Bis-RNAi compounds for CNS delivery

Abstract

One embodiment of the present invention relates to a molecule that targets two or more target nucleic acid sequences and exhibits efficacy in tissues of the CNS of a subject upon contact. Another embodiment of the present invention relates to a circular small interfering RNA (sciRNA) for regulating one or more target mRNAs in the central nervous system (CNS) of a subject. Other embodiments of the present invention relate to pharmaceutical compositions, methods for inhibiting the expression of one or more target mRNAs in the CNS of a subject, and methods for treating or preventing a disease or disorder of the CNS in a subject using the molecule and sciRNA.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 219,930, filed July 9, 2021, and U.S. Provisional Patent Application No. 63 / 220,232, filed July 9, 2021, which are incorporated by reference herein in their entireties.

[0002] The present invention relates generally to the field of RNA interference using bis-RNAi compounds, which are particularly useful for modulating gene expression of multiple targets in cells and tissues of the central nervous system (CNS). [Background technology]

[0003] Chemical modifications of the nucleobase, ribose sugar, and phosphate backbone have been used to improve the drug-like properties of therapeutic oligonucleotides and confer favorable pharmacological performance to GalNAc-siRNA conjugates in preclinical and clinical development.

[0004] However, relatively few modifications have been made at the level of the three-dimensional structure of siRNA. A few examples of the above RNAi structures have been reported, including hairpin siRNA (Yu et al., "RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells," 99:6047-52 (2002)), dumbbell-shaped nanocircular siRNA (Abe et al., "Dumbbell-shaped nanocircular RNAs for RNA interference," J. Am. Chem. Soc., 129:15108-09 (2007)), siRNA nanosheets (Kim et al., "Generation of siRNA Nanosheets for Efficient RNA Interference," Sci. Rep., 6:25146 (2016)), branched siRNA (Avino et al., "Branched RNA: A new architecture for RNA interference," J. Nucleic Acids, 2011:586935 (2011)), and caged circular siRNA for photomodulation of gene expression (Zhang et al. al., “Caged circular siRNAs for photomodulation of gene expression in cells and mice,” Chem. Sci., 9:44-51 (2018)), circular single-stranded RNA as siRNA precursor (Kimura et al., “Intracellular build-up RNAi with single-strand circular RNAs as siRNA precursors,” Chem. Commun., 10.1039 / C1039CC04872C (2019)), and circular siRNA with reduced off-target effects (Abe et al., “Synthesis and characterization of small circular double-stranded RNAs,” Chem. Commun.,47:2125-2127(2011); Zhang et al., “Circular siRNAs for reducing off-target effects and enhancing long-term gene silencing in cells and mice,” Mol. Ther. - Nucleic Acids,10:237-44(2018)).

[0005] However, these reports have been based on natural ribonucleotides and phosphodiester bonds, lacked therapeutically relevant siRNA chemical modifications, employed inefficient circularization strategies such as peptide coupling or T4 ligation, and / or achieved only limited success. Furthermore, some of these reports have demonstrated that circularization of the antisense (guide) strand into circular siRNA abolishes silencing activity (see, e.g., Zhang et al., “Caged circular siRNAs for photomodulation of gene expression in cells and mice,” Chem. Sci., 9:44–51 (2018)). This is likely because the circular antisense strand cannot be loaded onto the Argonaute 2 (Ago2) protein, preventing the formation of an active RNA-induced silencing complex (RISC), the driving component of RNA interference-mediated mRNA silencing. Furthermore, some of these reports also discourage the use of chemical modifications, such as 2'OMeRNA, locked nucleic acids, unlocked nucleoside analogs, 5-nitroindole-modified nucleotides, terminal methylation, and backbone phosphothioates, suggesting that these modifications may inhibit the loading and processing of the sense strand RNA to reduce siRNA off-target effects (see, e.g., Zhang et al., "Circular siRNAs for reducing off-target effects and enhancing long-term gene silencing in cells and mice," Mol. Ther. - Nucleic Acids, 10:237-44 (2018)). Reports describing in vivo applications do not address systemic administration or targeted exogenous gene expression, nor do they address therapeutically relevant delivery agents used for locally administered high-dose siRNA.

[0006] Thus, there is a continuing need for new and improved designs of three-dimensional siRNA duplex structures that achieve and enhance the therapeutic capabilities, e.g., increased potency, metabolic stability, and off-target performance, of RNAi agents, particularly molecules that can modulate gene expression of multiple target nucleic acids while achieving efficient delivery and efficacy in one or more tissues, particularly cells or tissues of the central nervous system (CNS). There is also a need in the art for molecules that can target multiple target nucleic acids while achieving efficient delivery and efficacy in one or more tissues of the central nervous system (CNS) of a subject. Summary of the Invention

[0007] The present disclosure provides molecules designed to target multiple target nucleic acids, or to target the same target nucleic acid more than once within the same agent, or to target two or more distinct target RNA sequences within one or more target nucleic acids, and that exhibit surprising efficacy upon contact with CNS tissue of a subject. Pharmaceutical compositions, methods, and other related aspects are also provided.

[0008] One embodiment of the invention provides a nucleic acid composition for modulating one or more target RNAs in a subject's central nervous system (CNS), the nucleic acid composition comprising one or more distinct RNA sequences, the nucleic acid composition having a first double-stranded RNA (dsRNA) molecule and a single-stranded nucleic acid agent or a second dsRNA molecule, the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule being connected together by a linker and not overlapping with each other, the first dsRNA comprising at least one conjugated lipophilic moiety, and, if present, the second dsRNA molecule comprising at least one conjugated lipophilic moiety, and each of the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule of the nucleic acid composition being capable of modulating the activity or expression of one or more target RNAs in CNS tissue of the subject by at least 15% relative to a suitable control.

[0009] In certain embodiments, the first dsRNA molecule and the second dsRNA molecule are connected together by a linker.

[0010] In certain embodiments, the first dsRNA molecule and the single-stranded nucleic acid agent are connected together by a linker.

[0011] In certain embodiments, the single-stranded nucleic acid agent is an inhibitory single-stranded oligonucleotide or a single-stranded small interfering RNA (ss-siRNA).

[0012] In certain embodiments, the nucleic acid composition is capable of inhibiting the activity or expression of one or more target RNAs in a CNS tissue of a subject. Optionally, the nucleic acid composition inhibits the activity or expression of one or more distinct target RNAs in a CNS tissue of a subject.

