Multi-targeted single entity conjugates

Abstract

To provide, in general, compounds, compositions and methods useful for modulating gene expression of multiple target nucleic acids by a single chemical entity.SOLUTION: A multi-targeted molecule comprises a first double-stranded siRNA molecule and a second double-stranded siRNA molecule, where the first siRNA and the second siRNA are covalently linked to each other, and where at least one ligand is conjugated with the multi-targeted molecule.SELECTED DRAWING: Figure 3A

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 194,003, filed on July 17, 2015, under 35 U.S.C.§119(e), the content of which is hereby incorporated by reference in its entirety into this specification.

Technical Field

[0002] The present disclosure generally relates to compounds, compositions, and methods useful for modulating the gene expression of multiple targets.

Background Art

[0003] There is a need in the art for molecules capable of targeting two or more targets. The present disclosure provides some solutions to that need.

Summary of the Invention

Means for Solving the Problems

[0004] In one aspect, multi - target molecules are provided herein. Generally, a multi - target molecule comprises at least two nucleic acid - based effector molecules covalently or non - covalently bonded to each other. Without limitation, any nucleic acid - based effector molecule capable of modulating the gene expression of a target can be included in the multi - target molecules disclosed herein.

[0005] The term "nucleic acid-based effector molecule" means a modified or unmodified single-stranded or double-stranded nucleic acid molecule capable of regulating the gene expression of a target gene. Exemplary nucleic acid-based effector molecules capable of regulating the gene expression of a target gene include, but are not limited to, double-stranded and single-stranded RNA interference agents (such as siRNA and shRNA, and those referred to herein as dsRNA agents), antisense oligonucleotides, microRNA, anti-microRNA or anti-mir, supermir, antagomir, ribozymes, triple helix-forming oligonucleotides, decoy oligonucleotides, RNA activators, U1 adapters, guide RNA (gRNA) of CRISPR Cas, etc.

[0006] Note 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. Thus, the multi-target molecule disclosed herein is different from a molecule in which one effector molecule is directed to two different targets, for example, a double-stranded effector molecule in which each strand is directed to a different target or an effector molecule containing a sequence at least partially complementary to or capable of hybridizing with two different target sequences.

[0007] In some embodiments, the multi-target molecule or the effector molecule in the multi-target molecule does not regulate non-specific gene expression by two different mechanisms. For example, the multi-target molecule or the effector molecule in the multi-target molecule does not regulate gene expression by RNA interference and targeting of the seed region of microRNA.

[0008] In some embodiments, each nucleic acid-based effector molecule in the multi-target molecule can regulate the gene expression of the target nucleic acid. Without limitation, each effector molecule in the multi-target molecule can be directed to the same target gene, different target genes, different positions by the same target gene, or different transcripts of the same target gene. Further, 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, the effector molecules included in the multi-target molecules described herein have equivalent gene expression regulatory activity compared to the gene expression regulatory activity when the effector molecules are not part of the multi-target molecule. In other words, the effector molecules have similar gene expression regulatory activity when they are part of the multi-target molecules disclosed herein as compared to when they are not part of the multi-target molecule. In some embodiments, the effector molecules included in the multi-target molecules described herein can independently regulate the 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 regulation of gene expression when they are not part of the multi-target molecule. In some embodiments, one of the effector molecules in the multi-target molecule regulates gene expression at a higher level compared to other effector molecules in the multi-target molecule. In some embodiments, the at least two effector molecules in the multi-target molecule regulate gene expression at similar levels (e.g., within a range of 10%, 7.5%, 5%, 2.5% or less of each other).

[0010] The inventors have found that multi-target molecules conjugated with ligands are particularly effective in regulating gene expression. Thus, in some embodiments, at least one ligand is conjugated to a multi-target molecule. Accordingly, a multi-target molecule conjugated with at least one ligand is also referred to herein as a "conjugated multi-target molecule". Without limitation, the ligand can be present in any of the effector molecules in the multi-target molecule. Further, the ligand can be present at any position of the effector molecule and / or the multi-target molecule. For example, the ligand can be conjugated at the 5'-end, 3'-end, internal position, or a combination thereof of the effector molecule in the multi-target molecule. In some embodiments, at least two ligands are conjugated to a multi-target molecule. The at least two ligands can be the same, different, or any combination of the same and different. The two ligands can be independently conjugated at any position in the multi-target molecule. In some embodiments, at least one ligand is bound to at least two effector molecules in the multi-target molecule. Without intending to be bound by a particular theory, the ligand can improve the delivery or pharmacokinetic profile of the conjugated multi-target molecule.

[0011] At least two effector molecules in the multi-target molecules disclosed herein can be covalently bonded 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, two effector molecules are bonded to each other via a nucleotide-based linker. In some other embodiments, two effector molecules are bonded to each other via a non-nucleotide-based linker.

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

[0013] As disclosed herein, at least two effector molecules in the multi-target molecules described herein can be covalently bound to each other via a nucleotide-based linker. Without limitation, the nucleotide-based linker that links the effector molecules can be all DNA, all RNA, or a mixture of DNA and RNA. In some embodiments, the nucleotide-based linker that links the two effector molecules is all DNA. RNA and DNA can be natural and modified. Further, the nucleotide-based linker that links the two effector molecules can be unmodified or can include one or more nucleic acid modifications described in this disclosure. Thus, in some embodiments, the nucleotide-based linker that links the effector molecules includes at least one modification selected from the group consisting of modified nucleoside linkages, modified nucleobases, modified sugars, and any combination thereof.

[0014] Nucleotide-based linkers that connect effector molecules can comprise one or two nucleic acid strands, and can be single-stranded, double-stranded, or can contain single-stranded and double-stranded regions. In some embodiments, the nucleotide-based linker that connects the effector molecule 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 that connects the effector molecule comprises two nucleic acid strands, one of the strands containing all DNA and the other strand containing a mixture of DNA and 2'-O alkyl modifications. In some embodiments, the linker that connects the effector molecule comprises the nucleotide sequence uuu or (dT)n, where n is from 1 to 20. In some embodiments, the linker that connects the effector molecule is -(CH2) 12 -(C12 linker or Q50), -(CH2)6-S-S-(CH2)6-(C6-S-S-C6 linker or Q51), Q151, Q173, -CH2CH2O-(CH2CH2) n -CH2CH2O-CH2CH2O-, where n is 0 or from 1 to 20; -(CH2)9-(CH2) n -CH2-, where n is 0 or from 1 to 20; mono-, di-, tri-, tetra-, penta- or polyprolinol optionally conjugated to a ligand; a molecule selected from the group consisting of mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinol optionally conjugated to a ligand. In some embodiments, the linker that connects the effector molecule comprises a molecule selected from those shown in FIGS. 16-23 and 26. In some embodiments, the linker comprises a monomer selected from the monomers described hereinafter in the section entitled "Exemplary Ligand Monomers". For example, monomers 1-30 described hereinafter in paragraphs

[0316] -

[0365] . Exemplary linkers are also described in the Examples section of the present disclosure, for example, Examples 1-23. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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DETAILED DESCRIPTION OF THE INVENTION

[0016] It should be understood that neither the foregoing summary nor the following detailed description is intended to limit the invention, but rather to exemplify and explain it, as set forth in the claims. In this specification, the use of the singular includes the plural unless otherwise stated. As used herein, the use of "or" means "and / or" unless otherwise stated. Further, the use of the terms "comprising," "including," and other forms thereof is not limiting. Also, the terms such as "element" or "component" include both elements and components that include one unit and elements and components that include two or more subunits, unless otherwise stated.

[0017] The section headings used in this specification are for purposes of organization only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, such as, 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 aspects described herein are based on multi-target molecules that each contain at least two nucleic acid-based effector molecules capable of regulating the gene expression of a target gene. Without limitation, each effector molecule in the multi-target molecule can be directed to the same target gene, different target genes, or different positions by the same target gene. Generally, the multi-target molecule contains at least two effector molecules.

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

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

[0021] In some embodiments, the siRNA comprises a first strand and a second strand. Thus, in some embodiments, a portion of one of the strands in the first siRNA hybridizes to a portion of one of the strands in the second siRNA. The hybridizing strands of the first and second siRNAs can both be sense strands, both be antisense strands, or one be a sense strand and the other be an 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 in 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 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 in 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, a portion of a strand in a first siRNA that is complementary to a portion of a strand of a 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, a portion of a strand in a second siRNA that is complementary to a portion of a strand of 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. Note that the two portions need not be of the same length. Thus, one can be shorter than the other.

[0025] Without limitation, the length complementary between the strands of the first siRNA and the 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, perfect complementarity between the strands of the first siRNA and the second siRNA may not be required. Thus, the complementary portion can include one or more (e.g., 1, 2, 3, 4, 5 or more) nucleotide mismatches, bulges, or loops.

[0026] The portion of the strand in the first siRNA that has complementarity to the strand of the second siRNA can be all DNA, all RNA, or a mixture of DNA and RNA. The RNA and DNA can be natural and modified. Thus, the portion of the strand in the first siRNA that has complementarity to the strand of the second siRNA can be unmodified or can include one or more of the nucleic acid modifications described herein.

[0027] Similarly, the strand portions in the second siRNA having complementarity to the strand of the first siRNA can all be DNA, all be RNA, or a mixture of DNA and RNA. The RNA and DNA can be natural and modified. Thus, the strand portions in the second siRNA having complementarity to the strand of the first siRNA can be unmodified or can include one or more of the nucleic acid modifications described herein.

[0028] In some embodiments, all of the strand portions in the first siRNA that are complementary to a portion of the strand of the second siRNA are RNA. In some embodiments, all of the strand portions in the first siRNA that are complementary to a portion of the strand of the second siRNA are DNA. Further, the complementary region can be unmodified or can include one or more of the nucleic acid modifications described in this disclosure. Thus, in some embodiments, the complementary region includes 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 strand portion in the first siRNA having complementarity to the strand of the second siRNA is all RNA, and the strand portion in the second siRNA having complementarity to the strand of the first siRNA is all DNA.

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

[0031] In some embodiments, the multi-target molecule is assembled from two separate siRNA molecules, where at least one ligand is attached to each siRNA, and where 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-target molecule is assembled from two separate siRNA molecules, with at least one ligand attached to each siRNA, where a portion of the antisense strand of the first siRNA hybridizes to a portion of the sense strand of the second siRNA molecule. In still other embodiments, the multi-target molecule is assembled from two separate siRNA molecules, where at least one ligand is attached to each siRNA, and where 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-target molecule is assembled from two separate siRNA molecules, where at least one ligand is attached to each siRNA, and where 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 the multi-target molecule, where at least two siRNAs each having at least one ligand are non-covalently bound to each other, the sense strand of the siRNA in the multi-target molecule can include a single-stranded overhang at its 3' end, together with the 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 can include from about 1 nucleotide to about 25 nucleotides. For example, the single-stranded overhang 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.

[0033] In various embodiments of a multi-target molecule in which at least two siRNAs each having at least one ligand are non-covalently bound to each other, the antisense strand of the siRNA in the multi-target molecule may include a single-stranded overhang at its 3' end, together with the 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 include from about 1 nucleotide to about 25 nucleotides. For example, the single-stranded overhang 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.

[0034] Without limitation, the single-stranded overhangs in the sense strand and / or the antisense strand can all be DNA, all be 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 overhang can be unmodified or can include one or more nucleic acid modifications described in this disclosure. Thus, in some embodiments, the single-stranded overhang includes at least one modification selected from the group consisting of modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof.

[0035] In some embodiments, the single-stranded overhangs in the sense strand of the first siRNA are all RNA, and the complementary single-stranded overhangs in the antisense strand of the second siRNA are all DNA. In another embodiment, the single-stranded overhangs in the antisense strand of the first siRNA are all RNA, and the complementary single-stranded overhangs in the antisense strand of the second siRNA are all DNA. In some embodiments, the single-stranded overhangs in the first siRNA and the single-stranded overhangs 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 the complementary strand of the second siRNA. Further, at the 5' end of the single-stranded overhang that does not have complementary nucleobases in the single-stranded overhangs of other sequences, there can be 0, 1, 2, 3, 4, 5 or more nucleobases. Thus, when two siRNAs in a multi-target molecule are assembled together, there can be a gap of 0 (e.g., a break), 1, 2, 3, 4, 5 or more nucleobases between the 3' ends of the single-stranded overhangs of the sense strand of the first siRNA and the sense strand of the second siRNA. Similarly, when two siRNAs in a multi-target molecule are assembled together, there can be a gap of 0 (e.g., a break), 1, 2, 3, 4, 5 or more nucleobases between the 3' ends of the single-stranded overhangs of the antisense strand of the first siRNA and the sense strand of the second siRNA.

[0037] In various embodiments of a multi-target molecule in which at least two siRNAs each having at least one ligand are non-covalently bound to each other, the at least two ligands can be the same or they can be different. Further, the at least ligands can be conjugated at any position of each siRNA independently. For example, one ligand can be bound to the sense strand of the first siRNA, and the other ligand can be bound to the sense strand of the second siRNA, or one ligand can be bound to the sense strand of the first siRNA, and the other ligand can be bound to the antisense strand of the second siRNA, or one ligand can be bound to the antisense strand of the first siRNA, and the other ligand can be bound to the antisense strand of the second siRNA. Without limitation, the first ligand can be bound independently at the 5' end, 3' end or an internal position of one strand (sense or antisense) of the first siRNA. Similarly, the second ligand can be bound independently at the 5' end, 3' end or an 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 at an 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 at an 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 at an 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 at an internal position of the sense strand of the second siRNA. In some embodiments, one ligand is conjugated at an internal position of the antisense strand of the first siRNA and another ligand is conjugated at an 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 another 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 another 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 another 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 another 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 another 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 another ligand is conjugated at an internal position of the second sense strand. In some embodiments, one ligand is conjugated at an internal position of the first sense strand and another ligand is conjugated at 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 another 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 another 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 another 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 another 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 another ligand is conjugated at an internal position of the second antisense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first antisense strand and another ligand is conjugated at 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 another 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-target molecule can be covalently linked to each other via nucleotide-based linkers or non-nucleotide-based linkers as are generally known in the art and as described herein. Thus, in some embodiments, at least two effector molecules in a multi-target 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] Note that a nucleotide-based linker can form part of one or both of the effector molecules being joined together. What this means is that at least a portion of the nucleotide sequence of the linker is required for one function of the effector molecule. In preferred embodiments, 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 that regulates 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 the target gene for activity, the linker may or may not be part of the effector molecule required for complementarity with the target sequence. In some embodiments, the linker has no complementarity with the target sequence (e.g., less than 5% complementarity) or hybridizes to the target sequence.

[0042] In some embodiments, the first strand of the double-stranded nucleotide-based linker that links two effector molecules comprises a nucleotide sequence that is substantially complementary to the second strand of the 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 that links two effector molecules.

[0043] Without limitation, nucleotide-based linkers that link effector molecules can be all DNA, all RNA, or a mixture of DNA and RNA. In some embodiments, the nucleotide-based linkers that link two effector molecules are all DNA. RNA and DNA can be natural and modified. Thus, in some embodiments, the nucleotide-based linker that links effector molecules comprises at least one modification selected from the group consisting of modified nucleoside internucleotide linkages, modified nucleobases, modified sugars, and any combination thereof. Exemplary modifications of the linker include, but are not limited to, locked nucleic acids (e.g., LNA, ENA, and BNA), 2'-O-alkyl nucleosides, 2'-halo nucleosides (such as 2'-F nucleotides), 2'-amino nucleosides, 2'-S-alkyl nucleosides, abasic nucleosides, 2'-cyano nucleosides, 2'-mercapto nucleosides; 2'-MOE nucleosides, acryloyl nucleosides, (S)-cEt monomers, and modified internucleotide linkages (phosphodiesters, phosphotriesters, hydrogen phosphonates, alkyl or aryl phosphonates, phosphoramidates, phosphorothioates, phosphorodithioates, methylene methylimino, thiodiesters, thiocarbamates, N,N'-dimethylhydrazine, phosphoroselenates, borano phosphates, borano phosphate esters, amides, hydroxyamines, siloxanes, dialkylsiloxanes, carboxamides, carbonates, carboxymethyl, carbamates, carboxylic acid esters, thioethers, ethylene oxide linkers, sulfides, sulfonates, sulfonamides, sulfonate esters, thioformacetals, formacetals, oximes, methyleneimino, methylenecarbonylamino, methylene methylimino, methylenehydrazo, methylene dimethylhydrazine, methyleneoxymethylimino, ethers, thioethers, and thioacetamides, etc.). Nucleic acid modifications are described in more detail below in the present disclosure.

[0044] In some embodiments, at least one of the internucleoside linkages between the linker that links the effector molecules and the effector molecules is a modified internucleoside linkage. In some embodiments, the internucleoside linkage that links 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 that links 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 that links the effector molecules is a modified internucleoside linkage. In 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 that links the effector molecules includes at least one (e.g., one, two, three, four, five, six or more) modified internucleoside linkage at an internal position of the linker.

[0047] Without limitation, the nucleotide-based linker that links the effector molecules can be of any desired length. For example, the nucleotide-based linker that links 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 that links the effector molecules can range in length from 1 nucleotide to 5 nucleotides. In certain embodiments, the nucleotide-based linker that links two effector molecules is 4 nucleotides in length.

[0048] When a nucleotide-based linker that links effector molecules contains nucleic acid modifications, such modifications can be located at any position in the linker. For example, the modification can be in the 5'-nucleotide, 3'-nucleotide, or internal nucleotide of the linker. In some embodiments, the first (e.g., 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 nucleic acid modifications. In some embodiments, one, two, three, four, five or more internal nucleotides of the linker contain nucleic acid modifications. In some embodiments, the internal nucleotides of the linker contain all of the 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 links effector molecules can contain 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 that links effector molecules contains two nucleic acid strands that do not form a double-stranded structure. In other words, the nucleotide-based linker contains two strands that do not hybridize to each other.

