligands include:

[0353] In some embodiments, the multitargeting molecule has the structure: [ka] The monomers are:

[0354] 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.

[0355] Linking groups or bifunctional linking moieties, such as those known in the art, are suitable for the compounds provided herein. Linking groups are useful for attaching chemical functional groups, conjugate groups, reporter groups, and other groups to selective sites in a parent compound, such as an oligomeric compound. Generally, bifunctional linking moieties comprise a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest, and the other is selected to essentially bind to any selected group, such as a chemical functional group or conjugate group. In some embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophilic groups for reacting with nucleophilic groups and nucleophilic groups for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties comprise amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), and the like. Some non-limiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl, with a non-limiting list of preferred substituents including hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0356] In some embodiments, the ligand is conjugated to the multi-targeting molecule via a linker.

[0357] As used herein, the term "linker" means an organic moiety that connects two parts of a compound. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR 1, C(O), C(O)NH, SO, SO2, SO2NH, or a unit such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkyl and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, and the like, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 1 )2, C(O), a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; 1 is hydrogen, acyl, aliphatic or substituted aliphatic.

[0358] In some embodiments, the linker is -[(P-Q''-R) q -X-(P'-Q'''-R')q’ ] q’’ -T-, wherein P, R, T, P', R', and T are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, CHO; NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CH=NO, [ka] or heterocyclyl; Q" and Q"' are each independently for each occurrence absent, -(CH2) n -, -C(R 1 )(R 2 )(CH2) n -, -(CH2) n C(R 1 )(R 2 )-, -(CH2CH2O) m CH2CH2- or -(CH2CH2O) m CH2CH2NH-; X is absent or a cleavable linking group; R a is H or an amino acid side chain; R 1 and R 2 is, independently for each occurrence, H, CH, OH, SH, or N(R N )2; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; q, q', and q'' are each independently for each occurrence 0 to 20, and the repeat units may be the same or different; n is, independently for each occurrence, 1 to 20; m is, independently for each occurrence, 0 to 50.

[0359] In some embodiments, the linker comprises at least one cleavable linking group.

[0360] In some embodiments, the linker is a branched linker. The branch point of a branched linker may be at least trivalent, but may also be a tetravalent, pentavalent, or hexavalent atom or group exhibiting such multiple valencies. In some embodiments, the branch point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC; where Q is, independently for each occurrence, H or an optionally substituted alkyl. In some embodiments, the branch point is glycerol or a glycerol derivative.

[0361] A cleavable linking group is one that is sufficiently stable outside a cell, but upon entering a target cell, is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least 10 times faster, and preferably at least 100 times faster, inside the target cell or under first reference conditions (which can, e.g., be selected to mimic or represent conditions inside the cell) than in the subject's blood or serum, or under second reference conditions (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0362] Cleavable linking groups 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, that can degrade redox-cleavable linking groups by reduction; esterases; amidases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; general acids, peptidases (which may be substrate-specific), and proteases, as well as enzymes that can act as phosphatases to hydrolyze or degrade acid-cleavable linking groups.

[0363] The linker can comprise a cleavable linking group that can be cleaved by a specific enzyme.The type of cleavable linking group incorporated into the linker can vary depending on the cell to be targeted.For example, a liver targeting ligand can be linked to a cationic lipid via a linker that comprises an ester group.Hepatocytes are rich in esterase, and therefore, the linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex, and testicular cells.

[0364] When targeting cell types that are rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0365] In some embodiments, the cleavable linking group 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) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). In some embodiments, the cleavable linking group 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) compared to inside cells (or under in vitro conditions selected to mimic intracellular conditions).

[0366] Exemplary cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., -SS- and -C(R)2-SS-, where R is H or C1-C6 alkyl and at least one R is C1-C6 alkyl, e.g., CH3 or CH2CH3); phosphate-based cleavable linking groups (e.g., -OP(O)(OR)-O-, -OP(S)(OR)-O-, -OP(S)(SR)-O-, -SP(O)(OR)-O-, -OP(O)(OR)-S-, -SP(O)(OR)-S-, -OP(S)(ORk)-S-, -SP(S)(OR)-O-, -OP(O)(R)-O-, -OP(S)(R)-O-, -SP(O)(R)-O-, -SP(S)(R)-O-, -SP(O)(R)-S-, -OP(S)(R)-S-, -OP(O) (OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(O -OP(S)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-, where R is an optionally substituted straight or branched C1-C 10 alkyl); acid-cleavable linking groups (e.g., hydrazones, esters, and esters of amino acids, -C=NN- and -OC(O)-); ester-based cleavable linking groups (e.g., -C(O)O-); peptide-based cleavable linking groups (e.g., linking groups that are cleaved by enzymes such as intracellular peptidases and proteases, e.g., -NHCHR A C(O)NHCHR B C(O)-, where R A and R B is an R group composed of two adjacent amino acids. Peptide-based cleavable linker groups comprise two or more amino acids. In some embodiments, the peptide-based cleavable bond comprises an amino acid sequence that is a substrate for a peptidase or protease found in cells.