[0013] In some embodiments, the nucleic acid composition is capable of inhibiting the activity or expression of two or more target RNAs in a CNS tissue of a subject. Optionally, the nucleic acid composition inhibits the activity or expression of two or more distinct target RNAs in a CNS tissue of a subject.

[0014] In certain embodiments, a single stranded nucleic acid agent includes at least one conjugated lipophilic moiety.

[0015] In some embodiments, the multi-target molecule does not regulate gene expression by two different mechanisms.

[0016] In certain embodiments, each nucleic acid-based effector molecule in a multi-target molecule can regulate gene expression of a target nucleic acid. Without limitation, each effector molecule in a multi-target molecule can be directed to the same target gene, different target genes, different locations within the same target gene, or different transcripts of the same target gene. Furthermore, the effector molecules included in the multi-target molecules disclosed herein can include any of the nucleic acid modifications, motifs, or structures described herein or known in the art.

[0017] Furthermore, effector molecules contained in the multi-target molecules described herein have comparable gene expression modulating activity compared to the gene expression modulating activity of the effector molecule when not part of the multi-target molecule. In other words, an effector molecule has similar gene expression modulating activity when it is part of a multi-target molecule disclosed herein compared to when it is not part of the multi-target molecule. In some embodiments, effector molecules contained in the multi-target molecules described herein can independently modulate gene expression of each target nucleic acid by at least 15%, optionally at least 20%, optionally at least 25%, optionally at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% (e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) compared to gene expression modulation when not part of the multi-target molecule. In some embodiments, one of the effector molecules in the multi-target molecule modulates gene expression at a higher level compared to other effector molecules in the multi-target molecule. In some embodiments, at least two effector molecules in the multi-target molecule modulate gene expression at similar levels (eg, 10%, 7.5%, 5%, 2.5% or less of each other).

[0018] In some embodiments, each effector molecule of a multi-targeting molecule of the present disclosure is capable of inhibiting expression of a target mRNA in the CNS of a subject by at least 15% relative to a suitable control (e.g., compared to an untreated or placebo-treated subject; or compared to a reference value comprising the level of target mRNA or protein in a treated subject, e.g., measured before treatment with the multi-targeting molecule is administered). In related embodiments, a multi-targeting molecule of the present disclosure is capable of inhibiting expression of a target mRNA in the CNS of a subject by at least 20%, optionally at least 25%, optionally at least 30%, optionally at least 35%, optionally at least 40%, optionally at least 45%, optionally at least 50%, optionally at least 55%, optionally at least 60%, optionally at least 65%, optionally at least 70%, optionally at least 75%, optionally at least 80%, optionally at least 85%, optionally at least 90%, and optionally at least 95% relative to a suitable control.

[0019] In some embodiments, the multi-targeting molecule modulates gene expression of at least two target nucleic acids by at least 75%, respectively, compared to when the effector molecules are not linked together.

[0020] In some embodiments, one of the at least two effector molecules regulates gene expression of a first target nucleic acid and another of the at least two effector molecules regulates gene expression of a second nucleic acid. In certain embodiments, the first target nucleic acid and the second target nucleic acid are located on different transcripts or in different genes. In some embodiments, the first target nucleic acid and the second target nucleic acid are located in the same nucleic acid.

[0021] In related embodiments, the multi-targeting molecule can inhibit expression of a target mRNA throughout a subject's CNS or within a location within the subject's CNS. In certain embodiments, the multi-targeting molecule can inhibit expression of a target mRNA in one or more of the following CNS locations of a subject: right hemisphere, left hemisphere, cerebellum, striatum, brainstem, and spinal cord. In some embodiments, CNS cell types are specifically targeted, including neurons, oligodendrocytes, microglia, and astrocytes.

[0022] It has been found herein that a multi-targeting molecule conjugated with at least one lipophilic ligand on each effector molecule / component is particularly effective in regulating gene expression. Accordingly, in some embodiments, at least two lipophilic ligands are conjugated to the multi-targeting molecule (distributed such that at least one lipophilic ligand is located on each effector molecule / component). The two ligands may be independently conjugated to any position in the multi-targeting molecule, provided that each effector molecule / component carries a lipophilic ligand. In such embodiments, at least two effector molecules in the multi-targeting molecule have at least one lipophilic ligand attached thereto. Therefore, a multi-targeting molecule conjugated with at least two lipophilic ligands is also referred to herein as a "conjugated multi-targeting molecule." Without limitation, each ligand may be located at any position on the effector molecule and / or the multi-targeting molecule. For example, each ligand can be conjugated at the 5'-terminus, 3'-terminus, internal (non-terminal) position of the effector molecule, or a combination thereof in the multi-targeting molecule. The at least two ligands can be the same, different, or any combination of the same and different. Without wishing to be bound by theory, it has surprisingly been determined that including at least one lipophilic ligand in each effector molecule / component improves the delivery or pharmacokinetic profile of the conjugated multi-targeting molecule when administered to the CNS of a subject, whereas testing of multi-targeting molecules bearing only one lipophilic ligand has not determined the effector molecule to be effective.

[0023] In some embodiments, provided herein is a multi-target molecule for modulating two or more distinct target RNAs in a subject's central nervous system (CNS), the multi-target molecule having a first double-stranded RNA (dsRNA) molecule and a second double-stranded RNA molecule, wherein: the first dsRNA molecule and the second dsRNA molecule are connected together by a linker and do not overlap with each other, and the first dsRNA and the second dsRNA each comprise at least one conjugated lipophilic moiety, and the multi-target molecule is capable of inhibiting the activity or expression of the two or more distinct target RNAs, each by at least 15%, in the subject's CNS tissue, relative to a suitable control.

[0024] In certain embodiments, the lipophilicity of each lipophilic moiety is determined by the log K ow is measured and exceeds zero.

[0025] In one embodiment, the hydrophobicity of the multiple target molecule, as measured by the unbound fraction in a plasma protein binding assay of the multiple target molecule, is greater than 0.2.

[0026] In some embodiments, each lipophilic moiety is one or more of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0027] In certain embodiments, at least one lipophilic moiety is a saturated or unsaturated C4-C 30 It contains a hydrocarbon chain and an optional functional group that is a hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne. Optionally, at least one lipophilic moiety is a saturated or unsaturated C-C 18Optionally, at least one lipophilic moiety comprises a saturated or unsaturated C 16 or C 22 Contains a hydrocarbon chain.