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

[0051] Without limitation, a nucleotide-based linker that links effector molecules can include one or more nucleic acid modifications disclosed herein. When a nucleotide-based linker that links effector molecules includes two nucleic acid strands, each strand can independently be unmodified or can include one or more nucleic acid modifications disclosed herein. Thus, in some embodiments, a nucleotide-based linker that links effector molecules includes two nucleic acid strands, where each strand is unmodified. In some embodiments, a nucleotide-based linker that links effector molecules includes two nucleic acid strands, where one strand is unmodified and the other strand includes at least one modification selected from the group consisting of modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof. In some embodiments, a nucleotide-based linker that links effector molecules includes two nucleic acid strands, and both strands include at least one modification independently selected from the group consisting of modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof.

[0052] In some embodiments, a nucleotide-based linker that links effector molecules includes two nucleic acid strands, where one strand consists entirely of DNA and the other strand includes a mixture of DNA and 2'-O alkyl modifications.

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

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

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

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

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

[0058] In some embodiments, the linker that connects 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 hereinafter 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 within the central position or within 1, 2, or 3 monomers or units of the center of the linker. In some embodiments, the ligand having a monomer serves as a linker.

[0059] In some embodiments, the multi - target molecule is assembled from two siRNAs, where the two siRNAs are covalently bonded to each other via a nucleotide - based or non - nucleotide - based linker. In some embodiments, the linker that connects the two siRNAs comprises the nucleotide sequence uuu or (dT)n, where n is from 1 to 20. In some embodiments, the linker that connects the effector molecules is -(CH2) 12 -(C12 linker or Q50), -(CH2)6 - S - S - (CH2)6 - (C6 - S - S - C6 linker or Q51), Q151, Q173, -CH2CH2O-(CH2CH2) n -CH2CH2O - CH2CH2O-(where n is 0 or from 1 to 20); -(CH2)9-(CH2) n-CH2- (where n is 0 or 1 to 20); a mono-, di-, tri-, tetra-, penta- or polyprolinol optionally conjugated to a ligand; a molecule selected from the group consisting of a 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 FIGS. 16-23 and 26. In some embodiments, the linker comprises a monomer selected from the monomers described hereinafter in the section entitled "Exemplary Ligand Monomers". For example, monomers 1-30 described hereinafter in paragraphs

[0316] -

[0365] . Exemplary linkers are also described in the Examples section of the present disclosure, for example, Examples 1-23.

[0060] In some embodiments, the multi-target molecule is assembled from two siRNAs, where the sense strand of the first siRNA is covalently linked to the sense strand of the second siRNA. Without limitation, the two sense strands can be joined to each other in any orientation. For example, the 3' end of the first sense strand can be joined to the 5' end of the second sense strand; the 3' end of the first sense strand can be joined to the 3' end of the second sense strand; or the 5' end of the first sense strand can be joined to the 5' end of the second sense strand.

[0061] In some embodiments, the multi-target molecule is assembled from two siRNAs, where the antisense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA. Without limitation, the two antisense strands can be joined to each other in any orientation. For example, the 3' end of the first antisense strand can be joined to the 5' end of the second antisense strand; the 3' end of the first antisense strand can be joined to the 3' end of the second antisense strand; or the 5' end of the first antisense strand can be joined to the 5' end of the second antisense strand.

[0062] In some embodiments, the multi-target molecule is assembled from two siRNAs, where 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 the sense strand can be linked to the 5' end of the antisense strand; the 3' end of the sense strand can be linked to the 3' end of the antisense strand; or the 5' end of the sense strand can be linked to the 5' end of the antisense strand.

[0063] In some embodiments, the multi-target molecule is assembled from two siRNAs, where 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-target molecule is assembled from two siRNAs, where 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 molecule Those skilled in the art are well aware that double-stranded oligonucleotides containing a double-helical structure of 20-23, particularly 21 base pairs, are particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, it has 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 siRNA. In some embodiments, the multi-target molecule comprises at least siRNA. As used herein, the term "siRNA" refers to a substance that mediates the targeted cleavage of RNA transcripts. These substances bind to a cytoplasmic multi-protein complex known as the RNA interference-induced silencing complex (RISC). Substances effective in inducing RNA interference are also referred to herein as siRNA, RNAi agents, or iRNA agents. As used herein, the term siRNA includes microRNA and pre-microRNA. 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. Generally, the double-helical structure is 15 to 35, more generally 18 to 25, even more generally 19 to 24, and most generally 19 to 21 base pairs in length. In some embodiments, longer double-stranded oligonucleotides with a length of 25 to 30 base pairs are preferred. In some embodiments, shorter double-stranded oligonucleotides with a length of 10 to 15 base pairs 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 complementary region that is complementary to at least a portion of the target sequence, and the double-helical region is 14 to 30 nucleotides in length. Similarly, the complementary region to the target sequence is 14 to 30, more generally 18 to 25, even more generally 19 to 24, and most generally 19 to 21 nucleotides in length.

[0068] As used herein, the phrase "antisense strand" refers to an oligomeric compound that is substantially complementary or 100% complementary to a target sequence of interest. The phrase "antisense strand" includes the antisense regions of both oligomeric compounds formed from two separate strands, as well as single molecule compounds that can form hairpin or dumbbell-shaped structures. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0069] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence that is all or partially the same as a target sequence such as a messenger RNA or DNA sequence. The terms "sense strand" and "passenger strand" are used interchangeably herein.

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

[0073] In some embodiments, one strand has at least one stretch of 1 to 10 single-stranded nucleotides within a double-stranded region. By "stretch of single-stranded nucleotides within a double-stranded region" is meant that there is at least one nucleotide within the double-stranded region that does not form a base pair with another nucleotide. When a stretch of single-stranded nucleotides is present within the interior of the double-stranded region, at least one nucleotide base pair may be present at both ends of the single-stranded stretch. When present 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 composed of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. When both strands have one stretch composed of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides may be opposite each other (e.g., a stretch of mismatches), or they may be arranged such that the second strand does not contain non-base-paired nucleotides opposite the single-stranded oligonucleotides of the first strand, and vice versa (e.g., a single-stranded loop) is also possible. In some embodiments, the single-stranded nucleotides are present within 8 nucleotides from either end, e.g., 8, 7, 6, 5, 4, 3, or 2 nucleotides from either the 5' or 3' end of the complementary region 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, 20, 21, 22, 23, 24, or 25 nucleotide pairs or more. The double-helical region can be of a length of 200, 100, or 50 or less. In some embodiments, the double-helical region ranges in length from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0075] In some embodiments, the nucleic acid-based effector molecule is a hairpin oligonucleotide that, in some embodiments, may have a single-stranded overhang or terminal unpaired region at the 3' end and, in some embodiments, on the antisense side of the hairpin. In some embodiments, the overhang was 1 to 4, more typically 2 to 3 nucleotides in length. Hairpin oligonucleotides capable of inducing RNA interference are also referred to herein as "shRNA".

[0076] In some embodiments, the two oligonucleotide strands specifically hybridize 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, when there is sufficient complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences.

[0077] As used herein, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which an antisense compound hybridizes to its target sequence but hybridizes to other sequences to a minimum extent. Stringent conditions are sequence-dependent and vary in different circumstances, and the "stringent conditions" under which an antisense compound hybridizes to its target sequence are determined by the nature and composition of the antisense compound and the assay in which they are tested.

[0078] In the art, it is understood that by incorporating nucleotide affinity modifications, more mismatch numbers can be achieved compared to unmodified activation. Similarly, certain oligonucleotide sequences may be more resistant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art can determine an 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 of ordinary skill in the art. For example, by the techniques described by Freier et al. (Nucleic Acids Research, 1997, 25, 22:4429-4443), one of ordinary skill in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA and RNA:RNA double helices.

[0079] In some embodiments, the effector molecule is a double-stranded RNA (dsRNA) agent, i.e., siRNA, for inhibiting the expression of a target gene. It is understood that dsRNA, siRNA, and oligonucleotide can be used synonymously unless otherwise stated. The dsRNA agent includes a sense strand and an antisense strand, each having 14 to 40 nucleotides. The dsRNA agent has the formula (I):

Chemical formula

[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 thermally labile nucleotide located at a site opposite to the seed region of the antisense strand (i.e., positions 2 to 8 at the 5' end of the antisense strand). For example, C1 is at one position of the sense strand that pairs with the nucleotides at positions 2 to 8 at the 5' end of the antisense strand. The C1 nucleotide carries a thermally labile modification, which can include abasic modification; mismatch with the opposite nucleotide in the double helix; and sugar modification such as 2'-deoxy modification or acyclic nucleotides, for example, unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA). In some embodiments, C1 is i) a mismatch with the opposite nucleotide of the antisense strand; ii)

Chemical formula

Chemical formula

Chemical formula

[0082] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that confers a steric bulk less than or equal to that of the 2'-OMe modification to the nucleotide. The modification may be at the 2'-position of the ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, acyclic nucleotide, or the nucleotide backbone, which modification is similar or equivalent to the 2'-position of the ribose sugar and confers a steric bulk less than or equal to that of the 2'-OMe modification to the nucleotide. For example, T1, T1', T2', and T3' each independently are 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 is independently 0 to 10 nucleotides in length.

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

[0088] Alternatively, 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, where there are two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 23 of the antisense strand (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, when the sense strand is 19 to 22 nucleotides in length and n 4 is 1, C1 is at positions 14 to 17 of the 5' end of the sense strand.

[0093] In some embodiments, T3' starts 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 q 6 is equal to 1.

[0094] In some embodiments, T1' starts 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 q 2 is equal to 1.

[0095] In some embodiments, T1’ and T3’ are separated by 11 nucleotides (i.e., the T1’ and T3’ nucleotides are not counted).

[0096] In some embodiments, T1’ is at the 14th position from the 5’ end of the antisense strand. In one example, T1’ is at the 14th position from the 5’ end of the antisense strand, and q 2 is equal to 1, and the non-ribose, acyclic or one or more 2’-position modifications in the backbone impart a steric bulk smaller than that of the 2’-OMe ribose modification.

[0097] In some embodiments, T3’ is at the 2nd position from the 5’ end of the antisense strand. In one example, T3’ is at the 2nd position from the 5’ end of the antisense strand, and q 6 is equal to 1, and the non-ribose, acyclic or one or more 2’-position modifications in the backbone impart a steric bulk smaller than that of the 2’-OMe ribose modification.

[0098] In some embodiments, T1 is at the cleavage site of the sense strand. In one example, when the sense strand is 19 - 22 nucleotides in length and n 2 is 1, T1 is at the 11th position from the 5’ end of the sense strand.

[0099] In some embodiments, T2’ starts at the 6th position 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 q 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, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1.

[0101] In some embodiments, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, q 7 is 1; having two phosphorothioate internucleotide linkage modifications within positions 1 to 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 to 23 of the antisense strand (counting from the 5’ end of the antisense strand).

[0102] In some embodiments, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, 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; having two phosphorothioate internucleotide bond modifications within positions 1 to 5 of the sense strand (counting from the 5’ end), two phosphorothioate internucleotide bond modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide bond modifications within positions 18 to 23 (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, there are two phosphorothioate internucleotide bond modifications within positions 1 to 5 of the sense strand (counting from the 5’ end of the sense strand), two phosphorothioate internucleotide bond modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide bond modifications within positions 18 to 23 of the antisense strand (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, within positions 1 to 5 of the sense strand, there are two phosphorothioate internucleotide bond modifications (counting from the 5'-end of the sense strand), and within positions 1 and 2 of the antisense strand, there are two phosphorothioate internucleotide bond modifications and within positions 18 to 23, there are two phosphorothioate internucleotide bond modifications (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 present invention are modified.

[0109] In some embodiments, each of the sense and antisense strands of the 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'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

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

[0111] In some embodiments, the dsRNA agent of formula (I) further comprises a 3' and / or 5' overhang that is 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' end overhang at the 5'-end of the sense strand.

[0112] In some embodiments, the dsRNA agents of the present invention do not contain any 2'-F modifications.

[0113] In some embodiments, the dsRNA agent of the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, the effector molecule of the present invention comprises 9 or 10 2'-F modifications.

[0114] In some embodiments, the sense strand and / or the antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate nucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate nucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate nucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate nucleotide linkages are separated by 16 to 18 phosphate nucleotide linkages.

[0115] In some embodiments, each of the sense and antisense strands of the dsRNA agent has 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 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 at the 5'-end of the antisense strand in the double helix 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 the dsRNA agent of the present invention is 100% complementary to the target RNA, hybridizes with it, and inhibits its expression by RNA interference. In another embodiment, the antisense strand of the 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 the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, and each strand has 14 to 40 nucleotides. The sense strand contains at least one thermally labile nucleotide, where at least one of the thermally labile nucleotides is located at or near a site opposite to the seed region of the antisense strand (i.e., positions 2 to 8 at the 5'-end of the antisense strand). For example, when the sense strand is 21 nucleotides in length, the thermally labile nucleotide is located at positions 14 to 17 at the 5'-end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification. Preferably, the two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification are separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 at the 5'-end of the antisense strand.

[0119] In some embodiments, the sense strand sequence of the dsRNA agent is of formula (Is):

Chemical formula

[0120] In some embodiments, the sense strand sequence of the dsRNA agent that is 19, 20, 21, or 22 nucleotides in length has the formula (Is):

Chemical formula

[0121] In some embodiments, the dsRNA agent of formula (Is) further comprises 3' and / or 5' overhangs that are 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 thermally labile nucleotide at the 14th, 15th, 16th, or 17th position 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 the 11th position from the 5'-end of the sense strand.

[0124] In some embodiments, the dsRNA agent of the invention comprises a sense strand (Is), wherein 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 the 11th position from the 5'-end of the sense strand.

[0125] In some embodiments, the antisense strand of the dsRNA agent has the formula (Ia):

Chemical formula

[0126] In some embodiments, the antisense strand sequence of the dsRNA agent that is 19, 20, 21, 22, 23, 24, or 25 nucleotides in length has the formula (Ia):

Chemical formula

[0127] In some embodiments, the dsRNA of formula (Ia) further comprises 3' and / or 5' overhangs that are 1 to 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 a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

Chemical formula

[0129] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent has sense and antisense strands, each strand having 14 to 40 nucleotides:

Chemical formula

[0130] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each having 15 to 30 nucleotides:

Chemical formula

[0131] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each having 19 to 23 nucleotides:

Chemical formula

[0132] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent has a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

Chemical formula

[0133] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

Chemical formula

[0134] In some embodiments, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides:

Chemical formula

[0135] In some embodiments, at least one of the effector molecules in the multi-target molecule disclosed herein is a microRNA. In some embodiments, the multi-target molecule comprises at least microRNAs that are covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure, or are non-covalently linked to each other. 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 not translated into proteins. Pre-microRNAs are processed into miRNAs. The processed microRNAs are incorporated into the RNA-induced silencing complex (RISC) and are single-stranded ~17 - 25 nucleotide (nt) RNA molecules that have been identified as major 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. RISC mediates the downregulation of gene expression by translational inhibition, transcript cleavage, or both. RISC is also involved in transcriptional silencing in the nuclei of a wide range of eukaryotes.

[0136] MicroRNAs are also involved in the regulation of pathogens in the host. See, for example, Jopling, C.L., et al., Science (2005) vol. 309, pp 1577-1581. While not intending to be bound by any particular theory, administration of microRNAs, microRNA mimics, and / or anti-microRNA oligonucleotides results in regulation of pathogen survival, growth, development, and / or replication. In some embodiments, the oligonucleotide is a microRNA, a microRNA mimic, and / or an anti-microRNA, where the microRNA is a host microRNA.

[0137] The number of miRNA sequences identified to date is large and continues to increase, exemplary examples of which are, for example, "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 found at http: / / microrna.dot.sanger.dot.ac.dot.uk / sequences / on the World Wide Web.

[0138] Mature miRNAs are generally characterized by a "seed region" that includes 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 in computer-based target prediction. Hundreds of target mRNAs are predicted for each miRNA, although relatively few targets have been experimentally verified to date. Recent deep sequencing techniques have led to changes in current miRNA databases, miRNA *is implicated as a valid miRNA molecule. Further, in some miRNA stem-loops such as mir-302b, both the 5’ and 3’ stem-loop sequences are 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-target molecules disclosed herein is a ribozyme. In some embodiments, the multi-target molecule comprises at least two ribozymes that are covalently bound to each other or non-covalently bound to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure.

[0140] A ribozyme is an oligonucleotide having a specific catalytic domain with endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S 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, an enzymatic nucleic acid acts by first binding to a target RNA. Such binding is effected by the target-binding portion of the enzymatic nucleic acid that is held in close proximity to the enzymatic portion of the molecule that serves to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA by complementary base pairing, and upon binding to the appropriate site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA will disrupt its ability to directly synthesize the encoded protein. After the enzymatic nucleic acid binds to and cleaves its RNA target, it can be released from that RNA to search for another target, bind repeatedly to a new target, and cleave it.

[0141] Methods for producing ribozymes targeted to any target sequence are known in the art. Ribozymes are designed as described in International Patent Application Publication No. WO 93 / 23569 and International Patent Application Publication No. WO 94 / 02595 (each incorporated herein by reference in its entirety), and can be synthesized as described in these references and tested in vitro and in vivo.

[0142] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is an aptamer. In some embodiments, the multi-target molecule comprises at least two aptamers that are covalently bound to each other or non-covalently bound to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in the present disclosure. An aptamer is a nucleic acid or peptide molecule that binds to a corresponding specific molecule with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). A large number of various entities from large proteins have been successfully produced as DNA or RNA aptamers that bind 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 modified to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms by repeating in vitro selection or equivalently SELEX (systematic evolution of ligands by exponential enrichment) multiple times. 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. Further, as fully described herein, the term "aptamer" specifically includes "secondary aptamers" that contain a consensus sequence 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 having the consensus binding sequences of specific transcription factors can be used as tools for manipulating gene expression in living cells. This approach involves the intracellular delivery of such "decoy oligonucleotides," which are then recognized and bound by the target factor. Thus, in some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is a decoy oligonucleotide. In some embodiments, the multi-target molecule comprises at least two decoy oligonucleotides that are covalently bound to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure, or are non-covalently bound to each other.