[0367] In some embodiments, the acid-cleavable linking group is cleavable in an acidic environment having a pH of about 6.5 or less (e.g., about 6.-, 5.5, 5.0, or less), or by an agent such as an enzyme that can act as a general acid.

[0368] A linker that is not an oligonucleotide or does not contain a nucleotide or nucleoside is also referred to as a non-nucleotide-based linker.

[0369] motif The present invention also includes multitarget 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 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 multitarget 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 multitarget 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.

[0370] 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.

[0371] 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 similar modification as 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.

[0372] When both strands of a double-stranded molecule contain alternating modification patterns, nucleotides in one strand may be in 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 modifications in the second strand. Preferably, this shift is such that the similarly modified nucleotides in the first and second strands are not in complementary positions to each other.

[0373] 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.

[0374] When both strands of a double-stranded oligonucleotide contain alternating 2'-O-methyl modifications, such 2'-modified nucleotides may 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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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" may be an unmodified nucleoside.

[0379] 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.

[0380] In some embodiments, the 5'-terminal monomer of a compound, e.g., a multi-targeting 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] In some embodiments, such alternating regions include: (2'-F)-(PS)-(2'-OMe)-(PO) is.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] 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.

[0390] 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.

[0391] 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.

[0392] 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 Including, wherein A is a first type of 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 as each other; w and z are 0 to 15; x and y are 1 to 15.

[0393] 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.

[0394] In some embodiments, all of the linkages in the 2-2-3 motif are modified linkages. In some embodiments, all of the linkages are phosphorothioate linkages. In some embodiments, the linkage at the 3' end of each first type of modification is a phosphodiester.

[0395] 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 may include a terminal group attached to the 3'-terminal nucleoside. Such a terminal group may include another nucleoside. Such another nucleoside is typically a non-hybridizing nucleoside.

[0396] 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.

[0397] Combination motifs It should be understood that some of the motifs and modifications described above may be combined. A motif may contain only a small number of nucleotides, and therefore a particular oligonucleotide may contain two or more motifs. As a non-limiting example, in some embodiments, an oligonucleotide in a multi-target molecule may have two or more nucleotide motifs selected from LNA, phosphorothioate linkages, 2'-OMe, and conjugated ligands.

[0398] 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.

[0399] 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.

[0400] 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., the 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.

[0401] 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 may be the same, different, or some combination of the same and different.

[0402] 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, without limitation, specific examples herein, such as 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 are intended to be interpreted in this manner.

[0403] 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.

[0404] In some embodiments, the oligonucleotides in the multi-target molecule comprise two or more chemically distinct regions and have the structure described in International Application PCT / US Ser. No. 09 / 038433, filed Mar. 26, 2009, the contents of which are incorporated herein in their entirety.

[0405] 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).

[0406] Nucleic acids, such as oligonucleotides, can be conveniently and routinely prepared 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.

[0407] 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.

[0408] 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 may 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.

[0409] 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.

[0410] 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 backbone-modified oligonucleotides and their preparation by reductive coupling. U.S. Patent No. 5,386,023 relating to modified nucleobases based on 3-deazapurine ring systems and methods for their synthesis; U.S. Patent No. 5,457,191 relating to modified nucleobases based on N-2 substituted purines; U.S. Patent No. 5,521,302 relating to methods for preparing oligonucleotides with chiral phosphorus linkages; U.S. Patent No. 5,539,082 relating to peptide nucleic acids; U.S. Patent No. 5,554,746 relating to 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, which relates to methods for preparing oligonucleotides having N-2 substituted purines; U.S. Patent No. 5,587,469, which relates to oligonucleotides having 3-deazapurines; U.S. Patent No. 5,223,168 and U.S. Patent No. 5,608,046, both of which relate to conjugated 4'-desmethylnucleoside analogs; U.S. Patent Nos. 5,602,240 and 5,610,289, which relate to 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.