[0028] In some embodiments, each lipophilic moiety is a saturated or unsaturated C4-C 30 Contains a hydrocarbon chain and an optional functional group that is a hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne. Optionally, each lipophilic moiety is a saturated or unsaturated C-C 18 Optionally, each lipophilic moiety comprises a saturated or unsaturated C 16 or C 22 Contains a hydrocarbon chain.

[0029] In some embodiments, at least one lipophilic moiety is conjugated to multiple targeting molecules via a monovalent linker, or a branched bivalent or trivalent linker.

[0030] In one embodiment, at least one lipophilic moiety is conjugated to one or more nucleotides in the multi-targeting molecule, as shown in formula (I) below: [ka] wherein B is a nucleotide base or nucleotide base analog, the n-hexadecyl chain (C16 ligand) is a lipophilic moiety, and optionally B is adenine, guanine, cytosine, thymine, or uracil. The modification shown in formula (I) is referred to herein as "2'-C16."

[0031] In some embodiments, one or more non-terminal nucleotide positions in the sense strand of the first dsRNA molecule and the second dsRNA molecule have the 2'-C16 structure of Formula (I), where B is a nucleotide base or nucleotide base analog, optionally B is adenine, guanine, cytosine, thymine, or uracil, and the n-hexadecyl chain is a lipophilic moiety. Optionally, one or more non-terminal nucleotide positions in the sense strand of the first dsRNA molecule and one or more non-terminal nucleotide positions in the sense strand of the second dsRNA molecule, if present, have the following structure: [ka] wherein B is a nucleotide base or nucleotide base analog, optionally B is adenine, guanine, cytosine, thymine, or uracil, and the n-hexadecyl chain is a lipophilic moiety.

[0032] In some embodiments, the multi-target molecule comprises a first effector molecule that is an RNAi agent and a second effector molecule that is an RNAi agent.In certain embodiments, the first effector molecule is a first dsRNA, and the second effector molecule is a second dsRNA.Optionally, the first effector molecule is a first double-stranded siRNA molecule, and the second effector molecule is a second double-stranded siRNA molecule.

[0033] In some embodiments, the sense strand of the first dsRNA is covalently linked to the sense strand of the second dsRNA.Alternatively, the sense strand of the first dsRNA is covalently linked to the antisense strand of the second dsRNA.Furthermore, the antisense strand of the first dsRNA is covalently linked to the sense strand of the second dsRNA.

[0034] In certain embodiments, each dsRNA comprises a lipophilic ligand, for example, a C16 ligand (also referred to herein as "2'-C16" as described above), and the ligand is conjugated to a residue that is six nucleotides from the 5'-end of the sense strand of the dsRNA (i.e., when counting nucleotide residues from the 5'-end of the sense strand, the 5'-end nucleotide is nucleotide number 1, and the lipophilic ligand is attached to nucleotide number 6). In alternative embodiments, the lipophilic ligand is optionally conjugated to the 3'-end of the sense strand via a monovalent linker, or a branched bivalent or trivalent linker. It is specifically anticipated that the lipophilic ligand can be included in or conjugated to any of the nucleotide positions of the multiple target molecules provided in the present application.

[0035] In certain embodiments, at least one lipophilic moiety / ligand is an aliphatic, alicyclic, or polyalicyclic compound. Optionally, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0036] In some embodiments, the lipophilic moiety is a saturated or unsaturated C4-C 30 It contains a hydrocarbon chain and an optional functional group that is a hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

[0037] In certain embodiments, at least one lipophilic moiety / ligand is a saturated or unsaturated C6-C 18Optionally, the lipophilic moiety / ligand comprises a saturated or unsaturated C 16 It contains a hydrocarbon chain. In related embodiments, at least one lipophilic moiety / ligand is conjugated via a carrier that replaces one or more nucleotides of the multiple targeting molecule. In certain embodiments, the carrier is a cyclic group that is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl, or is an acyclic moiety of a serinol backbone or a diethanolamine backbone system.

[0038] In some embodiments, a lipophilic moiety is independently conjugated to the sixth position of the sense strand of each dsRNA molecule, counting from the 5' end of the sense strand of each dsRNA molecule, and optionally the lipophilic moiety is a saturated or unsaturated C 16 or C 22 The lipophilic moiety comprises a hydrocarbon chain, and optionally the lipophilic moiety comprises a saturated or unsaturated C 16 or C 22 It is a hydrocarbon chain.

[0039] In some embodiments, the lipophilic moiety is conjugated via a bio-cleavable linker. Optionally, the bio-cleavable linker is or includes DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized mono- or oligosaccharides, or combinations thereof.

[0040] In some embodiments, saturated or unsaturated C 16 The hydrocarbon chain is conjugated to the sixth position counting from the 5' end of the chain.

[0041] In certain embodiments, the sense strand of at least one of the at least two dsRNAs is 21 nucleotides in length.

[0042] In some embodiments, the first dsRNA and the second dsRNA each have a sense strand length of 19 to 30 nucleotides. Optionally, the first dsRNA and the second dsRNA each have a sense strand length of 21 to 25 nucleotides. Optionally, the first dsRNA and the second dsRNA each have a sense strand length of 21 nucleotides.

[0043] In some embodiments, the first dsRNA and the second dsRNA each have an antisense strand 19-30 nucleotides in length. Optionally, the first dsRNA and the second dsRNA each have an antisense strand 21-25 nucleotides in length. Optionally, the first dsRNA and the second dsRNA each have an antisense strand 23 nucleotides in length. Optionally, the 3' end of the antisense strand forms a two-nucleotide 3' overhang relative to the 5' end of the sense strand.

[0044] In some embodiments, one or more lipophilic moieties are conjugated to one or more of the following internal (non-terminal) positions of one or more dsRNAs: non-terminal positions except positions 9-12 of the sense strand, and all non-terminal positions of the antisense strand. Optionally, one or more lipophilic moieties are conjugated to one or more of the following internal (non-terminal) positions of one or more dsRNAs: positions 4-8 and 13-18 of the sense strand, and positions 6-10 and 15-18 of the antisense strand, counting from the 5'-end of each strand. Optionally, one or more lipophilic moieties are conjugated to one or more of the following non-terminal positions: positions 5, 6, 7, 15, and 17 of the sense strand, and positions 15 and 17 of the antisense strand, counting from the 5'-end of each strand. In certain embodiments, the sense strand is 21 nucleotides in length, and the antisense strand is 23 nucleotides in length.

[0045] Optionally, the lipophilic moiety is conjugated to position 21, 20, 15, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0046] In certain embodiments, the lipophilic moiety is conjugated to position 21, 20, 15, or 6 of the sense strand.