[0144] Occupation of the DNA binding site of a transcription factor by a decoy prevents the transcription factor from subsequently binding to the promoter region of the target gene. Decoys can be used as therapeutic agents to inhibit the expression of genes activated by transcription factors or to upregulate genes repressed by transcription factor binding. An example of the use of decoy oligonucleotides is found in Mann et al., J. Clin. Invest., 2000, 106:1071-1075, which is hereby incorporated by reference in its entirety.

[0145] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is a miRNA mimic. In some embodiments, the multi-target molecule comprises at least two miRNA mimics that are covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure, or are non-covalently linked to each other. MicroRNA mimics (miRNA mimics) represent a class of molecules that can be used to mimic the gene regulatory activity of one or more miRNAs. Thus, the term "microRNA mimic" refers to a synthetic non-coding RNA that can enter the RNAi pathway and regulate gene expression (i.e., the miRNA is not obtained by purification from a source of endogenous miRNA). miRNA mimics can be designed as mature molecules (e.g., single-stranded) or mimic precursors (e.g., pri- or pre-miRNA). In one design, the miRNA mimic is a double-stranded molecule (e.g., having a double-stranded region about 16 to about 31 nucleotides in length) and contains one or more sequences having identity to the mature strand of a given miRNA. Double-stranded miRNA mimics have a design similar to that described above for double-stranded oligonucleotides.

[0146] In some embodiments, the miRNA mimic comprises a double-stranded region of 16 to 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains nucleotide 1 and 2 (counting from the 5' end of the sense oligonucleotide), and all 2'-O-methyl modifications of Cs and Us; the antisense strand modification can include all 2'F modifications of Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and a stabilized internucleotide linkage conjugated to a 2-nucleotide 3' overhang.

[0147] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is a supermir. In some embodiments, the multi-target molecule comprises at least two supermirs that are covalently bonded to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in the present disclosure, or non-covalently bonded to each other. A supermir refers to an oligonucleotide that is substantially identical to an miRNA and has a nucleotide sequence that is antisense to its target, for example, a single-stranded, double-stranded, or partially double-stranded oligonucleotide. This term also includes oligonucleotides that contain at least one non-natural moiety that functions similarly. In a preferred embodiment, the supermir does not contain a sense strand, and in another preferred embodiment, the supermir does not self-hybridize to a significant extent. The supermir that is the subject of the present invention may have a secondary structure, but it is substantially single-stranded under physiological conditions. A supermir that is substantially single-stranded 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 double-strand with itself. A supermir can include a hairpin segment. For example, the sequence preferably self-hybridizes at the 3' end to form a double-helical region, for example, a double-helical region of at least 1, 2, 3, or 4 nucleotides, preferably less than 8, 7, 6, or 5 nucleotides, for example, a double-helical region of 5 nucleotides. The double-helical region can be linked by a linker, such as a nucleotide linker, such as 3, 4, 5, or 6 dT, for example, a modified dT. In another embodiment, the supermir forms a double-strand with a shorter oligo, for example, 5, 6, 7, 8, 9, or 10 nucleotides in length, at one or both of the 3' and 5' ends or at one end and a non-terminal or central portion of the supermir.

[0148] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is an anti-miR. In some embodiments, the multi-target molecule comprises at least two anti-miRs that are covalently bound to each other or non-covalently bound to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers 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 a particular miRNA. The inhibitor can take various forms including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplex), and hairpin designs. Generally, a microRNA inhibitor comprises 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, the miRNA inhibitor can further comprise additional sequences located 5' and 3' of the sequence that is reverse complementary to the mature miRNA. The additional sequences can be reverse complementary to the sequences adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequences can be random sequences (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences are random sequences capable of forming a hairpin. Thus, in some embodiments, the sequence that is reverse complementary to the miRNA has hairpin structures disposed on the 5' side and 3' side. A microRNA inhibitor, when double-stranded, can include nucleotide mismatches in the reverse strand. Additionally, the microRNA inhibitor can be conjugated to a conjugate moiety to facilitate uptake of the inhibitor into cells.

[0149] MicroRNA inhibitors containing 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 International Publication Nos. WO 2007 / 095387 and WO 2008 / 036825 (each of which is incorporated herein by reference in its entirety). One of ordinary skill in the art can select sequences from a database of desired miRNAs and design inhibitors useful in the methods disclosed herein.

[0150] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is an antagomir. In some embodiments, the multi-target molecule comprises at least two antagomirs that are covalently bonded to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure, or non-covalently bonded to each other. Antagomirs are RNA-like oligonucleotides with various modifications due to pharmacological properties such as RNAse protection and improved tissue and cell uptake. They differ from normal RNA, for example, in the complete 2'-O-methylation of sugars, phosphorothioate sugar linkages, and cholesterol moieties, 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 sugar linkages at the first two positions at 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 a duplex comprising the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of miRNA silencing mediated by an antagomir is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689 (which is hereby expressly incorporated by reference in its entirety).

[0151] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is a U1 adapter. In some embodiments, the multi-target molecule comprises at least two U1 adapters that are covalently bound to each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure, or are non-covalently bound to each other. The U1 adapter is a bifunctional oligonucleotide having a "U1 domain" that inhibits the polyA site and binds to a target domain complementary to a site in the terminal exon of the target gene and to the smaller nuclear RNA component of U1 of the U1 snRNP. See, for example, International Patent Application Publication No. WO 2008 / 121963 Pamphlet and Goraczniak, et al., 2008, Nature Biotechnology, 27(3), 257-263, each of which is hereby expressly incorporated by reference in its entirety. The U1 snRNP is a ribonucleoprotein complex that functions primarily to direct the initial stages in spliceosome formation by binding to the pre-mRNA exon-intron boundary (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49:77-95).

[0152] In some embodiments, the U1 adapter comprises at least one annealing domain (targeting domain) bound to at least one effector domain (U1 domain), wherein the annealing domain hybridizes to the target gene sequence and the effector domain hybridizes to the U1 snRNA of the U1 snRNP. In some embodiments, the U1 adapter comprises one annealing domain. In some embodiments, the U1 adapter comprises one effector domain.

[0153] While not intending to be bound by any particular theory, annealing domains are 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 can 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 the 3'UTR and the polyadenylation signal sequence (e.g., via the polyadenylation site). In another embodiment, 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 with U1 snRNA, particularly the 5'-end, and more specifically, nucleotides 2-11. In another embodiment, the U1 domain is completely complementary to nucleotides 2-11 of 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 U1 domain to include base pairing to stem 1 and / or base pairing to position 1 of U1 snRNA improves the affinity of the U1 adapter for U1 snRNP.

[0155] Annealing of the U1 adapter and the effector domain can be coupled such that the effector domain is at the 5'-end and / or 3'-end of the annealing domain. The two domains can be coupled such that the 3'-end of one domain is coupled to the 5'-end of the other domain, or the 3'-end of one domain is coupled to the 3'-end of the other domain, or the 5'-end of one domain is coupled to the 5'-end of the other domain. The annealing and effector domains can be coupled directly to each other or by a nucleotide-based or non-nucleotide-based linker. If the linker is nucleotide-based, the linker can comprise 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 wishing to be bound by a particular theory, the multivalent linker can be used to couple a single annealing domain with multiple adapter domains.

[0157] It should be understood that the U1 adapter can include any of the oligonucleotide modifications described herein. Exemplary modifications for the U1 adapter 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, i.e., a mechanism called "small RNA-induced gene activation" or RNAa (activated RNA) has been discovered. See, e.g., Li, L.C. et al. Proc Natl Acad Sci U S A. (2006), 103(46):17337-42 and Li L.C. (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 the associated genes. Endogenous miRNAs that cause 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. The induction of gene expression was found to last for more than 10 days. The long-term effects of RNAa may be due to epigenetic changes at the dsRNA target site. In some embodiments, the RNA activator can increase the expression of a gene. In some embodiments, the increased gene expression inhibits survival, growth and / or replication.

[0160] Thus, in some embodiments, at least one of the effector molecules in the multi-target molecule disclosed herein is an activating RNA. In some embodiments, the multi-target molecule comprises at least two activating RNAs that are covalently bound to each other or non-covalently bound to each other via a nucleotide-based or non-nucleotide-based linker, such as the linker described in this disclosure.

[0161] Accordingly, in some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is a triple helix-forming oligonucleotide (TFO). In some embodiments, the multi-target molecule comprises at least two TFOs that are covalently linked to each other or non-covalently associated with each other via a nucleotide-based or non-nucleotide-based linker, such as the linkers described in this disclosure. Recent studies have shown that triple helix-forming oligonucleotides can be designed to sequence-specifically recognize and bind to polypurine / polypyrimidine regions in double-stranded DNA. These recognition rules are outlined in Maher III, L.J., et al., Science (1989) vol. 245, pp 725-730; Moser, H.E., et al., Science (1987) vol. 238, pp 645-630; Beal, P.A., et al., Science (1992) vol. 251, pp 1360-1363; Conney, M., et al., Science (1988) vol. 241, pp 456-459 and Hogan, M.E., et al., EP Publication 375408. Modifications of oligonucleotides, such as the introduction of intercalators and sugar backbone substitutions, and optimization of binding conditions (pH and cation concentration) help to 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 (see Seidman and Glazer, J Clin Invest 2003;l 12:487-94 for a recent review). Generally, triple helix-forming oligonucleotides have the following sequence correspondence. Oligo 3'-A G G T Duplex 5'-A G C T Duplex 3'-T C G A

[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, Sep 12, Epub). The same authors have demonstrated that TF0s designed according to the A-AT and G-GC rules do not form non-specific triplexes, indicating that triplex formation is indeed sequence-specific.

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

[0164] The formation of triple helix structures by the target DNA induces conformational and functional changes, inhibits the initiation and elongation of transcription, allows the introduction of desired sequence changes in the endogenous DNA, and results in specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include the knockout of 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 the sequence-specific and target-specific downregulation of the expression of the Ets2 transcription factor, which is important in the etiology of prostate cancer (Carbone, et al., Nucl Acid Res. 2003;31:833-43), and the inflammation-inducible 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 kinase (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0165] Furthermore, TFOs designed according to the above principles can induce site-directed mutagenesis capable of performing DNA repair and thus can provide both down-regulation and up-regulation of the expression of endogenous genes (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., and U.S. Patent Application Publication Nos. 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 by reference in their entirety.

[0166] Nucleic acid modification In some embodiments, the multi-target molecule comprises at least one nucleic acid modification described herein. For example, at least one modification selected from the group consisting of modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the multi-target molecule. For example, the modification may be present in an effector molecule or in a linker that links two effector molecules.

[0167] Nucleic acid modification (nucleobase) The naturally occurring base moieties of nucleosides are typically heterocyclic bases. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. In the case of nucleosides containing a pentofuranosyl sugar, a phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, these phosphate groups covalently bond adjacent nucleosides to each other to form a linear polymeric compound. Within an oligonucleotide, the phosphate groups generally refer to those that form the internucleoside backbone of the oligonucleotide. The naturally occurring linkages or backbones of RNA and DNA are 3'-5' phosphodiester linkages.

[0168] In addition to the "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimics well-known to those skilled in the art can be used in the compounds described herein. The unmodified or natural nucleobases can be modified or substituted to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using any one of these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nebularine, isoguanosine, or tubercidin) and the oligomer modifications described herein. Alternatively, substitution or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is substituted with a non-natural and / or universal base, herein this nucleotide is said to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include conjugate moieties, e.g., natural, non-natural and universal bases containing ligands described herein. Preferred conjugate moieties for conjugation to nucleobases contain a cationic amino group, which can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0169] The oligomeric compounds described herein can also include nucleobase (often simply referred to as "base" in the art) 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, nebularine, isoguanosine, 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, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(Acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(Methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)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, 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-(aminocarbonylethenyl)-pseudouracil, 1-(aminocarbonylethenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-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)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxo)-naphthalene, inosine, xanthine, hypoxanthine, nubraline, tubercidin, isoguanicin, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl) isocarbostyrylyl, 5-(methyl) isocarbostyrylyl, 3-(methyl)-7-(propynyl) isocarbostyrylyl, 7-(aza) indolyl, 6-(methyl)-7-(aza) indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyridinyl, isocarbostyrylyl, 7-(propynyl) isocarbostyrylyl, propynyl-7-(aza) indolyl, 2,4,5-(trimethyl) phenyl, 4-(methyl) indolyl, 4,6-(dimethyl) indolyl, phenyl, naphthalenyl, anthracenyl, phenanthrenyl, 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 pyrimidine, N, 2 -substituted purine, N6 - Replacement purine, O 6 - Replacement purine, replacement 1,2,4-triazole, 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-yl, 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 and other synthetic or natural nucleobases, or any O-alkylated or N-alkylated derivatives thereof. Alternatively, substituted or modified analogs of any of the bases listed above 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 melting behavior, recognition by intracellular enzymes, or the activity of an oligonucleoside double helix. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrylyl, 5-methylisocarbostyrylyl, 3-methyl-7-propynylisocarbostyrylyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyridinyl, isocarbostyrylyl, 7-propynylisocarbostyrylyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0171] Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in International Application PCT / US Patent Application No. 09 / 038425, filed Mar. 26, 2009; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858 - 85, Kroschwitz, J.I., 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 by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289 - 302, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993. The entire contents of these documents are hereby incorporated by reference into this specification.

[0172] In some embodiments, the modified nucleobase is a nucleobase that has a substantially similar structure to a parent nucleobase, such as, for example, 7 - deazapurine, 5 - methylcytosine, or a G - clamp. In some embodiments, the nucleobase analog includes a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase analog. Methods for preparing the modified nucleobases described above are well known to those of ordinary skill in the art.

[0173] Nucleic acid modification (sugar) The multi-target molecule provided in the present invention may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers containing a nucleoside or nucleotide having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, such as, but not limited to, addition of substituents, formation of locked nucleic acids or bicyclic nucleic acids by cross-linking of two non-geminal ring atoms. In some embodiments, the 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 locked nucleic acids, 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 -O-N(R1)-,-C(R1)=C(R2)-O-,-C(R1)=N-,-C(R1)=N-O-,-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)- and is linked to the 4'-position by a linker independently selected therefrom; wherein, 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, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, a substituted heteroaryl, a C5-C7 alicyclic radical, a substituted C5-C7 alicyclic 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, a heterocyclic radical, a substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.

[0175] In some embodiments, each of the linkers of the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]n-O-, -C(R1R2)-N(R1)-O- or -C(R1R2)-O-N(R1)-. In another embodiment, 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-O-N(R1)-2’ and 4’-CH2-N(R1)-O-2’, where each R1 is independently H, a protecting group or C1-C12 alkyl.

[0176] Some LNAs have been prepared and disclosed in patent literature as well as 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. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Patent 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] LNA is also provided herein 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. Patent Nos. 6,268,490 and 6,670,461). The linkage may 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 this bicyclic moiety; when 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 (Tm = +3 to +10 °C) with complementary DNA and RNA, stability against 3'-exonuclease degradation, and good solubility. Potent and non-toxic antisense oligonucleotides containing BNA are described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

[0178] Also taken up is the isomer of methyleneoxy(4’-CH2-O-2’)LNA, which is α-L-methyleneoxy(4’-CH2-O-2’)LNA, which has been shown to have excellent stability against 3’-exonuclease. α-L-methyleneoxy(4’-CH2-O-2’)LNA has been incorporated into antisense gapmers and chimeras, which showed 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 of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil have been described together with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation are also described in WO 98 / 39352 pamphlet and WO 99 / 14226 pamphlet.

[0180] Analogues 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 analogues containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (WO 99 / 14226 pamphlet by Wengel et al.). Furthermore, the synthesis of 2’-amino-LNA, a novel conformationally locked high-affinity oligonucleotide analogue, has also been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2’-amino- and 2’-methylamino-LNA have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has already been reported.

[0181] Modified sugar moieties are well known and can be used to modify, typically increase, the affinity of an antisense compound for its target and / or to increase nuclease resistance. Representative lists of preferred modified sugars include, but are not limited to, bicyclic modified sugars including methyleneoxy (4’-CH2-O-2’) LNA and ethyleneoxy (4’-(CH2)2-O-2’ bridging) 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. Further, the sugar can be substituted, inter alia, with sugar mimics. Methods for preparing modified sugars are well known to those of skill in the art. Some representative patents and publications teaching 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; 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 International Publication No. 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(CH2CH2O) nCH2CH2OR, where n = 1 - 50; a "locked" nucleic acid (LNA) in which the furanose moiety 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] As the "deoxy" modification, hydrogen (i.e., deoxyribose sugar particularly related to the protruding single strand); halo (e.g., fluoro); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) n 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 optionally be substituted, for example, with an amino functional group.

[0184] Other suitable 2'-modifications, such as modified MOE, are described in U.S. Patent Application Publication No. 20130130378, the content of which is incorporated herein by reference.

[0185] The modification at the 2'-position can exist in the arabinose configuration. The term "arabinosyl configuration" refers to the arrangement of the substituent on C2' of ribose having the same configuration as the 2'-OH in arabinose.

[0186] The sugar can contain two different modifications on the same carbon of the sugar, for example, a gem modification. The sugar group can also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, the oligomeric compound can include, as a sugar, one or more monomers containing, for example, arabinose. The monomer may have an α-bond, for example, an α-nucleoside, at the 1'-position of the sugar. The monomer may also have an opposite configuration at the 4'-position, for example, the C5' and H4' or the substituents replacing them are exchanged. When the C5' and H4' or the substituents replacing them are exchanged, this sugar is said to be modified at the 4'-position.