[0411] Compositions and methods for formulating pharmaceutical compositions The 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 formulating a pharmaceutical composition will depend on several criteria, including but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0412] Multitargeting molecules can be used as 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 antisense compound and / or an antidote compound and a pharmaceutically acceptable diluent are used in the methods described herein. In some embodiments, the pharmaceutically acceptable diluent is PBS.

[0413] 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 when administered 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.

[0414] 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.

[0415] 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., by inhalation or insufflation of powder or aerosol using a nebulizer; 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).

[0416] 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 may be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the multitargeting molecules may 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.

[0417] 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.

[0418] 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.

[0419] 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.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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).

[0424] 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 foreign gene was detected within the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0425] 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.

[0426] 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).

[0427] 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).

[0428] 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. An example of a sterically stabilized liposome is one 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).

[0429] 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.

[0430] 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 nonionic detergent containing a PEG moiety, 2C 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 a 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.

[0431] 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.

[0432] 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 produce 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.

[0433] 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.

[0434] 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.

[0435] 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. Each of the references, GenBank accession numbers, etc. cited in this application are incorporated herein by reference in their entirety.

[0436] 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 are described, 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.

[0437] Unless otherwise indicated, the following terms have the following meanings:

[0438] 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.

[0439] 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, of the mRNA levels found in a cell in the absence of the miRNA or RNA interference molecule. In one preferred embodiment, the 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%.

[0440] 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 the level of an RNA molecule or equivalent RNA molecule, such that the 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.

[0441] 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

[0442] 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).

[0443] As used herein, the terms "increase" or "up-regulate," 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 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, compared to a corresponding unmodulated control.

[0444] 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 10-100%, 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.

[0445] 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, for example, a decrease 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 a decrease of 100% or less (i.e., zero level compared to a reference sample), or any decrease between 10% and 100%.

[0446] "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, such as RNAi activity, to proceed. 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.

[0447] 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.

[0448] 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.

[0449] 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 may be modified with any of a variety of substituents.

[0450] As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase may include any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.

[0451] As used herein, the term "heterocyclic base moiety" refers to a nucleobase that comprises a heterocycle.

[0452] 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.

[0453] As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0454] 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.

[0455] As used herein, "internucleoside linkage" refers to the covalent bond between adjacent nucleosides.

[0456] As used herein, "naturally occurring internucleoside linkage" refers to a 3'-5' phosphodiester linkage.

[0457] 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.

[0458] As used herein, the term "control sample" refers to a sample that has not been contacted with a reporter oligomeric compound.

[0459] As used herein, the term "motif" refers to a pattern of unmodified and modified nucleotides in an oligomeric compound.

[0460] 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 may be independently modified or unmodified, and may be the same or different.

[0461] 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.

[0462] 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.

[0463] As used herein, the term "target protein" refers to a protein whose modulation is desired.

[0464] As used herein, the term "target gene" refers to a gene that encodes a target protein.

[0465] 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.

[0466] 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.

[0467] 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.

[0468] 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.

[0469] 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 may 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.

[0470] 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.

[0471] As used herein, "design" or "designed" refers to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

[0472] As used herein, the term "modulation" refers to a 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, i.e., either an increase (stimulation or induction) or a decrease (inhibition or suppression). As another example, modulation of expression can include a perturbation of splice site selection in pre-mRNA processing.

[0473] 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.

[0474] 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.

[0475] 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.

[0476] 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 to 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.

[0477] 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).

[0478] As used herein, unless otherwise indicated, the term "methyleneoxyLNA" refers solely to β-D-methyleneoxyLNA.

[0479] As used herein, the term "MOE" refers to a 2'-O-methoxyethyl substituent.

[0480] 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.

[0481] 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., a drug) within the body or its cells by the action of endogenous enzymes or other chemicals and / or conditions.

[0482] 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.

[0483] As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification incorporated at either end of an antisense compound.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent that confers a therapeutic benefit on an animal.

[0489] 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.

[0490] 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.

[0491] 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.

[0492] 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, e.g., in a test tube or reaction vessel, in a cell culture, etc. As used herein, the term "ex vivo" refers to cells that have been 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).

[0493] 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, e.g., Rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and sport animals include cattle, horses, pigs, deer, bison, water buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, avian species, e.g., chickens, emus, ostriches, and fish, e.g., 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.

[0494] 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.

[0495] 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, whether male or female, as well as fetuses. 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.

[0496] 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.

[0497] 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.