[0047] In some embodiments, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

[0048] In some embodiments, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

[0049] In some embodiments, a lipophilic moiety is conjugated to position 6 of each sense strand.

[0050] In some embodiments, the lipophilic moiety is conjugated to position 16 of the antisense strand.

[0051] In some embodiments, the lipophilic moiety is conjugated to the dsRNA of the multiple target molecule via a linker containing an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction, or a carbamate.

[0052] In one embodiment, the lipophilic moiety / ligand is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0053] In one embodiment, the multiple target molecule comprises at least one modified nucleotide that is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), or a nucleotide comprising a vinyl phosphonate. Optionally, the multiple target molecule, or each dsRNA of the multiple target molecule, comprises at least one of each of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), and a nucleotide comprising a vinyl phosphonate.

[0054] In a related embodiment, each dsRNA of the multiple target molecule comprises at least one modified nucleotide that is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide containing glycol nucleic acid (GNA), or a nucleotide containing vinyl phosphonate. Optionally, the multiple target molecule comprises at least one of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-C16 moiety, and a phosphorothioate internucleoside linkage.

[0055] In some embodiments, each dsRNA further comprises at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. Optionally, each dsRNA comprises two to eight phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages. Optionally, the phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage is located at the last and penultimate internucleoside linkages in each dsRNA at one or more of the following positions: the 5'-end of the sense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, the 3'-end of the antisense strand, and combinations thereof. Optionally, each dsRNA comprises six phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages located at the last and penultimate internucleoside linkages at the 5'-end of the sense strand, the 3'-end of the sense strand, and the 3'-end of the antisense strand.

[0056] In certain embodiments, all or substantially all of the nucleotides of each dsRNA are 2'-O-methyl modified, 2'-fluoro modified, or 2'-C6-C 18 It includes at least one modification which is a hydrocarbon chain modification.

[0057] In another embodiment, the multi-target molecule comprises the patterns of modified nucleotides provided herein (e.g., Figures 3A, 4A, 5A, 6A, 7A, 8A, and 9A), optionally with the positions of the 2'-C16 (or other lipophilic moiety / ligand), 2'-O-methyl, phosphorothioate, and 2'-fluoro modifications independent of the individual nucleotide base sequences of the presented RNAi agents.

[0058] In some embodiments, the sense strand of the first dsRNA has a 5' end and is connected at its 3' end to a linker, which is connected to the 5' end of the single-stranded nucleic acid agent or the second dsRNA molecule. Optionally, the linker is connected to the 5' end of the sense strand of the single-stranded nucleic acid agent or the second dsRNA molecule.

[0059] In certain embodiments, the sense strand of the dsRNA is 21 nucleotides in length and is connected by a linker.

[0060] In some embodiments, the linker that connects the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is a nucleic acid linker, or a carbohydrate linker or other organic polymer linker.Optionally, the linker is cleavable.

[0061] In certain embodiments, the linker connecting the effector molecules comprises one or more of the following: -(CH2) 12 -("C12 linker" or "Q50"), -(CH2)6-SS-(CH2)6- ("C6-SS-C6 linker" or "Q51"), ·Q151, ·Q173, -CH2CH2O-(CH2CH2) n -CH2CH2O-CH2CH2O-, where n is 0 or 1 to 20; -(CH2)9-(CH2) n -CH2-, where n is 0 or 1 to 20; mono-, di-, tri-, tetra-, penta-, or poly-prolinol, optionally conjugated to a ligand; or mono-, di-, tri-, tetra-, penta-, or poly (e.g., mono-, di-, tri-, tetra-, penta-, or poly-[4-hydroxyprolinol]) optionally conjugated to a ligand.

[0062] In some embodiments, the linker connecting the effector molecule (e.g., the dsRNA, or the first dsRNA and the single-stranded nucleic acid agent) is an organic polymer linker, such as, for example, an aliphatic saturated or unsaturated alkyl chain, a (poly)ethylene glycol chain including diethylene glycol, triethylene glycol, tetra-, penta-, hexa-, hepta-, octa-, nona-, and / or deca-ethylene glycol.

[0063] In certain embodiments, the linker is a biocleavable linker that is or comprises DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, or mannose functionalized mono- or oligosaccharides, or a combination thereof. In some embodiments, the biocleavable linker is a combination of organic polymer linkers, such as, for example, aliphatic saturated or unsaturated alkyl chains, (poly)ethylene glycol chains (including diethylene glycol, triethylene glycol, tetra-, penta-, hexa-, hepta-, octa-, nona-, and / or deca-ethylene glycol), and / or comprises DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, or mannose functionalized mono- or oligosaccharides, or a combination thereof.

[0064] In certain embodiments, the organic polymer linker comprises one or more of the following: aliphatic saturated or unsaturated alkyl chains, as well as diethylene glycol, triethylene glycol, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-ethylene glycol, and glycerol, and / or aminoalkyl ethers thereof.

[0065] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) comprises a moiety selected from the group consisting of: [ka]

[0066] In some embodiments, the organic polymer linker comprises DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, or mannose functionalized mono- or oligosaccharides, or combinations thereof.

[0067] In certain embodiments, the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is selected from the following: -(CH2) 12 -(C12 linker or Q50), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker or Q51), Q151, Q173, -CH2CH2O-(CH2CH2) n -CH2CH2O-CH2CH2O-, where n is 0 or 1 to 20; -(CH2)9-(CH2) n -CH2-, where n is 0 or 1 to 20; mono-, di-, tri-, tetra-, penta-, or poly-prolinol, optionally conjugated to a ligand; mono-, di-, tri-, tetra-, penta-, or poly-hydroxyprolinol, optionally conjugated to a ligand.

[0068] In some embodiments, the biocleavable linker is selected from the following: [ka] where n=1-12 and m=1-12, various carbohydrates (galactose, galactosamine, glucose, glucosamine, mannose, mannosamine derivatives, or pentose derivatives). [ka] where n=1-12 and m=1-12, various modified carbohydrates (galactose, galactosamine, glucose, glucosamine, mannose, mannosamine derivatives, or pentose derivatives). [ka] where n=1-12 and m=1-12, various modified carbohydrates (disaccharides or trisaccharides of galactose, galactosamine, glucose, glucosamine, mannose, mannosamine derivatives). [ka] where n=1-12 and m=1-12, various modified carbohydrates (disaccharides or trisaccharides of galactose, galactosamine, glucose, glucosamine, mannose, mannosamine derivatives).