[0187] The multi-target molecules disclosed herein can also include abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group in place of the nucleobase at C1'. See, for example, U.S. Patent No. 5,998,203, the content of which is incorporated herein by reference in its entirety. These abasic sugars can also further include modifications on one or more constituent sugar atoms. The multi-target molecule can contain one or more sugars that are L-isomers, for example, L-nucleosides. Modifications to the sugar group can also include substitution of 4'-O with sulfur, optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0188] The sugar modification can also include acyclic nucleotides in which there is no C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4' or O4') is not present in the nucleotide, either independently or in combination. In some embodiments, the acyclic nucleotide is

Chemical formula

[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 having 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 modification described herein can be placed at the 3'-position of the sugar for that particular nucleotide, e.g., the nucleotide to which it is linked through its 2'-position. The modification at the 3'-position can be present in the xylose configuration. The term "xylose configuration" refers to the placement of a substituent on C3' of ribose in the same configuration as the 3'-OH of xylose sugar.

[0191] The hydrogen atom attached to C4’ and / or C1’ can be replaced by a linear or branched, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, and the skeletons 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’), phosphorus-containing bonds, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, where R’ is hydrogen, acyl, or optionally substituted aliphatic, and Z’ is OR 11 、COR 11 、CO2R 11 、

Chemical Structure

[0192] In some embodiments, C4’ and C5’ together form an optionally substituted heterocyclic, preferably containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, where M is, for each occurrence, independently, an alkyl metal or transition metal with a total charge of +1; Y is O, S, or NR’, where R’ is hydrogen, optionally substituted aliphatic. This modification is preferably at the 5’-end of the oligonucleotide.

[0193] In some embodiments, the LNA comprises a bicyclic nucleoside having the formula: [Chemical formula] wherein: In the formula: Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C1-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 amide.

[0194] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with an optionally protected substituent 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 independently mono- or polysubstituted with a substituent 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 C1-C6 alkyl substituted with one or more Xx, 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 another embodiment, the Z group is C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-), substituted alkoxy or azide.

[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 another embodiment, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-) or azide.

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

Chemical formula

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

Chemical formula

[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, and a more preferred hydroxyl protecting group is T1 which is 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-target molecule has the formula:

Chemical formula

Chemical formula

Chemical formula

[0203] In some embodiments, each of the substituted groups is independently an optionally protected substituent 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), and is mono- or polysubstituted.

[0204] In some embodiments, each of the substituted groups is independently a substituent 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), and is mono- or polysubstituted.

[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 (for example, at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxys). In another embodiment, each substituent is independently C1-C6 alkoxy (for example, each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxys).

[0207] In some embodiments, at least one C1-C6 alkoxy substituent is CH3O- (for example, at least one Z is CH3OCH2-). In another embodiment, each C1-C6 alkoxy substituent is CH3O- (for example, each Z is CH3OCH2-).

[0208] In some embodiments, at least one substituent is a halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogens). In some embodiments, each substituent is independently a 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 a hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyls). In some embodiments, each substituent is independently a hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyls). In some embodiments, at least one Z is HOCH2-. In another embodiment, each Z is HOCH2-.

[0210] In some embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F- or HOCH2-. In some embodiments, each Z is independently CH3-, CH3CH2-, CH2OCH3-, CH2F- or HOCH2-.

[0211] In some embodiments, at least one Z group is C1-C6 alkyl substituted with one or more Xx, 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 another embodiment, at least one Z group is C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-) or azide.

[0212] In some embodiments, each Z group is independently C1-C6 alkyl substituted with one or more Xx, 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 another embodiment, each Z group is independently C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-) or azide.

[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., CH3O-) or azide.

[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 another embodiment, each Z group is independently -CH2Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O-) or azide.

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

[0216] In some embodiments, the Z group of at least one monomer has the formula:

Chemical formula

Chemical formula

Chemical formula

[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:

Chemical formula

Chemical formula

Chemical formula

[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 another embodiment, T3 is a nucleoside, nucleotide or internucleoside linking group attached to a monomer subunit. In some embodiments, T4 is a nucleoside, nucleotide or internucleoside linking group attached to a 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 oligomer compound. In some embodiments, T4 is an internucleoside linking group attached to an oligomer compound. In some embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from a phosphodiester or phosphorothioate.

[0221] In some embodiments, the multi-target molecule has the formula:

Chemical formula

Chemical formula

Chemical formula

[0222] In some such embodiments, as LNA, but not limited to,

Chemical formula

[0223] In some embodiments, the multi-target molecule comprises at least two regions of at least two consecutive monomers of the above formula. In some embodiments, the multi-target molecule comprises a gap motif. In some embodiments, the multi-target molecule comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In some embodiments, the multi-target molecule comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0224] In some embodiments, the multi-target molecule has the formula:

Chemical formula

[0225] In some embodiments, the monomer comprises a sugar mimic. In some such embodiments, the mimic is used in place of the sugar or sugar-nucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. Representative examples of sugar mimics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimics of sugar-nucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) linked by uncharged achiral bonds and morpholino groups. In some cases, a mimic is used in place of the nucleobase. Representative nucleobase mimics 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 mimics are well known to those of skill in the art.

[0226] Nucleic acid modification (inter-sugar linkage) This specification describes linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) to each other, thereby forming oligomeric compounds, such as oligonucleotides. Such linking groups are also referred to as internucleoside 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, phosphate diesters (P=O), phosphate triesters, methyl phosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-O-C(O)-S-), thiocarbamates (-O-C(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to the natural phosphate diester linkage, modified linkages can be used to modify, typically increase, the nuclease resistance of oligonucleotides. In some embodiments, linkages having chiral atoms can be prepared as individual enantiomers or as a racemic mixture. 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 of skill in the art.

[0227] The phosphate group in the linking group can be modified by substituting one of the oxygens with a different substituent. One result of this modification can be an increase in the resistance of the oligonucleotide to nuclease degradation. Examples of modified phosphate groups include phosphorothioates, phosphorosenates, 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 substituted with any of S, Se, BR3 (where R is hydrogen, alkyl, aryl), C (i.e., an alkyl group, aryl group, etc.), H, NR2 (where R is hydrogen, optionally substituted alkyl, aryl), or OR (where R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, substitution of one of the non-bridging oxygens by one of the above atoms or atomic groups renders the phosphorus atom chiral. In other words, the phosphorus atom in the phosphate group thus modified is a stereocenter. The stereogenic phosphorus atom can have either an "R" configuration (Rp herein) or an "S" configuration (Sp herein).

[0228] Phosphorodithioates have both non-bridging oxygens substituted by sulfur. The phosphorus center in phosphorodithioates is achiral, precluding the formation of oligonucleotide diastereomers. Thus, while not intending to be bound by any particular theory, modification of both non-bridging oxygens may be desirable as it precludes chiral centers, such as phosphorodithioate formation, from being able to generate a mixture of diastereomers. Accordingly, the non-bridging oxygen can independently be any of O, S, Se, B, C, H, N, or OR (where R is alkyl or aryl).

[0229] The phosphate linker can also be modified by substitution of the bridging oxygen (i.e., the oxygen that links the phosphate to the monomeric sugar) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate). This substitution can be carried out on either or both of the 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] A modified phosphate bond in which at least one of the oxygens linked to the phosphate is substituted or the phosphate group is substituted by a non-phosphorus group is also referred to as a "non-phosphate diester sugar-sugar bond" or a "non-phosphate diester linker".

[0231] In some embodiments, the phosphate group can be substituted by a non-phosphorus-containing connector, such as a dephospho linker. A dephospho linker is also referred to herein as a non-phosphate diester linker. Without intending to be bound by any particular theory, it is believed that substitution of the charged phosphate diester group, which is the reaction center for nucleic acid degradation, with a neutral structural mimic confers enhanced nuclease stability. Also without intending to be bound by any particular theory, in some embodiments, it may be desirable to introduce a modification that substitutes the charged phosphate group with a neutral moiety.

[0232] Examples of moieties that can replace the phosphate group include, but are not limited to, the following: 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, sulfonic acid ester, thioformacetal (3’-S-CH2-O-5’), formacetal (3’-O-CH2-O-5’), oxime, methyleneimino, 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’-O-P(O)-O-SS-C5’, C3’-CH2-NH-NH-C5’, 3’-NHP(O)(OCH3)-O-5’ and 3’-NHP(O)(OCH3)-O-5’ and nonionic linkages containing mixed N, O, S and CH2 moieties. See, for example, Carbohydrate Modifications in Antisense Research; Y.S.Sanghvi and P.D.Cook Eds.ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate and ethylene oxide linker.

[0233] One of ordinary skill in the art will fully recognize that in many cases, modification of the unbridged oxygen may require modification of the 2’-OH, e.g., modification that does not participate in cleavage of adjacent sugar linkages, e.g., arabinose sugars, 2’-O-alkyl, 2’-F, LNA and ENA, since in some cases replacement of the unbridged oxygen can result in enhanced cleavage of the sugar linkage by the adjacent 2’-OH.

[0234] Preferred non-phosphate diester sugar 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, aminoalkyl phosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N-alkyl phosphoramidates), and boranophosphonates.

[0235] In some embodiments, the multi-target molecule comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and including each numerical value and less than it) modification or non-phosphate diester bond. In some embodiments, the multi-target molecule comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and including each numerical value and less than it) phosphorothioate bond.

[0236] It is also possible to construct multi-target molecules in which the phosphate linker and sugar are replaced by nuclease-resistant nucleosides or nucleotide surrogates. Without intending to be bound by a particular theory, the absence of a repeatedly charged backbone is thought to weaken binding to proteins (e.g., nucleases) that recognize polyanions. Also without intending to be bound by a particular theory, in some embodiments, it may be desirable to introduce modifications in which the base is 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 the PNA surrogate.

[0237] The multi-target molecules described herein contain one or more asymmetric centers and thus can generate enantiomers, diastereomers, and other configurations, which can be defined as (R) or (S) in the case of sugar anomers, for example, or as (D) or (L) in the case of amino acids, with respect to absolute stereochemistry. The multi-target molecules provided herein include all such possible isomers, as well as their racemic and optionally pure forms.

[0238] Nucleic acid modification (terminal modification) The terminals of the multi-target molecule or effector molecule contained in the multi-target molecule can be modified. Such modifications are possible at one or both terminals. For example, the 3’ and / or 5’ terminals of an oligonucleotide can be conjugated with a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dye), or other functional molecular 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 bonding atom of the phosphate group, or the C-3’, 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 disposed between the two strands of a double-stranded oligomeric compound, this array can replace the hairpin loop in a hairpin-type oligomeric compound.

[0240] Useful terminal modifications for modulating activity include modification of the 5' end of the oligonucleotide with a phosphate or phosphate analog. In some embodiments, the 5' end of the oligonucleotide is phosphorylated or contains a phosphoryl analog. Exemplary 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Modification of the 5' end can also be useful in stimulating or inhibiting the immune system of a subject. In some embodiments, the 5' end of the oligomeric compound is modified

Chemical formula

[0241] Exemplary 5'-modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); 5'-α-thiotriphosphate; 5'-β-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'). Other 5'-modifications include 5'-alkylphosphonates (R(OH)(O)P-O-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)P-O-5', R = alkyl ether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those where Z is optionally substituted alkyl at least once, e.g., ((HO)2(X)P-O[-(CH2) a -O-P(X)(OH)-O] b -5’, ((HO)2(X)P-O[-(CH2) a -P(X)(OH)-O] b -5’, ((HO)2(X)P-[-(CH2) a -O-P(X)(OH)-O] b -5’; dialkyl terminal phosphates and phosphate mimics: HO[-(CH2) a -O-P(X)(OH)-O] b -5’, H2N[-(CH2) a -O-P(X)(OH)-O] b -5’, H[-(CH2) a -O-P(X)(OH)-O] b -5’, Me2N[-(CH2) a -O-P(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’ is exemplified, where a and b are each independently 1 to 10. Other embodiments include substitution of oxygen and / or sulfur by BH3, BH3 - and / or Se.

[0242] Terminal modifications can also be useful in monitoring distribution, in which case preferred groups to add include fluorophores, such as fluorescein or Alexa dyes, such as Alexa 488. Terminal modifications can also be useful in enhancing uptake, and useful modifications for this purpose include targeting ligands. Terminal modifications can also be useful in cross-linking an oligonucleotide to another moiety; useful modifications for this purpose include mitomycin C, psoralen, and their derivatives.

[0243] Thermal destabilizing modifications By introducing thermally destabilizing nucleotides into the sense strand at a site opposite the seed region of the antisense strand (i.e., positions 2 to 8 at the 5’ end of the antisense strand), the tendency of the dsRNA double helix to dissociate or melt (reducing the free energy of double helix association) can be enhanced, thereby optimizing effector molecules such as siRNA or dsRNA agents for RNA interference. This modification can enhance the tendency of the double helix to dissociate or melt within the seed region of the antisense strand.

[0244] Thermal destabilizing modifications can include abasic modifications; mismatches with opposite nucleotides in the opposite strand; and sugar modifications such as 2’-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA).

[0245] The exemplified abasic modifications are as follows. [Chemical formula]

[0246] The exemplified sugar modifications are as follows. [Chemical formula]

[0247] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, wherein any of the bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4') is independently or in combination deleted from the nucleotide. In some embodiments, the acyclic nucleotide is [Chemical formula] wherein B is a modified or unmodified nucleobase, R 1 and R 2is, independently, H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term "UNA" refers to unlocked nucleic acids, in which any of the sugar linkages are removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) is removed (see Mikhailov et.al., Tetrahedron Letters, 26(17):2059(1985); and Fluiter et al., Mol.Biosyst., 10:1039(2009); these are incorporated herein by reference in their entirety). The acyclic derivatives confer 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 glycol nucleic acids, polymers that are similar to DNA or RNA but differ in the composition of their "backbones" in that they are composed of repeating glycerol units linked by: [Chemical formula] the indicated linkages.

[0249] Thermal destabilizing modifications may be mismatches (i.e., non-complementary base pairs) between thermally destabilizing nucleotides within the dsRNA double helix and opposite nucleotides on the opposite strand. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatched base pairs known in the art are also suitable for the present invention. Mismatches can occur between any nucleotides, whether naturally occurring nucleotides or modified nucleotides. That is, mismatched base pairing can occur between nucleobases from each nucleotide, regardless of modifications to the ribose sugar of the nucleotide. In some embodiments, effector molecules such as siRNA or dsRNA agents contain at least one nucleobase in a mismatched pairing, which is a 2'-deoxy nucleobase; for example, the 2'-deoxy nucleobase is present in the sense strand.

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

[0251] Thermal destabilizing modifications can also include universal bases with reduced or abrogated ability to form hydrogen bonds with opposite bases, and even phosphate modifications.

[0252] Nucleobase modifications with impaired or completely abrogated ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of the dsRNA double helix, as described in WO 2010 / 0011895 pamphlet, which is hereby incorporated by reference in its entirety. Exemplary nucleobase modifications are

Chemical formula

[0253] Exemplary phosphate modifications that have been found to reduce the thermal stability of the dsRNA double helix compared to natural phosphodiester linkages are [Chemical formula] .

[0254] In some embodiments, the effector molecule in the multi-target molecule comprises 2'-5' linkages (with 2'-H, 2'-OH, and 2'-OMe, and with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance, or inhibit the binding of the sense strand and the antisense strand, or the above modifications can be used at the 5' end of the sense strand to avoid sense strand activation by RISC.

[0255] In another embodiment, the effector molecule in the multi-target molecule comprises L sugars (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 the binding between the sense strand and the antisense strand, or the above modifications can be used at the 5' end of the sense strand to avoid sense strand activation by RISC.

[0256] In one embodiment, the dsRNA agent of the present invention is conjugated to a ligand via a carrier, and the carrier may be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0257] In some embodiments, at least one strand of at least one effector molecule in the multi-target molecules disclosed herein is 5'-phosphorylated or contains a phosphoryl mimic at the 5'-prime end. The 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include the following: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (methylated or non-methylated at the 7-position) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-dithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); oxygen / sulfur-substituted monophosphate, diphosphate, and triphosphate (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoroamidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-), 5'-alkylvinylphosphonate (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-), any combination thereof.

[0258] target gene Without limitation, as the target gene of the siRNA, although not limited, genes that promote unnecessary cell proliferation, growth factor genes, growth factor receptor genes, gene expression kinases, adapter protein genes, genes encoding G protein superfamily molecules, genes encoding transcription factors, genes mediating angiogenesis, viral genes, genes necessary for viral replication, cellular genes mediating viral functions, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes mediating unnecessary immune responses, genes mediating pain processing, genes mediating neurological diseases, allene genes found in cells characterized by loss of heterozygosity, or one allege gene of polymorphic genes may be mentioned.