[0498] As used herein, the term "subcutaneous administration" refers to administration just below the skin. "Intravenous administration" means administration into a vein.

[0499] 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.

[0500] 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.

[0501] As used herein, the term "active pharmaceutical ingredient" refers to the substance in a pharmaceutical composition that imparts a desired effect.

[0502] 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, increased bilirubin may indicate hepatotoxicity or liver function abnormalities.

[0503] 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" includes 1 to about 6 carbon atoms. As used herein, alkyl groups may optionally include one or more additional substituents.

[0504] 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 may optionally contain one or more additional substituents.

[0505] 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.

[0506] 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.

[0507] 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, e.g., polyalkylene glycols, polyamines, and polyimines. The aliphatic groups used herein may optionally contain other substituents.

[0508] 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 may 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 may 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.

[0509] 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 may optionally include other substituents.

[0510] 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 may optionally include additional substituents attached to the alkyl, aryl, or both groups that form the radical group.

[0511] 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. Heterocyclic groups as used herein 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 may optionally include additional substituents.

[0512] 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 additional substituents on either or both of the heteroaryl or alkyl portions.

[0513] 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 can contain ring atoms selected from C, N, O, and S, thereby forming heterocyclic rings, as well as rings containing only C ring atoms, which can be present in mixed motifs, such as benzimidazole, where one ring has only carbon ring atoms and the fused ring has 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.

[0514] 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 may optionally include other substituents.

[0515] 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.

[0516] 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 protected or unprotected and can be added to one available site or to 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. [Example]

[0517] Example 1: Synthesis of bis(siRNA) with parallel and antiparallel strand orientation [ka] As shown in S...

Claims

1. A multi-targeting molecule comprising a first double-stranded siRNA molecule and a second double-stranded siRNA molecule, wherein the first siRNA and the second siRNA are 【Chemistry 1】 The multi-targeting molecules are covalently linked to each other via a non-nucleotide based linker selected from the group consisting of:

2. The multitargeting molecule of claim 1 , wherein the sense strand of the first siRNA molecule is covalently linked to the sense strand of the second siRNA molecule.

3. The multitargeting molecule of claim 1 , wherein the sense strand of the first siRNA molecule is covalently linked to the antisense strand of the second siRNA molecule.

4. The multitargeting molecule of claim 1 , wherein the antisense strand of the first siRNA molecule is covalently linked to the antisense strand of the second siRNA molecule.

5. The multitargeting molecule of any one of claims 1 to 3, wherein the first and second siRNAs independently modulate gene expression of their respective target nucleic acids by at least 70% compared to when the first siRNA and the second siRNA are not part of the multitargeting molecule.

6. The multitargeting molecule of any one of claims 1 to 5, wherein the first siRNA regulates gene expression of a first target nucleic acid and the second siRNA regulates gene expression of a second nucleic acid.

7. The multitarget molecule of claim 6 , wherein the first target nucleic acid and the second target nucleic acid are the same.

8. The multitargeting molecule of claim 7 , wherein the first siRNA and the second siRNA target the same nucleic acid sequence.

9. The multitargeting molecule according to any one of claims 1 to 8, comprising at least one ligand conjugated to said multitargeting molecule.

10. The multitargeting molecule of claim 9 , wherein the ligand is conjugated to one of the sense strand or the antisense strand.

11. The multitargeting molecule of claim 10, wherein the ligand is conjugated at the 3' end of one of the sense strands.

12. The multitargeting molecule of claim 10, wherein the ligand is conjugated at the 5' end of one of the sense strands.

13. The multitargeting molecule of claim 10, wherein the ligand is conjugated at the 3' end of one of the antisense strands.

14. The multitargeting molecule of claim 10, wherein the ligand is conjugated at the 5' end of one of the antisense strands.

15. The multitargeting molecule of any one of claims 1 to 14, comprising at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof.

16. The multitargeting molecule of claim 15 , wherein the at least one modification is comprised in the sense strand, the antisense strand, or the linker connecting the at least two siRNA molecules.

17. The method of claim 1, wherein the first siRNA and the second siRNA comprise a non-nucleotide based linker comprising: 【Chemistry 2】 A multitargeting molecule according to any one of claims 1 to 16, which are covalently linked to each other via:

18. The method of claim 1, wherein the first siRNA and the second siRNA comprise a non-nucleotide based linker: 【Chemistry 3】 A multitargeting molecule according to any one of claims 1 to 16, which are covalently linked to each other via:

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