[0069] In certain embodiments, the linker is a polynucleotide. Optionally, the linker is a polynucleotide comprising a deoxyribonucleotide sequence, a ribonucleotide sequence, or both. Optionally, the linker is a polynucleotide having a modified ribonucleotide sequence. Optionally, the linker is a polynucleotide having one or more of the following modifications: 2'-O-methyl ribonucleotides or 2'-fluoro-ribonucleotides; 2'-5'-linked nucleotides having 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro). Optionally, the linker is a polynucleotide having one or more of the following modifications: glycol nucleic acid (GNA), locked nucleic acid (LNA), hexanol nucleic acid (HNA), abasic ribose, abasic deoxyribose, abasic hydroxyprolinol. Optionally, all nucleic acid linker nucleotides are the same type of nucleotide. Optionally, the linker is comprised entirely of 2'-O-methyl nucleotides, entirely of 2'-fluoro nucleotides, or entirely of deoxyribonucleotides.

[0070] In some embodiments, the linker is n nucleotides in length. Optionally, the length of the longest strand of a multiple target molecule (i.e., the first strand, e.g., the combined / linked sense strands of each dsRNA) is equal to the length of the first dsRNA + n + the length of the second dsRNA. Optionally, if there are only two dsRNAs, each with a sense strand 21 nucleotides in length, connected by a polynucleotide linker n nucleotides in length, the total length of the longest strand of a multiple target molecule (i.e., the first strand) is 42 + n nucleotides. In certain embodiments, the polynucleotide linker is two or more nucleotides in length. Optionally, the polynucleotide linker is three or more nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length). In certain embodiments, the linker connecting the dsRNAs is a nucleic acid linker 1 to 15 nucleotides in length. Optionally, the linker is two to five nucleotides in length. Optionally, the linker is three or four nucleotides in length. Optionally, the linker is three nucleotides in length. In a related embodiment, the total length of the longest strand (i.e., the first strand) of the multi-target molecule is 45 nucleotides.

[0071] In certain embodiments, the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule comprises one or more of the following sequences: UUU, 2'-O-methyl-UUU (uuu), and 2'-fluoro-UUU (UfUfUf), and (dT)n, where n is 1 to 20, e.g., dTdTdT.

[0072] In some embodiments, the multi-target molecule comprises two nucleic acid dsRNAs, wherein the sense strand of each dsRNA is 21 nucleotides in length, the antisense strand of each dsRNA is 23 nucleotides in length, the linker connecting the dsRNAs is a three-nucleotide long nucleic acid linker connecting the sense strands of each dsRNA, and a lipophilic moiety is conjugated to position 6 of the sense strand of each dsRNA.

[0073] In some embodiments, the linker connecting two siRNAs comprises the nucleotide sequence UUU or (dT)n, where n is 1 to 20. In certain embodiments, the linker connecting the dsRNAs comprises one or more of the following sequences: dTdTdT, UUU, 2'-O-methyl-UUU(uuu), and 2'-fluoro-UUU(UfUfUf).

[0074] In certain embodiments, the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is a polynucleotide having a modified ribonucleotide sequence.Optionally, the linker is a polynucleotide having one or more of the following modifications: 2'-O-methyl ribonucleotide modification, 2'-fluoro-ribonucleotide modification, 2'-5'-linked nucleotides with different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, hexanol nucleic acid (HNA) modification, abasic ribose modification, abasic deoxyribose modification, and abasic hydroxyprolinol modification.

[0075] In some embodiments, all nucleic acid nucleotides of the linker that connects the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule are the same type of nucleotide.Optionally, the linker comprises entirely 2'-O-methyl nucleotides, entirely 2'-fluoro nucleotides, or entirely deoxyribonucleotides.

[0076] In some embodiments, the linker that connects the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is an endosomal cleavage linker or a protease cleavage linker.Optionally, the linker is a carbohydrate linker, and the linker is cleaved at least 1.25 times faster in cells (or under in vitro conditions that are selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions that are selected to mimic extracellular conditions).

[0077] In some embodiments, the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is selected from the following: [ka] [ka] [ka] [ka] [ka] [ka] wherein n=1 to 12, and m=1 to 12; [ka] [ka] (Y195), and [ka] (Y254).

[0078] In certain embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via phosphodiester bonds.

[0079] In some embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via a phosphotriester bond.

[0080] In certain embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via a phosphotriester bond, and the linking phosphorus atom is in either the Rp or Sp configuration.

[0081] In some embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via phosphorothioate diester bonds.

[0082] In certain embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via phosphorothioate diester bonds, with the linking phosphorus atom being in either the Rp or Sp configuration.

[0083] In some embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via phosphoramidate diester bonds.

[0084] In certain embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via phosphoramidate diester bonds, and the linking phosphorus atom is in either the Rp or Sp configuration.

[0085] In some embodiments, the nucleotide and / or non-nucleotide linkers are connected to the oligonucleotide chain via disulfide bonds.

[0086] In certain embodiments, the antisense strand of at least one dsRNA is 23 nucleotides in length.Optionally, each antisense strand of dsRNA is 23 nucleotides in length.In some embodiments, the last nucleotide and the penultimate nucleotide at the 3'-end of the antisense strand are not base-paired with the sense strand oligonucleotide, and optionally form a 3' overhang with respect to the 5'-end of the corresponding sense strand dsRNA.

[0087] In another embodiment, the multi-targeting molecule, or the effector of the multi-targeting molecule, further comprises a phosphate or phosphate mimetic at the 5'-end of the antisense strand. Optionally, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0088] In some embodiments, the multi-targeting molecule comprises a targeting ligand, e.g., a hydrophobic ligand, that targets a receptor that mediates delivery to CNS tissue. In certain embodiments, the targeting ligand is a C16 ligand.

[0089] In some embodiments, the multi-targeting molecule comprises a targeting ligand that targets brain tissue, such as the striatum.

[0090] In certain embodiments, the CNS tissue of interest is the right hemisphere, left hemisphere, cerebellum, striatum, brainstem, and / or spinal cord.

[0091] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker that is DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized mono- or oligosaccharide, or a combination thereof.

[0092] In some embodiments, one or more lipophilic moieties are conjugated to the nucleic acid composition by a linker containing a compound selected from the group consisting of an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction, or a carbamate.

[0093] In some embodiments, one or more lipophilic moieties are conjugated to positions in the nucleic acid composition selected from the group consisting of a nucleobase, a sugar moiety, and an internucleotide linkage.