[0259] Specific exemplary target genes of 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; BCL-2 gene; Cyclin D gene; VEGF gene; EGFR gene; Cyclin A gene; Cyclin E gene; WNT-1 gene; beta-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; Topoisomerase I gene; Topoisomerase II alpha gene; p73 gene; p21 (WAF1 / CIP1) gene, p27 (KIP1) gene; PPM1D gene; Caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor genes; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, such as MLL-AF9, BCR / ABL fusion gene; TEL / AML1 fusion gene; EWS / FLI1 fusion gene; TLS / FUS1 fusion gene; PAX3 / FKHR fusion gene; AML1 / ETO fusion gene; alpha v-integrin gene; Flt-1 receptor gene; Tubulin gene; Human Papilloma Virus gene, genes required for Human Papilloma Virus replication, Human Immunodeficiency Virus gene, genes required for Human Immunodeficiency Virus replication, Hepatitis A Virus gene, genes required for Hepatitis A Virus replication, Hepatitis B Virus gene, Hepatitis BGenes necessary for the replication of (Virus), Hepatitis C Virus genes, genes necessary for the replication of Hepatitis C Virus, Hepatitis D Virus genes, genes necessary for the replication of Hepatitis D Virus, Hepatitis E Virus genes, genes necessary for the replication of Hepatitis E Virus, Hepatitis F Virus genes, genes necessary for the replication of Hepatitis F Virus, Hepatitis G Virus genes, genes necessary for the replication of Hepatitis G Virus, Hepatitis H Virus genes, genes necessary for the replication of Hepatitis H Virus, Respiratory Syncytial Virus genes, genes necessary for the replication of Respiratory Syncytial Virus, Herpes Simplex Virus genes, genes necessary for the replication of Herpes Simplex Virus, herpes Cytomegalovirus genes, genes necessary for the replication of herpes Cytomegalovirus, Epstein Barr Virus genes, genes necessary for the replication of Epstein Barr Virus, Kaposi’s Sarcoma-associated Herpes Virus genes, genes necessary for the replication of Kaposi’s Sarcoma-associated Herpes Virus, JC Virus genes, JCHuman genes required for the replication of (Virus), mumps virus genes, genes required for the replication of mumps virus genes, rhinovirus genes, genes required for the replication of rhinovirus, coronavirus genes, genes required for the replication of coronavirus, West Nile Virus genes, genes required for the replication of West Nile Virus, St. Louis Encephalitis genes, genes required for the replication of St. Louis Encephalitis, Tick-borne encephalitis virus genes, genes required for the replication of Tick-borne encephalitis virus, Murray Valley encephalitis virus genes, genes required for the replication of Murray Valley encephalitis virus, dengue virus genes, genes required for the replication of dengue virus genes, Simian Virus 40 genes, genes required for the replication of Simian Virus 40, Human T Cell Lymphotropic Virus genes, genes required for the replication of Human T Cell Lymphotropic Virus, Moloney-Murine Leukemia Virus genes, genes required for the replication of Moloney-Murine Leukemia Virus, encephalomyocarditis virus genes, genes required for the replication of encephalomyocarditis virus, measles virus genes, genes required for the replication of measles virus, Varicella zoster virus genes, Varicella zosterGenes necessary for the replication of virus), adenovirus genes, genes necessary for adenovirus replication, yellow fever virus genes, genes necessary for yellow fever virus replication, poliovirus genes, genes necessary for poliovirus replication, poxvirus genes, genes necessary for poxvirus replication, plasmodium genes, genes necessary for plasmodium gene replication, Mycobacterium ulcerans genes, genes necessary for Mycobacterium ulcerans replication, Mycobacterium tuberculosis genes, genes necessary for Mycobacterium tuberculosis genes, Mycobacterium leprae genes, genes necessary for Mycobacterium leprae replication, Staphylococcus aureus genes, genes necessary for Staphylococcus aureus replication, Streptococcus pneumoniae genes, genes necessary for Streptococcus pneumoniae replication, Streptococcus pyogenes genes, genes necessary for Streptococcus pyogenes replication, Chlamydia pneumoniae genes, genes necessary for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, Mycoplasma pneumoniaeGenes necessary for the replication of pneumoniae), integrin genes, selectin genes, complement system genes, chemokine genes, chemokine receptor genes, GCSF genes, Gro1 genes, Gro2 genes, Gro3 genes, PF4 genes, MIG genes, preplatelet basic protein genes, MIP-1I genes, MIP-1J genes, RANTES genes, MCP-1 genes, MCP-2 genes, MCP-3 genes, CMBKR1 genes, CMBKR2 genes, CMBKR3 genes, CMBKR5v, AIF-1 genes, I-309 genes, genes of the components of ion channels, genes of neurotransmitter receptors, genes of 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 polymorphic genes, and combinations thereof.

[0260] Loss of heterozygosity (LOH) can cause hemizygosity of sequences, such as genes, in the region of LOH. This can result in significant genetic differences between normal cells and cells in a diseased state, such as cancer cells, which provides a useful difference between normal cells and cells in a diseased state, such as cancer cells. This difference can occur because a gene or other sequence is heterozygous in diploid cells but hemizygous in cells with LOH. The region of LOH often contains genes that promote unwanted proliferation by loss, such as tumor suppressor genes, and other sequences, such as other genes, which in some cases are genes essential for normal functions, such as growth. The method of the present invention utilizes, in part, the specific regulation of one allele of an essential gene by the composition of the present invention.

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

[0262] ligand In some embodiments, the multi-target molecule is modified by covalent attachment of one or more conjugate groups. Generally, the conjugate groups modify one or more properties of the attached multi-target molecule, such properties 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 linked to a parent compound, such as an oligomeric compound, either directly or via an optional linking moiety or linker group. A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholate moieties, folates, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, and dyes.

[0263] Preferred conjugate groups suitable for the present invention include the following: lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains such as dodecadiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-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); palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0264] In general, a very diverse array of entities, such as 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, the following: 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, polyphosphazene, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalators (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin 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) cholenic acid, dimethoxytrityl, or phenoxazine), peptide (e.g., α-helical peptide, amphiphilic peptide, RGD peptide, cell-penetrating peptide, endosomal-lytic / fusogenic peptide), alkylating agent, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption promoter (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraazamacrocycle), dinitrophenyl, HRP, AP, antibody, hormone and hormone receptor, lectin, carbohydrate, polyvalent carbohydrate, vitamin (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactor, lipopolysaccharide, activator of p38 MAP kinase, activator of NF-κB, taxol, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor α (TNFα), interleukin-1β, γ interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high density lipoprotein (HDL), and cell permeating agent (e.g., helical cell permeating agent).

[0265] Peptides and peptidomimetic ligands include natural or modified peptides, such as D- or L-peptides; α-, β-, or γ-peptides; N-methyl peptides; azapeptides; peptides having one or more amides, i.e., bonds substituted with one or more urea, thiourea, carbamate, or sulfonylurea bonds; or those having cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into a defined three-dimensional structure similar to that of natural peptides. The peptide or peptidomimetic ligand may be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0266] Exemplary amphiphilic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, bovicin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainine, 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 having endosomolytic properties. An endosomolytic ligand promotes the dissolution of the compositions or components of the present invention and / or the transport of the compositions or components of the present invention from intracellular compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular vesicles into the cytoplasm of the cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazole, poly- or oligoimidazole, linear or branched polyethyleneimine (PEI), linear and branched polyamines such as spermine, cationic linear or branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers having masked or unmasked cationic or anionic charges, dendrimers having masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0268] Exemplary endosome-lytic / fusogenic peptides include, but are not limited to, the following: AALEALAEALEALAEALEALAEAAAAGGC(GALA) (SEQ ID NO: 1); AALAEALAEALAEALAEALAEALAAAAGGC(EALA) (SEQ ID NO: 2); ALEALAEALEALAEA (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, where 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] While not intending to be bound by any particular theory, fusogenic lipids fuse with membranes and, as a result, destabilize them. Fusogenic lipids typically have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylcholine (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 having endosomal lysis 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 hereby incorporated by reference in their entirety.

[0271] Exemplary cell-permeable 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 (amphiphilic model peptide) (SEQ ID NO: 24); RRRRRRRRR (Arg9) (SEQ ID NO: 25); KFFKFFKFFK (bacterial cell wall permeable peptide) (SEQ ID NO: 26); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (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, for example, O-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, for example, O(CH2) nAMINE, for example, AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH2CH2NH) n Protonated amino groups obtained from CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino) are included.

[0273] As used herein, the term "targeting ligand" refers to any molecule that confers increased affinity for a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a cellular, tissue or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, 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, polyvalent galactose, N-acetyl-D-galactose (GalNAc), polyvalent GalNAc, such as GalNAc2 and GalNAc3; D-mannose, polyvalent mannose, polyvalent lactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent fucose, glycosylated polyamino acids and lectins. The term polyvalent indicates the presence of two or more monosaccharide units. Such monosaccharide subunits can be linked to each other via glycosidic bonds or to a scaffold molecule.

[0275] As ligands, several folates and folate analogs suitable for the present invention are described in U.S. Patent Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the contents of which are hereby incorporated by reference in their entirety.

[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 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 trans-thyretin-binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligomeric compounds containing some phosphorothioate sugar linkages are also known to bind to serum proteins and thus short oligomeric compounds, e.g., oligonucleotides containing from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and containing multiple phosphorothioate linkages in the backbone are also suitable for the invention as ligands (e.g., PK modulating ligands). 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 nucleotide linkages in the PK modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Additionally, aptamers that bind to serum components (e.g., serum proteins) are also suitable for the invention as PK modulating ligands. Binding to serum components (e.g., 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 two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties and the rest may have different properties. For example, the ligand can have targeting properties, endosomal lysis activity, or PK modulating properties. In a preferred embodiment, the ligands all have different properties.

[0278] The ligand or the tethered ligand may be present on the monomer when the monomer is incorporated into a component of the multi-target molecule (e.g., an effector molecule or a linker). In some embodiments, the ligand can be incorporated into a "precursor" monomer by coupling after the "precursor" monomer is incorporated into a component of the multi-target molecule (e.g., an effector molecule or a linker). For example, a monomer having an amino-terminal tether (i.e., no ligand is attached), e.g., monomer-linker-NH2, can be incorporated into a component of the multi-target molecule (e.g., an effector molecule or a linker). In a subsequent step, i.e., after incorporation of the precursor monomer into the component of the multi-target molecule (e.g., an effector molecule or a linker), a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or an aldehyde group, can be attached to the precursor monomer by coupling of the electrophilic group of the ligand and the terminal nucleophilic group of the tether of the precursor monomer.

[0279] In another example, a monomer having a chemical group suitable for participating in a click chemistry reaction, e.g., an azide or alkyne terminal tether / linker, can be incorporated. In a subsequent step, i.e., after incorporation of the precursor monomer into the chain, a ligand having a complementary chemical group, e.g., 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 linkage of the multi-target molecule. Conjugation to a purine nucleobase or its derivative can be carried out at any position including intra-ring and exo-ring atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can also be carried out at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be substituted with the conjugate moiety. When conjugating the ligand to the nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions necessary for base pairing.

[0281] Conjugation to the sugar moiety of the nucleoside can be carried out at any carbon atom. Exemplary carbon atoms of the sugar moiety to which the conjugate moiety can be attached are the 2’, 3’, and 5’ carbon atoms. Also, the 1’ position can be attached to a conjugate moiety, such as an abasic residue. The internucleoside linkage can also carry the conjugate moiety. In the case of a phosphorus-containing linkage (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 an amine- or amide-containing internucleoside linkage (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0282] There are many methods for preparing conjugates of oligonucleotides. Generally, the oligonucleotide is attached to the conjugate moiety by contacting a reactive group of the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) with a reactive group of the conjugate moiety. In some embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0283] For example, the electron-withdrawing group may be a carbonyl-containing functional group, and the nucleophilic group may be an amine or a thiol. Methods for conjugating nucleic acids and related oligomeric compounds with or without a linking group are well described, for example, in Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17 (which is hereby incorporated by reference in its entirety).

[0284] The ligand can bind to a multi-target molecule via a carrier monomer, for example, a ligand carrier. The carrier includes (i) at least one "backbone binding site", preferably two "backbone binding sites", and (ii) at least one "tethering binding point". As used herein, a "backbone binding site" refers to a functional group, such as a hydroxyl group, or generally, a backbone, such as a phosphate of an oligonucleotide, or a modified phosphate (e.g., sulfur-containing) backbone, and is available for the incorporation of a carrier monomer and refers to a suitable bond. A "tethering binding point" (TAP) refers to an atom of the carrier monomer, such as a carbon atom or a heteroatom (different from the atom conferring the backbone binding site), which links the selected moiety. The selected moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is linked to the carrier monomer by an intervening tether. Thus, the carrier often contains a functional group, such as an amino group, or generally provides a suitable bond for the incorporation or tethering of a ligand to another chemical entity, such as a constituent atom.

[0285] Representative U.S. patents teaching the preparation of nucleic acid conjugates include, but are not limited to, U.S. Patent No. 4,828,979; No. 4,948,882; No. 5,218,105; No. 5,525,465; No. 5,541,313; No. 5,545,730; No. 5,552,538; No. 5,578,717; No. 5,580,731; No. 5,580,731; No. 5,591,584; No. 5,109,124; No. 5,118,802; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,149,782; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928; No. 5,672,662;U.S. Patent 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; 6,559,279 are hereby incorporated by reference in their entirety.;

[0286] In some embodiments, the multi-target molecule comprises a ligand having the structure shown in

Chemical Formula

[0287] In some embodiments, the multi-target molecule is of formula (II), (III), (IV) or (V):

Chemical Formula

Chemical formula

[0288] In some embodiments, the multi-target molecule has the structure: [Chemical formula] and includes a ligand of

[0289] In some embodiments, the multi-target molecule has the structure: [Chemical formula] and includes a ligand of

[0290] In some embodiments, the multi-target molecule has the structure: [Chemical formula] and includes a ligand of

[0291] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0292] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0293] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0294] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0295] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0296] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0297] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the ligand of

[0298] In some embodiments, the multi-target molecule has the structure: [Chemical formula] comprises a ligand of

[0299] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0300] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0301] Exemplary ligand monomer In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0302] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0303] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0304] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0305] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0306] In some embodiments, the multi-target molecule has the structure:

Chem.

[0307] In some embodiments, the multi-target molecule has the structure:

Chem.

[0308] In some embodiments, the multi-target molecule has the structure:

Chem.

[0309] In some embodiments, the multi-target molecule has the structure:

Chem.

[0310] In some embodiments, the multi-target molecule has the structure:

Chem.

[0311] In some embodiments, the multi-target molecule has the structure:

Chem.

[0312] In some embodiments, the multi-target molecule has the structure:

Chem.

[0313] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0314] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0315] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0316] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0317] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0318] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0319] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0320] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the monomers of

[0321] In some embodiments, the multi-target molecule has the structure: [Chemical formula] contains the monomers of

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

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

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

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

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

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

[0332] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0333] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0334] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0335] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0336] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0337] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0338] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0339] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0340] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0341] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0342] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0343] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0344] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0345] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0346] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0347] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0348] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0349] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0350] In the aforementioned monomers, X and Y are each independently, for their respective existences, H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, -P(Z’)(Z’’)O-nucleoside, -P(Z’)(Z’’)O-oligonucleotide, a lipid, PEG, a steroid, a polymer, a nucleotide, a nucleoside, or an oligonucleotide; Z’ and Z’’ are each independently, for their respective existences, O or S.

[0351] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0352] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0353] In some embodiments, the multi-target molecule has the structure:

Chemical formula

[0354] The synthesis of the aforementioned ligands and monomers is described, for example, in U.S. Patent No. 8,106,022, the content of which is incorporated herein by reference in its 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 the attachment of chemical functional groups, conjugate groups, reporter groups, and other groups to selected sites in a parent compound such as an oligomeric compound. Generally, a bifunctional linking moiety includes a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or a compound of interest, and the other is selected to bind essentially to an arbitrarily selected group such as a chemical functional group or a conjugate group. In some embodiments, the linker includes a chain structure or oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups commonly used for 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, the bifunctional linking moiety includes 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, and a non-limiting list of preferred substituents includes 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-target molecule via a linker.

[0357] As used herein, the term "linker" means an organic moiety that connects two parts of a compound. A linker typically is a direct bond or an atom such as oxygen or sulfur, NR 1, units such as C(O), C(O)NH, SO, SO2, SO2NH, or 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, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, etc., including atomic chains, where one or more methylenes are O, S, S(O), SO2, N(R 1 )2, C(O), a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, which may be interrupted or terminated; where R 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 instance of their presence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, CH2O; NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CH=N-O,

Chemical formula

[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 the branched linker may be at least trivalent, but may also be a tetravalent, pentavalent or hexavalent atom, or a group representing such polyvalence. In some embodiments, the branch point is -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)O-C; wherein Q is, for each occurrence independently, H or optionally substituted alkyl. In some embodiments, the branch point is glycerol or a glycerol derivative.

[0361] The cleavable linking group is sufficiently stable outside the cell, but upon entry into the target cell, it is cleaved to release the two moieties that the linker holds together. In preferred embodiments, the cleavable linking group is cleaved at least 10-fold, preferably at least 100-fold faster inside the target cell or under first reference conditions (e.g., conditions that can be selected to mimic or represent intracellular conditions) than in the subject's blood or serum, or under second reference conditions (e.g., conditions that can be selected to mimic or represent conditions found in blood or serum).

[0362] The cleavable linking group is sensitive to a cleaving agent, such as pH, redox potential, or the presence of a degrading molecule. Generally, the cleaving agent is more pervasive, or found at a higher level or activity, inside the cell than in serum or blood. Examples of such degrading agents include redox agents selected for a particular substrate or redox agents without substrate specificity, such as reducing agents present in the cell, such as mercaptans, that can oxidatively or reductively cleave a cleavable linking group by reduction; esterases; amidases; agents that can create an endosomal or acidic environment, such as those that result in a pH of 5 or less; general acids, peptidases (which may be substrate specific) and proteases, and enzymes that can hydrolyze or degrade an acid-cleavable linking group by acting as phosphatases.

[0363] The linker can include a cleavable linking group that is cleavable 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 contains an ester group. Hepatocytes are rich in esterases, and thus the linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0364] When targeting peptidase-rich cell types such as hepatocytes and synoviocytes, a linker containing a peptide bond 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 intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). In some embodiments, the cleavable linking group is cleaved less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in blood (or under in vitro conditions selected to mimic extracellular conditions) compared to intracellularly (or under in vitro conditions selected to mimic intracellular conditions).

[0366] Exemplary cleavable linking groups include, but are not limited to, the following: redox-cleavable linking groups (e.g., -S-S- and -C(R)2-S-S-, where R is H or C1-C6 alkyl and at least one R is C1-C6 alkyl, such as CH3 or CH2CH3); phosphate-based cleavable linking groups (e.g., -O-P(O)(OR)-O-, -O-P(S)(OR)-O-, -O-P(S)(SR)-O-, -S-P(O)(OR)-O-, -O-P(O)(OR)-S-, -S-P(O)(OR)-S-, -O-P(S)(ORk)-S-, -S-P(S)(OR)-O-, -O-P(O)(R)-O-, -O-P(S)(R)-O-, -S-P(O)(R)-O-, -S-P(S)(R)-O-, -S-P(O)(R)-S-, -O-P(S)(R)-S-, -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, and -O-P(S)(H)-S-, where R is optionally substituted linear or branched C1-C 10 alkyl); acid-cleavable linking groups (e.g., hydrazone, ester, and ester of amino acid, -C=NN- and -OC(O)-); ester-based cleavable linking groups (e.g., -C(O)O-); peptide-based cleavable linking groups (e.g., linking groups 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 are R groups composed of two adjacent amino acids). Peptide-based cleavable linking groups contain two or more amino acids. In some embodiments, the peptide-based cleavage bond contains 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 6.0, 5.5, 5.0, or below), or by an agent such as an enzyme that can act as a general acid.

[0368] Linkers that are not oligonucleotides or that do not contain nucleotides or nucleosides are also referred to as non-nucleotide-based linkers.

[0369] Motif The present invention also includes multi-target molecules that are chimeric compounds. In the context of the present invention, a "chimeric" compound or "chimera" is a compound that includes two or more chemically distinct regions, each region being composed of at least one monomer unit, e.g., in the case of an oligonucleotide, a modified or unmodified nucleotide. Chimeric compounds can be described as having a particular motif. In some embodiments, motifs include, but are not limited to, alternating motifs, gap motifs, hemimer motifs, homogeneously and fully modified motifs, and position-modified motifs. As used herein, the phrase "chemically distinct region" refers to a region of a multi-target molecule that is different from other regions in that it has a modification not present elsewhere in the compound or in that it has no modification present elsewhere in the compound. A multi-target molecule can include two or more chemically distinct regions. As used herein, a region without a modification is also considered to be chemically distinct.

[0370] Chemically distinct regions can be repeated within a multi-target molecular compound. Thus, the pattern of chemically distinct regions within a multi-target molecule can be realized such that one or more second chemically distinct regions follow the first chemically distinct region. Such an arrangement of chemically distinct regions can be repeated one or more times. Preferably, the sequence is repeated two or more times. For example, both strands of a double-stranded effector molecule can include these sequences. Each of the chemically distinct regions may actually contain only a few monomers, for example, nucleotides. In some embodiments, each of the chemically distinct regions contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 monomers, such as nucleotides.

[0371] In some embodiments, alternating nucleotides contain the same modification, e.g., all odd nucleotides within a strand have the same modification and / or all even nucleotides within a strand have a modification similar to that of the first strand. In some embodiments, all odd nucleotides in a double-stranded effector molecule or multi-target molecule have the same modification, and all even nucleotides have a modification that is not present in the odd nucleotides, and vice versa.

[0372] When both strands of a double-stranded molecule contain an alternating modification pattern, the nucleotides of one strand may be in complementary positions relative to the similarly modified nucleotides of the second strand. In another embodiment, there is a phase shift between the modification patterns of the first strand relative to the similar modification pattern of the second strand. This shift preferably causes the similarly modified nucleotides of the first and second strands not to be in complementary positions with each other.

[0373] In some embodiments, the first strand has an alternating modification pattern in which alternating nucleotides contain a 2'-modification, e.g., a 2'-O-methyl modification. 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 a 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. Alternatively, such 2'-modified nucleotides may not be at complementary positions in the double-helical region.

[0375] In some embodiments, the oligonucleotide present in the multi-target molecule comprises 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 oligonucleotide present in the multi-target molecule comprises three chemically distinct regions. The central region is about 5 to 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 to 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 of 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 composed of linked monomer subunits, where the monomer subunits have two different types of sugar groups that alternate over substantially the entire sequence of the compound, including a continuous sequence. An oligonucleotide having an alternating motif can be represented by the formula: 5'-A(-L-B-L-A)n(-L-B)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 that is from about 9 to about 26 monomer subunits in length. Since longer and shorter compounds are also suitable for the present invention, this length range is not limiting. In some embodiments, one of A and B is a 2'-modified nucleoside provided herein.

[0378] As used herein, "type of modification" with respect to a "type" of nucleoside or nucleoside refers to a 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 part of a compound where all of the nucleosides and internucleoside linkages within the region contain the same type of modification; the nucleosides and / or internucleoside linkages of any adjacent portion contain at least one different type of modification. As used herein, the term "homogeneous fully modified motif" refers to an oligonucleotide containing a continuous sequence of linked monomer subunits each having the same type of sugar group. In some embodiments, the homogeneous fully modified motif includes a continuous sequence of the nucleosides of the present invention. In some embodiments, one or both of the 3' and 5' ends of the continuous sequence of nucleosides provided herein contain end groups such as one or more unmodified nucleosides.

[0380] In some embodiments, the 5' terminal monomer of a compound, such as a multi-target molecule or an effector molecule, includes a phosphorus-containing moiety at the 5' terminus. In some embodiments, the 5' terminal monomer includes 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 includes a 5'-modification. In some embodiments, the 5' terminal monomer includes both a 2'-modification and a 5'-modification. In some embodiments, the 5' terminal monomer is a 5'-stabilized nucleoside. In some embodiments, the modification of the 5' terminal monomer stabilizes the 5'-phosphate. In some embodiments, a compound comprising a modification of the 5' terminal monomer is resistant to exonuclease. In some embodiments, a compound comprising a modification of the 5' terminal monomer has improved gene expression modulating properties.

[0381] In some embodiments, the 5' terminal monomer is attached to the remainder of the compound by a modified linkage. In some such embodiments, the 5' terminal monomer is attached to the remainder of the compound by a phosphorothioate linkage.

[0382] In some embodiments, the oligomeric compounds of the invention include one or more regions of alternating modifications. In some embodiments, the oligomeric compounds include one or more regions of alternating nucleoside modifications. In some embodiments, the oligomeric compounds include one or more regions of alternating linkage modifications. In some embodiments, the oligomeric compounds include one or more regions of alternating nucleoside and linkage modifications.

[0383] In some embodiments, the oligomeric compounds of the invention include regions of one or more alternating 2'-F modified nucleosides and 2'-OMe modified nucleosides. In some such embodiments, such regions of alternating 2'F and 2'OMe modified nucleosides also include 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 are: (2'-F)-(PS)-(2'-OMe)-(PO) is.

[0385] In some embodiments, the oligomeric compound includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 such alternating regions. Such regions may be contiguous or may be interrupted by different modified nucleosides or linkages.

[0386] In some embodiments, one or more alternating regions in the alternating motif include multiple single nucleosides of a certain type. For example, the oligomeric compounds of the invention have the following nucleoside motifs: ABA; ABBA; AABA; AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA; ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA; It may include one or more regions of any of them, where A is a nucleoside of the first type and B is a nucleoside of the second type. 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'-CH2O-2'-LNA. In some embodiments, B is 4'-CH2O-2'-LNA. In some embodiments, A is DNA and B is 4'-CH2O-2'-LNA. In some embodiments, A is 4'-CH2O-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'-CH2O-2'-LNA. In some embodiments, A is 4'-CH2O-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'-CH2O-2'-LNA. In some embodiments, A is 4'-CH2O-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 5'-terminal nucleosides that include a phosphate stabilization modification. In some embodiments, oligomeric compounds having such alternating motifs also include 5'-terminal nucleosides that include a 2'-cationic modification. In some embodiments, oligomeric compounds having such alternating motifs also include 5'-terminal modifications.

[0392] 2-2-3 motif In some embodiments, the oligonucleotide in the multi-target molecule includes a region having a 2-2-3 motif. Such a region has 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 modifications different from A, but B, C, D, and E may have the same or different modifications from each other; w and z are from 0 to 15; x and y are from 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 linkages of the 2-2-3 motif are modified linkages. In some embodiments, all linkages are phosphorothioate linkages. In some embodiments, the linkage at the 3'-end of each modification of the first type is a phosphodiester.

[0395] In some embodiments, Z is 0. In such embodiments, the region of the 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 the three nucleosides of the first type. In some embodiments, an oligomeric compound comprising an oligonucleotide with Z being 0 may comprise a terminal group attached to the 3'-terminal nucleoside. Such a terminal group may comprise another nucleoside. Such another nucleoside is typically a non-hybridizing nucleoside.

[0396] In some embodiments, Z is from 1 to 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. Note that such non-hybridizing nucleosides may also be described as 3'-terminal groups with Z = 0 to avoid confusion.

[0397] Combination motif It should be understood that some of the foregoing motifs and modifications may be combined. Since a motif may contain only a few nucleotides, a particular oligonucleotide can contain two or more motifs. By way of non-limiting example, in some embodiments, an oligonucleotide in a multi-target molecule can have two or more nucleotide motifs selected from LNA, phosphorothioate linkages, 2'-OMe, conjugate ligands.

[0398] Without limitation, a multi-target molecule of the invention having any of the various nucleotide motifs described herein can also have any linkage motif. For example, in an oligonucleotide present in a multi-target molecule, the first 1, 2, 3, 4, or 5 sugar linkages at the 5' end can be modified sugar linkages, and the first 4, 5, 6, 7, or 8 sugar linkages at the 3' end can be modified sugar linkages. The central region of such a modified oligonucleotide can have sugar linkages based on any of the other motifs described herein, such as homogeneous, alternating, hemimer, gapmer, etc. In some embodiments, an oligonucleotide present in a multi-target molecule includes phosphorothioate linkages between the first and second monomers at the 5' end, phosphorothioate / phosphodiester alternating linkages in the central region, and 6, 7, or 8 phosphorothioate linkages at the 3' end.

[0399] It should be noted that the lengths of the regions defined by nucleotide motifs and the lengths of linkage motifs need not be the same.

[0400] In some embodiments, at least one strand of a single-stranded oligonucleotide or a double-stranded oligonucleotide includes at least one of the following motifs: (a) 5'-phosphorothioate or 5'-phosphorodithioate; (b) cationic modification of nucleotides 1 and 2 at the 5' end, where the cationic modification is located at the C5 position of pyrimidine and at C2, C6, C8, exocyclic N2, or exocyclic N6 of purine; (c) At least one G-clamp nucleotide and other nucleotides having a cationic modification at the first two terminal nucleotides at the 5'-end, wherein the cationic modification is located at the C5 position of pyrimidine or the C2, C6, C8, exocyclic N2 or exocyclic N6 position of 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 containing a nucleobase modification, preferably the methyl substituent is in an upward configuration, for example, in an arabinose configuration; (f) A 5'-PuPu-3' dinucleotide at the 3'-end, wherein each nucleotide contains a modified MOE at the 2'-position as described in US Patent Application Publication No. 20130130378 (the content of which is incorporated herein by reference in its entirety); (g) A 5'-PuPu-3' dinucleotide at the 5'-end, wherein each nucleotide contains a modified MOE at the 2'-position as described in US Patent Application Publication No. 20130130378; (h) A nucleotide at the 5'-end containing a modified MOE at the 2'-position as described in US Patent Application Publication No. 20130130378; (i) A nucleotide at the 5'-end having a 3'-F modification; (j) A 5'-end nucleotide containing a 4'-substituent; (k) A 5'-end nucleotide containing an O4' modification; (l) A 3'-end nucleotide containing a 4'-substituent; and (m) Combinations thereof.

[0401] In some embodiments, both strands of the double-stranded oligonucleotide independently contain at least one of the aforementioned motifs. In some other embodiments, both strands of the double-stranded oligonucleotide contain at least one of the aforementioned motifs, and these motifs may be the same or different, or may be some combinations of the same and different ones.

[0402] The foregoing examples are provided only to illustrate how the described motifs can be combined and used, and are not intended to limit the invention to the specific combinations or to the specific modifications used to exemplify the combinations. Further, without limitation, specific examples in this specification, such as those shown in the table above, are intended to encompass more general embodiments. For example, column A in the table above exemplifies a region of alternating 2'-OMe and 2'-F nucleosides. Accordingly, this same disclosure also exemplifies a region of another alternating 2'-modification. This also exemplifies a region of alternating 2'-O-alkyl and 2'-halogen nucleosides. This also exemplifies a region of alternating different modified nucleosides. All of the examples throughout this specification are to be considered in this inclusive sense.

[0403] It is also noted that the length of the oligonucleotide, for example, present in the multi-target molecule, can be readily manipulated by extending or shortening one or more of the described regions without breaking the motif.

[0404] In some embodiments, the oligonucleotide in the multi-target molecule comprises two or more chemically distinct regions and has a structure described in International Application PCT / US Patent Application No. 09 / 038433, filed Mar. 26, 2009, the contents of which are incorporated herein by reference in their entirety.

[0405] Synthesis, Purification and Analysis The oligomerization of modified and unmodified nucleosides and nucleotides can be routinely carried out according to the procedures described in the literature 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 produced by well-known techniques of solid-phase synthesis. Apparatus for such synthesis is sold by a number of suppliers including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be additionally or alternatively used. It is well known to prepare oligonucleotides such as phosphorothioate and alkylated derivatives using similar techniques. The present invention is not limited by the method of synthesis.

[0407] Methods for the purification and analysis of nucleic acids are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthetic and analytical methods can be carried out in multiwell plates. The methods of the present invention are not limited by the oligomer purification method.

[0408] Nucleic acids, such as oligonucleotides, can also be prepared using liquid or solid-phase organic synthesis or using enzymes by methods known in the art. Organic synthesis offers the advantage of being able to readily prepare oligonucleotide chains containing non-natural or modified nucleotides. Any other means for such synthesis known in the art may be used additionally or alternatively. Using similar techniques, it is also known to prepare other nucleic acids, such as those containing phosphorothioate, phosphorodithioate, and alkylated derivatives of sugar linkages. Double-stranded nucleic acids can be prepared using a two-step method. First, the individual strands of the double-stranded molecule are prepared separately. Next, the constituent strands are annealed.

[0409] Regardless of the method of synthesis, nucleic acids can be prepared in a solution suitable for formulation (e.g., water and / or organic solution). For example, the nucleic acid preparation can be precipitated and then redissolved in pure redistilled water and then lyophilized. Next, the dried nucleic acid can be resuspended in a solution suitable for the intended formulation method.

[0410] Instructions regarding the synthesis of specific modified nucleic acids can be found in the following U.S. patents or pending patent applications: U.S. Patent Nos. 5,138,045 and 5,218,105 regarding polyamine-conjugated oligonucleotides; U.S. Patent No. 5,212,295 regarding monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Patent Nos. 5,378,825 and 5,541,307 regarding oligonucleotides having modified backbones; U.S. Patent No. 5,386,023 regarding backbone-modified oligonucleotides and their preparation by reductive coupling; U.S. Patent No. 5,457,191 regarding modified nucleobases based on 3-deazapurine ring systems and methods for their synthesis; U.S. Patent No. 5,459,255 regarding modified nucleobases based on N-2 substituted purines; U.S. Patent No. 5,521,302 regarding methods for preparing oligonucleotides having chiral phosphorus linkages; U.S. Patent No. 5,539,082 regarding peptide nucleic acids; U.S. Patent No. 5,554,746 regarding oligonucleotides having β-lactam backbones; U.S. Patent No. 5,571,902 regarding methods and materials for the synthesis of oligonucleotides; U.S. Patent No. 5,578,718 regarding nucleosides having alkylthio groups (such groups can be used as linkers to other moieties attached at any of various positions of the nucleoside); U.S. Patent Nos. 5,587,361 and 5,599,797 regarding oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Patent No. 5,506,351 regarding methods for the preparation of 2'-O-alkylguanosine and related compounds (such as 2,6-diaminopurine compounds); U.S. Patent No. 5,587,469 regarding oligonucleotides having N-2 substituted purines; U.S. Patent No. 5,587,470 regarding oligonucleotides having 3-deazapurines; U.S. Patent No. 5,223,168 regarding conjugate 4'-desmethyl nucleoside analogs in each case, and U.S. Patent No. 5,608,046; U.S. Patent Nos. 5,602,240 and 5,610,289 regarding backbone-modified oligonucleotide analogs;U.S. Patent No. 6,262,241 and U.S. Patent No. 5,459,255, particularly relating to methods for synthesizing 2'-fluoro-oligonucleotides;

[0411] Compositions and methods for formulating pharmaceutical compositions Multitarget molecules can be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for formulating pharmaceutical compositions are influenced by several criteria, including but not limited to the route of administration, the degree of the disease, or the dose to be administered.

[0412] Multitarget molecules can be used as pharmaceutical compositions by combining such oligomeric compounds with suitable pharmaceutically acceptable diluents or carriers. A pharmaceutically acceptable diluent is 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 multitarget molecules include any pharmaceutically acceptable salts, esters, or salts of such esters. In some embodiments, pharmaceutical compositions comprising multitarget molecules can (directly or indirectly) confer one or more oligonucleotides that give rise to biologically active metabolites or residues thereof when administered to animals such as humans. 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 include the incorporation of another nucleoside at one or both ends of a multitarget molecule that is cleaved by endogenous nucleases in the body to form the active molecule.

[0415] The pharmaceutical composition of the present invention can be administered in various ways depending on whether local or systemic treatment is desired and on the site to be treated. Administration can be local (e.g., by transdermal patch), pulmonary, e.g., by inhalation or ventilation of powder or aerosol using a nebulizer, etc.; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, e.g., by an implantable device; or intracranial, e.g., intracerebral, intrathecal or intraventricular administration. The multi-target molecule can be delivered to target specific tissues such as the liver (e.g., hepatocytes of the liver).

[0416] Examples of pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, infusion solutions, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be required or may be desirable. Also, coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the multi-target molecule that is the subject of the present invention is 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., dioleoyl phosphatidylethanolamine DOPE, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidylglycerol DMPG), and cationic (e.g., dioleoyl tetramethylaminopropyl DOTAP and dioleoyl phosphatidylethanolamine DOTMA). The multi-target molecule that is the subject of the present invention may be encapsulated in liposomes or may form a complex with them, particularly cationic liposomes. Alternatively, the multi-target molecule 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, dicapric acid ester, tricapric acid ester, monoolein, dilaurin, glyceryl 1-monocapric acid ester, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 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] In addition to microemulsions that have been studied and used for drug formulation, there are many organized surfactant structures. 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 duration of action they offer. When used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged within a spherical bilayer or bilayer.