[0094] In certain embodiments, the multi-targeted molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs in the subject's CNS tissue by at least 20%, each compared to a suitable control. Optionally, the multi-targeted molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs in the subject's CNS tissue by at least 25%, each compared to a suitable control. Optionally, the multi-targeted molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs in the subject's CNS tissue by at least 30%, each compared to a suitable control. Optionally, the multi-targeted molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs in the subject's CNS tissue by at least 35%, each compared to a suitable control. Optionally, the multi-targeted molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs in the subject's CNS tissue by at least 40%, each compared to a suitable control. Optionally, the multi-targeted molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs in the subject's CNS tissue by at least 45%, each compared to a suitable control. Optionally, the multi-targeting molecule is capable of inhibiting the activity or expression of one or more distinct target RNAs, each by at least 50%, in a CNS tissue of a subject, relative to a suitable control.

[0095] In a related embodiment, a suitable control is an untreated subject.

[0096] In some embodiments, a suitable control is a baseline value. Optionally, the baseline value is a value obtained for a subject prior to administration of the multi-target molecule to the subject.

[0097] In certain embodiments, the multi-targeted molecule is formulated for intracerebroventricular (ICV) administration.

[0098] In some embodiments, one or more distinct target RNAs are mRNA. Optionally, two or more distinct target RNAs are mRNA.

[0099] In some embodiments, one or more distinct target RNAs are transcripts of genes associated with a disease or disorder of the CNS. Optionally, two or more distinct target RNAs are transcripts of genes associated with a disease or disorder of the CNS.

[0100] In some embodiments, the 3' end of the sense strand of the multiple-target molecule is protected via an end-cap that is an amine-bearing cyclic group, wherein the cyclic group is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0101] In some embodiments, the multiple-target molecule further comprises: a terminal chiral modification at the first internucleotide linkage at the 3' end of the antisense strand of one or more dsRNAs, the terminal chiral modification having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first internucleotide linkage at the 5' end of the antisense strand of one or more dsRNAs, the terminal chiral modification having a linking phosphorus atom in the Rp configuration; or a terminal chiral modification at the first internucleotide linkage at the 5' end of the sense strand of one or more dsRNAs, the terminal chiral modification having a linking phosphorus atom in either the Rp or Sp configuration.

[0102] In some embodiments, the multiple-target molecule further comprises: a terminal chiral modification at the first and second internucleotide linkages at the 3' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first internucleotide linkage at the 5' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Rp configuration; or a terminal chiral modification at the first internucleotide linkage at the 5' end of the sense strand of one or more dsRNAs, having a linking phosphorus atom in either the Rp or Sp configuration.

[0103] In certain embodiments, the multiple-target molecule further comprises: a terminal chiral modification at the first, second, and third internucleotide linkages at the 3' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first internucleotide linkage at the 5' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Rp configuration; or a terminal chiral modification at the first internucleotide linkage at the 5' end of the sense strand of one or more dsRNAs, having a linking phosphorus atom in either the Rp or Sp configuration.

[0104] In some embodiments, the multiple-target molecule further comprises: a terminal chiral modification at the first and second internucleotide linkages at the 3' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the third internucleotide linkage at the 3' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Rp configuration; a terminal chiral modification at the first internucleotide linkage at the 5' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Rp configuration; or a terminal chiral modification at the first internucleotide linkage at the 5' end of the sense strand of one or more dsRNAs, having a linking phosphorus atom in either the Rp or Sp configuration.

[0105] In some embodiments, the multiple-target molecule further comprises: a terminal chiral modification at the first and second internucleotide linkages at the 3' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first and second internucleotide linkages at the 5' end of the antisense strand of one or more dsRNAs, having a linking phosphorus atom in the Rp configuration; or a terminal chiral modification at the first internucleotide linkage at the 5' end of the sense strand of one or more dsRNAs, having a linking phosphorus atom in either the Rp or Sp configuration.

[0106] In certain embodiments, the nucleic acid composition comprises three or more linked dsRNAs, single-stranded nucleic acid agents, or combinations thereof.

[0107] Another aspect of the present disclosure provides a method for modulating one or more target RNAs in a central nervous system (CNS) of a subject, the target RNAs having one or more distinct target RNA sequences, the method comprising contacting CNS cells of the subject with a multi-target molecule having at least two nucleic acid-based effector molecules (at least one of which is dsRNA), the effector molecules being connected together by a linker and not overlapping with one another, each of the at least two effector molecules that are dsRNAs comprising at least one conjugated lipophilic moiety, wherein the multi-target molecule inhibits the activity or expression of one or more target RNAs in the CNS of the subject by at least 15% relative to a suitable control.

[0108] A further aspect of the present disclosure provides a method for treating or preventing a disease or disorder of the CNS in a subject having or at risk of developing such a disease or disorder, the method comprising administering a multi-targeted molecule disclosed herein to the CNS of the subject, thereby treating the subject.

[0109] Examples of CNS diseases or disorders that may be treated or prevented using the compositions or methods of the present disclosure include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease (PD), Alzheimer's disease, early-onset familial Alzheimer's disease (EOFAD), cerebral amyloid angiopathy (CAA), spinal muscular atrophy (SMA), Angelman syndrome, ataxic / neurodegenerative disorders of the nervous system (e.g., Friedreich's ataxia), Huntington's disease (Huntington's chorea), multiple sclerosis, amyotrophic lateral sclerosis (ALS)), depression, Down's syndrome, psychosis, schizophrenia, Creutzfeldt-Jakob disease, multiple system atrophy, dementia with Lewy bodies (LBD), pure autonomic dysfunction (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig disease, and tangle predominant dementia. dementia, argyrophilic granule disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, ribofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, vascular disorders (e.g., stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage), infections (e.g., meningitis, encephalitis, polio, epidural abscess), structural disorders (e.g., brain or spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, Guillain-Barré syndrome), and functional disorders (e.g., headache, epilepsy, dizziness, neuralgia). Other diseases or disorders of the CNS that may be treated or prevented using the compositions or methods of the present disclosure include, but are not limited to, spinal muscular atrophy (SMA), Angelman syndrome, and ataxic / neurodegenerative disorders of the nervous system (e.g., Friedreich's ataxia).

[0110] In one embodiment, treatment includes amelioration of at least one sign or symptom of a disease or disorder.

[0111] In certain embodiments, treatment includes prevention of the progression of a disease or disorder.