[0418] Liposomes are single - layer or multi - layer vesicles having a membrane formed from lipophilic substances and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage that they can fuse with cell walls. Non - cationic liposomes cannot efficiently fuse with cell walls but are taken up by macrophages in vivo.

[0419] Further advantages of liposomes are as follows: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water - and lipid - soluble drugs; liposomes can protect drugs encapsulated within their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and water content of the liposomes.

[0420] Liposomes are useful for the transport and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the liposomes begin to fuse with the cell membrane, and as the fusion of liposomes and 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. In the case of topical administration, there is increasing evidence that liposomes offer several advantages over other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, an increased accumulation of the administered drug at the desired target site, and the ability to administer a variety of hydrophilic and hydrophobic drugs into the skin.

[0422] Multiple reports have detailed the ability of liposomes to deliver drugs containing high molecular weight DNA into the skin. Compounds containing 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 can be 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 complex binds to the negatively charged cell surface and is internalized into the endosome. Since the interior of the endosome is at an acidic pH, the liposome is disrupted and its contents are released into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980 - 985).

[0424] pH - sensitive or negatively charged liposomes do not complex with DNA but rather entrap it. Since both DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. 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 liposomal composition contains phospholipids other than naturally derived phosphatidylcholine. Neutral liposomal compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposomal compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed, for example, 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] In several studies, the topical delivery of liposomal drug formulations to the skin has been evaluated. When liposomes containing interferon were applied to the skin of guinea pigs, reduction of cutaneous herpes was achieved, whereas delivery of interferon by other means (e.g., as a solution or an emulsion) was not effective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405 - 410). Further, in another study, the efficacy of interferon administered as part of a liposomal formulation was tested against administration of interferon using an aqueous system, and the conclusion was drawn that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259 - 265).

[0427] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been tested to determine their usefulness in drug delivery to the skin. Nonionic liposome formulations containing Novasome(™) I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome(™) II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver cyclosporin-A to the dermis of mouse skin. From the results, it was found that such nonionic liposome systems are effective in promoting the deposition of cyclosporin-A into various layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0428] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes containing one or more specialized lipids which, when incorporated into the liposome, result in an increased circulation lifetime as compared to liposomes without such specialized lipids. Examples of sterically stabilized liposomes include those in which a portion of the vesicle-forming lipid moiety of the liposome contains one or more glycolipids such as (A) monosialoganglioside G M1 and the like, or (B) those derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. Without intending to be bound by any particular theory, in the art it is thought that for sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids, the increased circulation lifetime of these sterically stabilized liposomes is due to reduced uptake by cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0429] A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactosylcerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings have been elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924 pamphlet (both by Allen et al.) disclose liposomes containing (1) sphingomyelin and (2) ganglioside G M1 or galactosylcerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 pamphlet (Lim et al.).

[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) reported a nonionic detergent containing a PEG moiety, 2C 1215GLiposomes containing it are described. Illum et al. (FEBS Lett., 1984, 167, 79) described that the hydrophilic coating of polystyrene particles with polymer glycol significantly increased the half-life in blood. Synthetic phospholipids modified by the binding of carboxylic acid groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Patent Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have a significant increase in the blood circulation half-life. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, such as DSPE-PEG formed from a combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having PEG moieties covalently bound to the outer surface are described in Fisher's European Patent No. 0 445 131 B1 and International Publication No. 90 / 04384 pamphlet. Liposome compositions containing 1 to 20 mole percent of PE derivatized with PEG and methods of using the same are described by Woodle et al. (U.S. Patent Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent Nos. 5,213,804 and European Patent No. 0 496 813 B1). Liposomes containing several other lipid-polymer conjugates are disclosed in International Publication No. 91 / 05545 pamphlet and U.S. Patent No. 5,225,212 (both by Martin et al.) and International Publication No. 94 / 20073 pamphlet (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in International Publication No. 96 / 10391 pamphlet (Choi et al.). U.S. Patent No. 5,540,935 (Miyazaki et al.) and U.S. Patent No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surface.

[0431] Some liposomes containing nucleic acids are known in the art. The international publication No. WO 96 / 40062 by Thierry et al. discloses a method for encapsulating high molecular weight nucleic acids in liposomes. The US Patent No. 5,264,221 by Tagawa et al. discloses protein-binding liposomes and claims that the contents of such liposomes can contain dsRNA. The US Patent No. 5,665,710 by Rahman et al. describes a specific method for encapsulating oligodeoxynucleotides in liposomes. The international publication No. WO 97 / 04787 by Love et al. discloses liposomes containing dsRNA targeted to the raf gene.

[0432] Transfersome is another type of liposome, which is a highly deformable lipid aggregate and an attractive candidate for drug delivery vehicles. Transfersome can be represented as lipid droplets, which, due to their high deformability, can easily penetrate pores smaller than the lipid droplets. Transfersomes can adapt to the environment in which they are used. For example, they can self-optimize (adapt to the shape of the pores in the skin), self-repair, and often reach their target without fragmentation and self-fill. To produce transfersomes, surface surfactants, usually detergents, can be added to the standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. It has been found that transfersome-mediated delivery of serum albumin is as effective as subcutaneous injection of a solution containing serum albumin.

[0433] Research tools In some cases, oligonucleotides capable of regulating gene expression are 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 cells or animals and observe phenotypic changes in the cells or animals. In some embodiments, the present invention provides a method for reducing the amounts 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, such techniques can be used by a tester to characterize a protein or non-translated nucleic acid. In some embodiments, such experiments are used to examine the kinetics and / or turnover of gene products and / or some cellular functions. In some embodiments, such experiments are used to examine the relationship or correlation between different genes or gene products.

[0434] Kit In some embodiments, the present invention provides a kit comprising one or more multi-target molecules. In some embodiments, such kits are intended for therapeutic use. In some embodiments, such kits are intended for research use.

[0435] Although specific compounds, compositions, and methods described herein have been described in detail according to some embodiments, the following examples are only used to illustrate the compounds described herein and are not intended to limit the present invention. The reference documents, GenBank accession numbers, etc. cited in this application are each incorporated herein by reference in their entirety.

[0436] Definitions Unless otherwise defined, the names, procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Some such techniques and procedures can be found, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 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”, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference herein for all purposes. To the extent permitted, all patents, applications, published applications, and other publications and other data mentioned in this disclosure are hereby incorporated by reference herein 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 modulated 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), cDNA obtained from such RNA, and miRNA. For example, the target nucleic acid may be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a particular disorder or condition. In some embodiments, the 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 the level of mRNA in a cell for 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%, and any integer percentage between these, up to 100%, of the mRNA level found in a cell in the absence of the miRNA or RNA interference molecule. In one preferred embodiment, the mRNA level is reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, and any integer percentage between 5% and 100%.

[0440] As used herein, the term "modulating gene expression" means upregulating or downregulating 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", but the use of the term "modulate" is not limited to this definition.

[0441] As used herein, gene expression regulation 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 % and / or fold difference can be calculated, for example, relative to a control or non-control, as follows. [Number]

[0442] As used herein, with respect to gene expression, the terms "inhibit", "down-regulate", or "reduce" 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% lower than the corresponding unregulated control, preferably 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, with respect to gene expression, the terms "increased" or "upregulated" 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 upregulated if 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 as compared to the corresponding unregulated control.

[0444] As used herein, the terms "increased" or "increasing" generally mean a statistically significant amount of increase; to avoid misunderstanding, "increased" means an increase of at least 10% compared to a reference level, for example, 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 100% or less increase or any increase between 10 - 100%, or an increase of 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 compared to the reference level, or any increase between 2-fold - 10-fold or more.

[0445] As used herein, the terms "reduced" or "reducing" generally mean a statistically significant amount of decrease. However, to avoid misunderstanding, "reduced" means a decrease of at least 10% compared to a reference level, e.g., 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 100% (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 drive the relevant functions of the nucleic acid, such as RNAi activity. The 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). The percentage of complementarity indicates the percentage of consecutive 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 out of 10, 6 out of 10, 7 out of 10, 8 out of 10, 9 out of 10, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" or 100% complementarity means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Less than complete complementarity refers to a situation where some (but not all) of the nucleoside units of the two strands can hydrogen bond with each other. "Substantial complementarity" refers to a polynucleotide strand that exhibits at least 90% complementarity, excluding regions of the polynucleotide strand that are selected to be non-complementary (e.g., overhangs). Specific binding requires a degree of complementarity sufficient to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions under which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0447] The terms "off-target" and the phrase "off-target effect" refer to any instance where an effector molecule against a given target causes an unintended effect by interacting, either directly or indirectly, with another target sequence, DNA sequence, 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 co-degradation of the other transcripts.

[0448] As used herein, the term "nucleoside" means a glycosylamine that includes a nucleobase and a sugar. Nucleosides include, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and / or sugar moieties.

[0449] As used herein, the term "nucleotide" refers to a glycosomine that includes a nucleobase and a sugar covalently bonded to a phosphate group. 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 the base of another nucleic acid.

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

[0452] As used herein, the term "oligomeric compound" refers to a polymeric structure that contains two or more substructures and is 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 a detoxifying agent compound. In some embodiments, the oligomeric compound contains a conjugate group.

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

[0454] As used herein, the term "oligonucleotide" refers to an oligomeric compound that includes a plurality of linked nucleosides. In certain embodiments, one or more nucleotides of the oligonucleotide are modified. In some embodiments, the oligonucleotide includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the oligonucleotide is composed of natural and / or non-natural nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.

[0455] As used herein, "internucleoside linkage" refers to a 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 terms "detecting siRNA activity" or "measuring siRNA activity" mean that a test for detecting or measuring siRNA activity is performed on a particular sample and compared to that of a control sample. Such detection and / or measurement may include a value of zero. Thus, even if the result of a test for detecting siRNA activity reveals no siRNA activity (zero siRNA activity), the step of "detecting siRNA activity" has still been performed.

[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 modified in a different manner compared to at least one other sugar, nucleobase, or internucleoside linkage within the same oligomeric compound. The remaining 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 modified in a different manner compared to at least one other sugar, nucleobase, or internucleoside linkage within the same oligonucleotide. The remaining sugars, nucleobases, and internucleoside linkages may be independently modified or unmodified, and may 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 regulation is desired.

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

[0465] As used herein, the term "targeting" or "being targeted" refers 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 nucleobases refer to the nucleobases of an antisense compound that can base pair with the nucleobases of its target nucleic acid. For example, if the nucleobase at a particular position of an antisense compound can hydrogen bond with the nucleobase at a particular position of a target nucleic acid, the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary in that nucleobase pair.

[0467] As used herein, the term "non-complementary nucleobases" refers to pairs of nucleobases that do not form hydrogen bonds with each other or do not support hybridization.

[0468] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize by nucleic acid base complementarity to another oligomeric compound or nucleic acid. In some embodiments, an oligomeric compound and its target are complementary to each other when a sufficient number of corresponding positions within each molecule are occupied by nucleobases that can bind to each other to allow for stable association of the antisense compound and the target. One of ordinary skill in the art will recognize that mismatches can be tolerated without excluding the ability of the oligomeric compound to maintain the association. Thus, oligomeric compounds (e.g., siRNAs, multi-target molecules, etc.) that contain up to about 20% nucleotides that are mismatches (i.e., are not nucleobases complementary to the corresponding nucleotides of the target) are described herein. Preferably, oligomeric compounds such as siRNAs and multi-target molecules contain 15% or less, more preferably 10% or less, and most preferably 5% or less mismatches, or contain no mismatches at all. The remaining nucleotides are either complementary nucleobases or do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art will recognize that the compounds provided herein are 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" means the pairing of complementary oligomeric compounds (e.g., the antisense strand of an siRNA with its target nucleic acid or the antisense and sense strands of an siRNA). Without being limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobases), which may be Watson-Crick type, Hoogsteen type or reversed Hoogsteen type hydrogen bonds. For example, the natural base adenine is a nucleobase that is complementary to the natural nucleobases thymidine and uracil that pair via 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 hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with a higher affinity than to hybridize to other nucleic acid sites. In some embodiments, the antisense strand of an siRNA specifically hybridizes to two or more target sites.

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

[0472] As used herein, the term "modulate" refers to a perturbation of a function or activity as compared to the level of the function or activity prior to modulation. For example, modulation includes either an increase (stimulation or induction) or a decrease (inhibition or repression) in gene expression. As another example, modulation of expression can include perturbation of splice site selection in pre-mRNA processing.

[0473] As used herein, the term "expression" refers to any function and step by which the encoded information of a gene is converted into a structure that exists and operates within a cell. Such structures include, but are not limited to, the products of transcription and translation.

[0474] As used herein, "variant" refers to an alternative RNA transcript that can be generated from the same genomic region of DNA. Variants include, but are not limited to, "pre-mRNA variants," which are transcripts generated from the same genomic DNA, differ from other transcripts generated from the same genomic DNA in either their start or stop position, and include both intron and exon sequences. Variants also include those having, but not limited to, an alternative splice site, or alternative start and stop codons.

[0475] As used herein, "high affinity modified monomer" refers to a monomer having at least one modified nucleobase, internucleoside linkage, or sugar moiety as compared to a naturally occurring monomer, where the modification enhances the affinity of an antisense compound containing the high affinity modified monomer for its target nucleic acid. High affinity modifications include, but are not limited to, monomers containing a 2'-modified sugar (e.g., nucleosides and nucleotides).

[0476] As used herein, the terms "2'-modified" or "2'-substituted" mean a sugar having 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-C 10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O-N(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn) on BNA and monomers (e.g., nucleosides and nucleotides), where each Rm and Rn is independently H or substituted or unsubstituted C1-C 10 alkyl. In some embodiments, the oligomeric compound has the formula 2'-O(CH2) nIt includes 2'-modified monomers without H (where n is from 1 to 6). In some embodiments, the oligomeric compound includes 2'-modified monomers without 2'-OCH3. In some embodiments, the oligomeric compound includes 2'-modified monomers without the above formula or, in another embodiment, without 2'-O(CH2)2OCH3.

[0477] As used herein, unless otherwise indicated, the terms "locked nucleic acid" or "LNA" or "locked nucleoside" or "locked nucleotide" refer to a nucleoside or nucleotide in which the furanose moiety of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic system. Locked nucleic acids are also referred to as bicyclic nucleic acids (BNA).

[0478] As used herein, unless otherwise indicated, the term "methyleneoxy LNA" alone refers to β-D-methyleneoxy LNA.

[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 that includes a central region ("gap") and regions on either side of the central region ("wings"), where the gap includes at least one modification that is different from each wing. Such modifications include nucleobases, monomer linkages, and sugar modifications, as well as the absence of modification (unmodified). Thus, in some embodiments, the nucleotide linkages in each wing are different from the nucleotide linkages within the gap. In some embodiments, each wing includes nucleotides having a high affinity modification, and the gap includes nucleotides that do not include that modification. In some embodiments, all of the nucleotides within the gap and within the wings include high affinity modifications, but the high affinity modifications within the gap are different from the high affinity modifications within the wings. In some embodiments, the modifications within the wings are the same as each other. In some embodiments, the modifications within the wings are different from each other. In some embodiments, the nucleotides within the gap are unmodified and the nucleotides within the wings are modified. In some embodiments, the modifications within each wing are the same. In some embodiments, the modifications within one wing are different from the modifications within the other wing. In some embodiments, the oligomeric compound is a gapmer having 2'-deoxynucleotides within the gap and nucleotides having a high affinity modification within 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) in the body or in its cells by the action of an endogenous enzyme or other chemical and / or 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 undesirable 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 "prevent" refers to delaying or preventing the onset or occurrence of a condition or disease over a period of hours to days, preferably weeks to months.

[0485] As used herein, the term "ameliorate" refers to reducing at least one activity or one measure of severity of a condition or disease. The severity of the measure can be determined by subjective or objective measures known to those of skill in the art.

[0486] As used herein, the term "treat" refers to administering a composition of the invention to effect a change or amelioration of a disease or condition. Prevention, amelioration, and / or treatment may require administration of multiple doses at regular intervals or prior to the onset of a condition or disease to effect a change in the course of a disease or condition. Further, a single agent may be used sequentially or simultaneously in an individual for prevention, amelioration, 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 provides a therapeutic benefit to 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, two or more such pharmaceutical agents are administered together. In some embodiments, two or more such pharmaceutical agents are administered separately. In some embodiments, two or more such pharmaceutical agents are administered simultaneously. In some embodiments, two or more such pharmaceutical agents are administered at different times. In some embodiments, two or more such pharmaceutical agents are administered by the same route of administration. In some embodiments, two or more such pharmaceutical agents are administered by different routes of administration. In some embodiments, two or more such pharmaceutical agents are contained in the same pharmaceutical formulation. In some embodiments, two or more such 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 can include an antisense oligonucleotide and a sterile aqueous solution. In some embodiments, a pharmaceutical composition includes a pharmaceutical agent and a diluent and / or a carrier.

[0492] As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal or a plant), but rather in an artificial environment, such as within a test tube or reaction vessel, within cell culture, and the like. As used herein, the term "ex vivo" refers to cells that are removed from a living organism and cultured outside the organism (e.g., within a test tube). As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, and / or a microorganism).

[0493] As used herein, the terms "subject" or "patient" refer to any organism to which the compositions disclosed herein can be administered for purposes such as, for example, experimentation, diagnosis, and / or treatment. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Usually, the animal is a vertebrate such as a primate, rodent, livestock, or game animal. Examples of primates include chimpanzees, squirrel monkeys, spider monkeys, and macaques, such as Rhesus monkeys. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of livestock and game animals include cows, horses, pigs, deer, bison, sheep, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as salmon, catfish, and trout. A patient or subject can be any subset of those listed above, for example, including 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, such as a primate, such as a human. The terms "patient" and "subject" are used synonymously herein. The subject can be male (male) or female (female).