[0112] One aspect of the present invention provides a pharmaceutical composition for inhibiting expression of one or more target genes having one or more distinct target RNA sequences, wherein at least one target gene is associated with a disease or disorder of the CNS, the pharmaceutical composition being formulated for administration to the CNS of a subject and comprising a multi-target molecule of the present disclosure and a pharmaceutically acceptable carrier.

[0113] In some embodiments, the present disclosure provides an injectable formulation formulated for CNS delivery comprising a pharmaceutical composition of the present disclosure.

[0114] A further aspect of the present disclosure provides a method for inhibiting expression of a target gene associated with a CNS disease or disorder in a CNS cell, the method comprising: (a) contacting the cell with a multi-targeted molecular agent of the present disclosure or a pharmaceutical composition of the present disclosure; and (b) maintaining the cell produced in step (a) for a period of time sufficient to allow degradation of mRNA transcripts of the target gene associated with a CNS disease or disorder, thereby inhibiting expression of the target gene associated with a CNS disease or disorder in the cell.

[0115] In one embodiment, the cell is in a subject. Optionally, the subject is a human.

[0116] In certain embodiments, the subject is a mammal. Optionally, the subject is a rhesus monkey, a cynomolgus monkey, a mouse, or a rat.

[0117] In some embodiments, expression of each target gene associated with a disease or disorder of the CNS is inhibited by at least 15%, optionally at least 20%, optionally at least 25%, optionally at least 30%, optionally at least 35%, optionally at least 40%, optionally at least 45%, optionally at least 50%.

[0118] In certain embodiments, the subject meets at least one diagnostic criterion for a disease or disorder of the CNS.

[0119] In certain embodiments, the human subject is diagnosed with a neurodegenerative disorder (e.g., Parkinson's disease (PD), Alzheimer's disease, early-onset familial Alzheimer's disease (EOFAD), cerebral amyloid angiopathy (CAA), spinal muscular atrophy (SMA), Angelman syndrome, ataxic / neurodegenerative disorders of the nervous system (e.g., Friedreich's ataxia), Huntington's disease (Huntington's chorea), multiple sclerosis, amyotrophic lateral sclerosis (ALS)), depression, Down's syndrome, psychosis, schizophrenia, Creutzfeldt-Jakob disease, multiple system atrophy, dementia with Lewy bodies (LBD), pure autonomic dysfunction (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig disease, tangle predominant dementia dementia, argyrophilic granule disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, ribofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, vascular disorders (e.g., stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage), infections (e.g., meningitis, encephalitis, polio, epidural abscess), structural disorders (e.g., brain or spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, Guillain-Barré syndrome), and functional disorders (e.g., headache, epilepsy, dizziness, neuralgia).

[0120] In some embodiments, the contacting step comprises administering to the subject an intrathecal injection or an intracerebroventricular (ICV) injection.

[0121] In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent or therapy. Exemplary additional therapeutic agents and therapies include, for example, sedatives, antidepressants, clonazepam, sodium valproate, analgesics, antiepileptic drugs, cholinesterase inhibitors, memantine, benzodiazepines, levodopa, COMT inhibitors (e.g., tolcapone and entacapone), dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, and lisuride), Treatments include MAO-B inhibitors (e.g., safinamide, selegiline, and rasagiline), surgery, amantadine, anticholinergics, modafinil, pimavanserin, doxepin, rasagline, antipsychotics, atypical antipsychotics (e.g., amisulpride, olanzapine, risperidone, and clozapine), riluzole, edaravone, deep brain stimulation, noninvasive ventilation (NIV), invasive ventilation, physical therapy, occupational therapy, speech therapy, dietary changes and swallowing techniques, feeding tubes, PEG tubes, probiotics, and psychotherapy.

[0122] In certain embodiments, the multi-targeted molecules of the present disclosure are administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0123] In some embodiments, the multi-targeted molecule of the present disclosure is administered intrathecally to a subject.

[0124] In one embodiment, the method reduces expression of a target gene associated with a CNS disorder or disorder in brain (e.g., striatum) or spinal tissue. Optionally, the brain or spinal tissue is the striatum, cortex, cerebellum, cervical spine, lumbar spine, or thoracic spine.

[0125] In some embodiments, the multiple target molecule further comprises at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. In related embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand, or optionally, at the 3'-end of at least one strand of each dsRNA of the multiple target molecule. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand. In related embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand, or optionally, at the 5'-end of at least one strand of each dsRNA of the multiple target molecule. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0126] In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5'-end and the 3'-end of one strand, or optionally at both the 5'-end and the 3'-end of at least one strand of each dsRNA of the multiple target molecule. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0127] In additional embodiments, the base pair at position 1 of the 5' end of the multiple-target molecule, or the antisense strand of the dsRNA of the multiple-target molecule, is an A:U base pair.

[0128] An additional aspect of the present disclosure provides a multi-target molecule for inhibiting expression of a target gene, wherein one or more dsRNAs targeting the target gene (each dsRNA of the multi-target molecule targets a different portion of the same gene or a different gene) comprise a sense strand and an antisense strand forming a double-stranded region, and the sense strand has at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% nucleotide sequence identity to the nucleotide sequence of the target gene of the dsRNA or the entire nucleotide sequence of the target gene of the dsRNA. The antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less (i.e., 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences having 9 or 100% identity, and the antisense strand is a nucleotide sequence having at least 90% nucleotide sequence identity, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to the complementary strand of any one of the nucleotide sequences of the target gene of the dsRNA, or the complementary strand of the entire nucleotide sequence of the target gene of the dsRNA. a sequence of at least 15 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from any one of the complementary nucleotide sequences provided in the target nucleotide sequence of the dsRNA, wherein any thymine to uracil substitution in the sequence of the target gene of the dsRNA (when compared to the alignment sequence) does not count as a difference that contributes to the dsRNA target nucleotide sequence differing by no more than 3 nucleotides from any one of the complementary nucleotide sequences provided in the target nucleotide sequence of the dsRNA; optionally, substantially all of the nucleotides in the sense strand of the one or more dsRNAs comprise a modification that is a 2'-O-methyl modification, a GNA, or a 2'-fluoro modification; optionally, the sense strand of the one or more dsRNAs comprise two phosphorothioate internucleotide linkages at the 5' end; optionally, substantially all of the nucleotides in the antisense strand of the one or more dsRNAs comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; optionally, the antisense strand of the one or more dsRNAs comprise two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end;Optionally, the sense strand of one or more dsRNAs is conjugated to one or more lipophilic ligands, such as, for example, C16. In certain embodiments, the sense strand of one or more dsRNAs comprises at least one 3'-terminal deoxythymidine nucleotide (dT), and optionally, the antisense strand of one or more dsRNAs comprises at least one 3'-terminal deoxythymidine nucleotide (dT).