[0494] Preferably, the subject is a mammal. The mammal can be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cows. Non-human mammals can be advantageously used as subjects for animal models of human diseases and disorders. Further, the compounds, compositions, and methods described herein can be used in livestock and / or pets.

[0495] In some embodiments, the subject is a human. In other embodiments, the subject is an experimental animal or a surrogate animal as a disease model. This term does not mean a specific age or gender. Thus, it is intended to include adult and neonatal subjects, as well as fetuses, regardless of whether male (male) or female (female). Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term "subject" is also intended to include transgenic species. In some embodiments, the subject may be of European descent. In some embodiments, the subject may be of African American descent. In some embodiments, the subject may be of Asian descent.

[0496] In jurisdictions that prohibit the patenting of methods practiced on the human body, the meaning of "administration" of a composition to a human subject will be limited to prescribing a regulated substance for self-administration by the human subject by any method (e.g., oral, inhalation, topical application, injection, insertion, etc.). The broadest and most reasonable interpretation that conforms to the laws or regulations defining the subject matter eligible for patenting is intended. In jurisdictions that do not prohibit the patenting of methods practiced on the human body, "administration" of a composition includes both methods practiced on the human body and the activities described above.

[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 under the skin. "Intravenous administration" means administration into a vein.

[0499] As used herein, the term "dose" refers to the specified amount of a pharmaceutical agent supplied in a single administration. In some embodiments, a single 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 supplied by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In some embodiments, a single dose may be administered in two or more injections to minimize the injection site reaction in an individual.

[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 a lyophilized antisense oligonucleotide. In some embodiments, the unit dosage form is a vial containing a reconstituted antisense oligonucleotide.

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

[0502] As used herein, the term "side effect" refers to a physiological reaction that is thought to be due to a treatment other than the desired effect. In some embodiments, side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and muscle disorders. For example, an increase in aminotransferase levels in the serum may indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin may indicate hepatotoxicity or abnormal liver function.

[0503] As used herein, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms, as used herein. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. An alkyl group typically contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl), and more preferably from 1 to about 6 carbon atoms. The term "lower alkyl" as used herein contains from 1 to about 6 carbon atoms. The alkyl groups used herein may optionally contain one or more additional substituents.

[0504] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon chain radical containing up to 24 carbon atoms and having at least one carbon-carbon double bond, as used herein. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and dienes such as 1,3-butadiene. An alkenyl group typically contains from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, and more preferably from 2 to about 6 carbon atoms. The alkenyl groups used herein may optionally contain one or more additional substituents.

[0505] As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon radical containing up to 24 carbon atoms and having at least one carbon-carbon triple bond, as used herein. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. An alkynyl group typically contains from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, and more preferably from 2 to about 6 carbon atoms. The alkynyl groups used herein may optionally contain one or more additional substituents.

[0506] As used herein, the term "aminoalkyl" refers to an amino-substituted alkyl radical as used herein. This term means that it includes a C1-C12 alkyl group having an amino substituent at any position, where the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and / or amino moieties 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 and having saturation between any two carbon atoms as a single bond, double bond or triple bond as used herein. The aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms, and more preferably from 1 to about 6 carbon atoms. The straight or branched chain of the aliphatic group may be interrupted by one or more heteroatoms including nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include, but are not limited to, polyalkoxy, such as polyalkylene glycol, polyamine, and polyimine. The aliphatic groups used herein may optionally contain another substituent.

[0508] As used herein, the term "alicyclic" or "alicyclic" refers to a ring system in which the ring is an aliphatic ring system. This ring system may 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. The alicyclic as used herein may optionally contain another substituent. As used herein, the term "alkoxy" 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. The alkoxy group as used herein may optionally contain another substituent. As used herein, the terms "halo" and "halogen" refer to atoms selected from fluorine, chlorine, bromine, and iodine as used herein.

[0509] As used herein, the terms "aryl" and "aromatic" refer to monocyclic or polycyclic carbocyclic radicals having one or more aromatic rings as used herein. 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 one or more rings. The aryl group as used herein may optionally contain another substituent.

[0510] As used herein, the terms "aralkyl" and "arylalkyl" refer to radicals 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. The aralkyl group as used herein may optionally contain another substituent attached to the alkyl, aryl, or both groups forming 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, is unsaturated, partially saturated or fully saturated, and thus includes heteroaryl groups. Heterocyclic also means including 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, but are not limited to, [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 another substituent. As used herein, the terms "heteroaryl", and "heteroaromatic" refer to radicals that include monocyclic or polycyclic aromatic rings, ring systems or fused ring systems in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl also means including fused ring systems, which includes systems in which one or more of the fused rings contain no 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. Heteroaryl radicals can be attached to the parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. Heteroaryl groups as used herein may optionally contain another substituent.

[0512] As used herein, the term "heteroarylalkyl" refers to a heteroaryl group as defined herein having an alkyl radical capable of bonding a heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, naphthyridinylpropyl, and the like. The heteroarylalkyl groups used herein may optionally contain another substituent on one or both of the heteroaryl or alkyl moieties.

[0513] As used herein, the term "monocyclic or polycyclic structure" encompasses all ring systems that are monocyclic or polycycl...

Claims

**Claim 1** A multi-target molecule comprising at least two effector molecules, wherein the effector molecules are linked together, at least one ligand is conjugated to the multi-target molecule, and the multi-target molecule regulates the gene expression of at least two target nucleic acids by at least 70% each as compared to the case where the effector molecules are not linked together, and the at least two effector molecules do not overlap with each other. **Claim 2** The multi-target molecule according to claim 1, wherein the at least two effector molecules are non-covalently linked to each other, and each of the at least two effector molecules is conjugated to at least one ligand. **Claim 3** The multi-target molecule according to claim 2, wherein the non-covalent bond is nucleotide hybridization between the at least two effector molecules. **Claim 4** The multi-target molecule according to claim 1, wherein the effector molecule is selected from siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA or anti-miR, super-miR, antagomiR, ribozyme, triple helix-forming oligonucleotide, decoy oligonucleotide, splice-switching oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adapter, CRISPR Cas, and combinations thereof. **Claim 5** The multi-target molecule according to claim 1, which regulates the gene expression of at least two target nucleic acids by at least 75% each as compared to the case where the effector molecules are not linked together. **Claim 6** The multi-target molecule according to claim 5, wherein one of the at least two effector molecules regulates the gene expression of a first target nucleic acid, and the other of the at least two effector molecules regulates the gene expression of a second nucleic acid. **Claim 7** The multi-target molecule according to claim 6, wherein the first target nucleic acid and the second target nucleic acid are the same. **Claim 8** The multi-target molecule according to claim 7, wherein the first target nucleic acid and the second target nucleic acid target the same nucleotide sequence. **Claim 9** The multi-target molecule according to claim 1, wherein the ligand is conjugated at the 3'-end of one of the at least two effector molecules. **Claim 10** The multi-target molecule according to claim 1, wherein the ligand is conjugated at the 5'-end of one of the at least two effector molecules.

11. A multi-target molecule comprising a first double-stranded siRNA molecule and a second double-stranded siRNA molecule, wherein the sense strand of the first siRNA comprises a single-stranded overhang at its 3'-end, and the antisense strand of the second siRNA comprises a single-stranded overhang at its 3'-end, the nucleic acid sequence of the single-stranded overhang of the sense strand being substantially complementary to the nucleotide sequence of the single-stranded overhang of the antisense strand, the two single-stranded overhangs forming a double strand, and the first siRNA and the second siRNA being conjugated to at least one ligand respectively.

12. The multi-target molecule according to claim 11, wherein the first and second siRNAs independently regulate the gene expression of their respective target nucleic acids by at least 70% compared to the case where the first siRNA and the second siRNA are not linked together.

13. The multi-target molecule according to claim 11 or 12, wherein the first siRNA regulates the gene expression of a first target nucleic acid, and the second siRNA regulates the gene expression of a second nucleic acid.

14. The multi-target molecule according to claim 13, wherein the first target nucleic acid and the second target nucleic acid are the same.

15. The multi-target molecule according to claim 14, wherein the first siRNA and the second siRNA target the same nucleic acid sequence.

16. The multi-target molecule according to any one of claims 11 to 15, wherein at least one ligand is conjugated to one of the sense strands.

17. The multi-target molecule according to claim 16, wherein at least one ligand is conjugated at the 3'-end, 5'-end or internal position of one of the sense strands.

18. The multi-target molecule according to any one of claims 11 to 16, wherein at least one ligand is conjugated to one of the antisense strands.

19. The multi-target molecule according to claim 18, wherein at least one ligand is conjugated at the 3'-end, 5'-end or internal position of one of the antisense strands.

20. The multi-target molecule according to any one of claims 11 to 19, wherein the first ligand is conjugated to the sense strand and the second ligand is conjugated to the antisense strand.

21. The multi-target molecule according to claim 20, wherein the sense strand conjugated to the first ligand is derived from the second siRNA, and the antisense strand conjugated to the second ligand is derived from the first siRNA.

22. The multi-target molecule according to claim 20, wherein the sense strand conjugated to the first ligand is derived from the first siRNA, and the antisense strand conjugated to the second ligand is derived from the second siRNA.

23. The multi-target molecule according to claim 20, wherein the sense strand conjugated to the first ligand is derived from the first siRNA, and the antisense strand conjugated to the second ligand is also derived from the first siRNA.

24. The multi-target molecule according to claim 20, wherein the sense strand conjugated to the first ligand is derived from the second siRNA, and the antisense strand conjugated to the second ligand is also derived from the second siRNA.

25. The multi-target molecule according to any one of claims 11 to 24, wherein all of the single-stranded overhangs of the sense strand contain all DNA, all RNA, or a mixture of DNA and RNA nucleotides, and the DNA and RNA can be natural or modified.

26. The multi-target molecule according to any one of claims 11 to 25, wherein all of the single-stranded overhangs of the antisense strand contain all DNA, all RNA, or a mixture of DNA and RNA nucleotides, and the DNA and RNA can be natural or modified.

27. The multi-target molecule according to any one of claims 11 to 26, wherein less than 5% of the double strand formed by the two single-stranded overhangs is cleaved by nuclease.

28. The multi-target molecule according to any one of claims 11 to 26, wherein at least 50% of the double strand formed by the two single-stranded overhangs is cleaved by nuclease.

29. The multi-target molecule according to any one of claims 11 to 18, 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.

30. The multi-target molecule according to claim 29, wherein the at least one modification is included in the sense strand, the antisense strand, a single-stranded overhang of the sense strand, a single-stranded overhang of the antisense strand, or any combination thereof.

31. A multi-target molecule comprising a first double-stranded siRNA molecule and a second double-stranded siRNA molecule, wherein the antisense strand of the first siRNA comprises a single-stranded overhang at the 3' end, and the antisense strand of the second siRNA comprises a single-stranded overhang at the 3' end, the nucleic acid sequences of the two single-stranded overhang nucleic acid sequences are substantially complementary to each other, and the two single-stranded overhangs form a double strand, the two single-stranded overhangs form a double strand, and the first siRNA and the second siRNA are each conjugated to at least one ligand.

32. The multi-target molecule according to claim 31, wherein the first and second siRNAs independently regulate the gene expression of their respective target nucleic acids by at least 70% compared to the case where the first siRNA and the second siRNA are not linked together.

33. The multi-target molecule according to claim 31 or 32, wherein the first siRNA regulates the gene expression of a first target nucleic acid, and the second siRNA regulates the gene expression of a second nucleic acid.

34. The multi-target molecule according to claim 33, wherein the first target nucleic acid and the second target nucleic acid are the same.

35. The multi-target molecule according to claim 34, wherein the first siRNA and the second siRNA target the same nucleic acid sequence.

36. The multi-target molecule according to any one of claims 31 to 35, wherein at least one of the ligands is conjugated to one of the sense strands.

37. The multi-target molecule according to claim 36, wherein at least one of the ligands is conjugated at the 3' end, 5' end, or an internal position of one of the sense strands.

38. The multi-target molecule according to any one of claims 31 to 36, wherein at least one of the ligands is conjugated to one of the antisense strands.

39. The multi-target molecule according to claim 38, wherein at least one of the ligands is conjugated at one of the 3'-end, 5'-end or internal position of one of the antisense strands.

40. The multi-target molecule according to any one of claims 31 to 39, wherein the first ligand is conjugated to the sense strand and the second ligand is conjugated to the antisense strand.

41. The multi-target molecule according to claim 40, wherein the sense strand conjugated to the first ligand is derived from the second siRNA, and the antisense strand conjugated to the second ligand is derived from the first siRNA.

42. The multi-target molecule according to claim 40, wherein the sense strand conjugated to the first ligand is derived from the first siRNA, and the antisense strand conjugated to the second ligand is derived from the second siRNA.

43. The multi-target molecule according to claim 40, wherein the sense strand conjugated to the first ligand is derived from the first siRNA, and the antisense strand conjugated to the second ligand is also derived from the first siRNA.

44. The multi-target molecule according to claim 40, wherein the sense strand conjugated to the first ligand is derived from the second siRNA, and the antisense strand conjugated to the second ligand is also derived from the second siRNA.

45. The multi-target molecule according to any one of claims 31 to 44, wherein all of the single-stranded overhangs of the antisense strand of the first siRNA contain all DNA, all RNA, or a mixture of DNA and RNA nucleotides.

46. The multi-target molecule according to any one of claims 31 to 45, wherein all of the single-stranded overhangs of the antisense strand of the second siRNA contain all DNA, all RNA, or a mixture of DNA and RNA nucleotides.

47. The multi-target molecule according to any one of claims 31 to 46, wherein less than 5% of the double-strand formed by the two single-stranded overhangs is cleaved by a nuclease.

48. The multi-target molecule according to any one of claims 31 to 46, wherein at least 50% of the double-strand formed by the two single-stranded overhangs is cleaved by a nuclease.

49. The multi-target molecule according to any one of claims 31 to 48, 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.

50. The multi-target molecule according to claim 39, wherein the at least one modification is included in the sense strand, the antisense strand, the single-stranded overhang of the antisense strand of the first siRNA, the single-stranded overhang of the antisense strand of the second siRNA, or any combination thereof.

51. A multi-target 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 bonded to each other, and at least one ligand is conjugated to the multi-target molecule.

52. The multi-target molecule according to claim 51, wherein the sense strand of the first siRNA molecule is covalently bonded to the sense strand of the second siRNA molecule.

53. The multi-target molecule according to claim 51, wherein the sense strand of the first siRNA molecule is covalently bonded to the antisense strand of the second siRNA molecule.

54. The multi-target molecule according to claim 51, wherein the antisense strand of the first siRNA molecule is covalently bonded to the antisense strand of the second siRNA molecule.

55. The multi-target molecule according to any one of claims 51 to 53, wherein the first and second siRNAs independently regulate the gene expression of their respective target nucleic acids by at least 70% compared to the case where the first siRNA and the second siRNA are not part of the multi-target molecule.

56. The multi-target molecule according to any one of claims 51 to 55, wherein the first siRNA regulates the gene expression of a first target nucleic acid, and the second siRNA regulates the gene expression of a second nucleic acid.

57. The multi-target molecule according to claim 56, wherein the first target nucleic acid and the second target nucleic acid are the same.

58. The multi-target molecule according to claim 57, wherein the first siRNA and the second siRNA target the same nucleic acid sequence.

59. The multi-target molecule according to any one of claims 51 to 58, wherein the ligand is conjugated to one of the sense strands.

60. The multi-target molecule according to claim 59, wherein the ligand is conjugated at the 3' end of one of the sense strands.

61. The multi-target molecule according to claim 59, wherein the ligand is conjugated at one 5' end of the sense strand.

62. The multi-target molecule according to claim 59, wherein the ligand is conjugated at one 3' end of the antisense strand.

63. The multi-target molecule according to claim 59, wherein the ligand is conjugated at one 5' end of the antisense strand.

64. The multi-target molecule according to any one of claims 51 to 60, wherein the first siRNA molecule and the second siRNA molecule are linked to each other via a nucleotide-based linker.

65. The multi-target molecule according to claim 64, wherein the linker is single-stranded.

66. The multi-target molecule according to claim 64, wherein the linker is double-stranded.

67. The multi-target molecule according to claim 66, wherein the double-stranded linker contains a single-stranded region.

68. The multi-target molecule according to any one of claims 51 to 60, wherein the first siRNA molecule and the second siRNA molecule are linked to each other via a non-nucleotide-based linker.

69. The multi-target molecule according to any one of claims 64 to 68, wherein the linker connecting the two sense strands is a cleavable linker.

70. The multi-target molecule according to any one of claims 64 to 68, wherein the ligand is conjugated to the linker.

71. The multi-target molecule according to any one of claims 51 to 70, comprising at least one modification selected from the group consisting of modified nucleoside internucleotide linkages, modified nucleobases, modified sugars, and any combination thereof.

72. The multi-target molecule according to claim 71, wherein the at least one modification is included in the sense strand, the antisense strand, or the linker connecting the at least two siRNA molecules.

73. The multi-target molecule according to claim 71 or 72, wherein the linker comprises at least one nucleic acid modification selected from the group consisting of locked nucleic acids, 2'-O-alkyl nucleosides, 2'-halo nucleosides, 2'-amino nucleosides, 2'-S-alkyl nucleosides, abasic nucleosides, 2'-cyano nucleosides, 2'-mercapto nucleosides; 2'-MOE nucleosides, acryloyl nucleosides, S-cEt nucleosides, and any combination thereof.

74. The linker contains at least one modified nucleotide internucleoside bond selected from the group consisting of phosphodiester, phosphotriester, hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate, phosphorothioate, methylenemethylimino, thiodiester, thiocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, boranophosphate, borano phosphate ester, amide, hydroxyamino, siloxane, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal, formacetal, oxime, methyleneimino, methylenecarbonylamino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether, thioether, thioacetamide, and any combination thereof, the multi-target molecule according to any one of claims 51 to 73.

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