[0129] In one embodiment, all of the nucleotides in the sense strand of one or more dsRNAs of a multiple target molecule are modified nucleotides, and optionally, all of the nucleotides in the antisense strand of one or more dsRNAs of a multiple target molecule are modified nucleotides.

[0130] In another embodiment, each strand of the one or more dsRNAs of the multiple target molecule has between 19 and 30 nucleotides.

[0131] In certain embodiments, the antisense strand of one or more dsRNAs of the multiple target molecule comprises at least one duplex-thermally destabilizing modification within the first 9 nucleotide positions of the 5' region or precursor thereof. Optionally, the duplex-thermally destabilizing modification is one or more of the following: [ka] wherein B is a nucleobase.

[0132] Another aspect of the present disclosure provides a cell containing a multi-target molecule of the present disclosure. Optionally, the cell is a cell of a CNS tissue of the subject.

[0133] An additional aspect of the present disclosure provides a pharmaceutical composition for inhibiting expression of a target gene, comprising a multi-target molecule of the present disclosure.

[0134] In one embodiment, the multi-targeted molecule is administered in an unbuffered solution. Optionally, the unbuffered solution is saline or water.

[0135] In another embodiment, the multi-target molecule is administered in a buffer solution. Optionally, the buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer solution is phosphate buffered saline (PBS).

[0136] Another aspect of the present disclosure provides a pharmaceutical composition comprising a multi-targeted molecule and a lipid formulation of the present disclosure.

[0137] In one embodiment, the lipid formulation comprises a lipid nanoparticle (LNP).

[0138] Another aspect of the present disclosure provides a kit for practicing the methods of the present disclosure, the kit comprising: a) a multi-target molecule of the present disclosure; and b) instructions for use; and c) optionally, a device for administering the multi-target molecule to a subject.

[0139] Additional embodiments of the present disclosure provide multiple target molecules, optionally including one or more of the following modifications within each dsRNA: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-alkyl-modified nucleotides, nucleotides comprising glycol nucleic acids (GNAs), phosphorothioates (PS), and vinyl phosphonates (VP). Optionally, the RNAi agent includes at least one of each of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-alkyl-modified nucleotides, nucleotides comprising glycol nucleic acids (GNAs), phosphorothioates, and vinyl phosphonates (VP).

[0140] In another embodiment, the multiple target molecule, or the dsRNA of the multiple target molecule, comprises four or more PS modifications, optionally 6 to 16 PS modifications, optionally 8 to 14 PS modifications, optionally 10 to 12 PS modifications, optionally six modifications in the dsRNA, and optionally 12 modifications in the multiple target molecule.

[0141] In a further embodiment, the sense and antisense strands of each dsRNA of the multiple target molecule possess a 5'-end and a 3'-end (linkers are excluded from consideration), and one or more dsRNAs of the multiple target molecule comprise six PS modifications at the final internucleotide bond and the penultimate internucleotide bond, respectively: the 5'-end and 3'-end of the sense strand of each dsRNA of the multiple target molecule, and the 3'-end of the antisense strand of each dsRNA of the multiple target molecule.

[0142] In a further embodiment, the sense and antisense strands of each dsRNA of the multiple target molecule possess a 5'-end and a 3'-end (linkers are excluded from consideration), and one or more dsRNAs of the multiple target molecule comprise eight PS modifications at the last and penultimate internucleotide linkages of each of the 3'-end and 5'-end of each of the sense and antisense strands of the multiple target molecule.

[0143] In another embodiment, each of the sense and antisense strands of each dsRNA of the multiple target molecule has a 5'-end and a 3'-end (linkers are excluded from consideration), and one or more of the dsRNAs of the multiple target molecule contains only one GNA-containing nucleotide. Optionally, the GNA-containing nucleotide is located on the antisense strand at the seventh nucleobase residue from the 5'-end of the antisense strand.

[0144] In additional embodiments, each of the sense and antisense strands of each dsRNA of the multiple target molecule possesses a 5'-end and a 3'-end (linkers are excluded from consideration), and one or more dsRNAs of the multiple target molecule comprise one to four 2'-alkyl-modified nucleotides. Optionally, the 2'-alkyl-modified nucleotides are 2'-C 16 Optionally, each of one or more dsRNAs of a multiple target molecule comprises one 2'-alkyl, e.g., C 16 Optionally, one 2'-alkyl, e.g., C 16The modified nucleotide is located on the sense strand at the sixth nucleobase position from the 5'-end of the sense strand.

[0145] In another embodiment, each of the sense strand and antisense strand of each dsRNA of the multiple target molecule has a 5'-end and a 3'-end (linkers are excluded from consideration), and one or more dsRNAs of the multiple target molecule comprise two or more 2'-fluoro modified nucleotides. Optionally, each of the sense strand and antisense strand of one or more dsRNAs of the multiple target molecule comprise two or more 2'-fluoro modified nucleotides. Optionally, the 2'-fluoro modified nucleotides are located on the sense strand at nucleobase positions 7, 9, 10, and 11 from the 5'-end of the sense strand, and on the antisense strand at nucleobase positions 2, 14, and 16 from the 5'-end of the antisense strand. In certain embodiments, the antisense strand of each dsRNA further comprises a 2'-fluoro modified nucleotide at one or more of nucleobase positions 6, 8, and 9 from the 5'-end of the antisense strand. In related embodiments, the 2'-fluoro modified nucleotides are located on the sense strand at nucleobase positions 7, 9, 10, and 11 from the 5'-end of the sense strand, and on the antisense strand at nucleobase positions 2, 6, 8, 9, 14, and 16 from the 5'-end of the antisense strand.

[0146] In additional embodiments, each of the sense and antisense strands of each dsRNA of the multiple target molecule possesses a 5'-end and a 3'-end (linkers excluded from consideration), and one or more dsRNAs of the multiple target molecule comprise one or more VP modifications. Optionally, one or more dsRNAs of the multiple target molecule comprise a VP modification at the 5'-end of the antisense strand.

[0147] In another embodiment, each of the sense and antisense strands of each dsRNA of the multiple target molecule possesses a 5'-end and a 3'-end (linkers are excluded from consideration), and one or more dsRNAs of the multiple target molecule comprise two or more 2'-O-methyl modified nucleotides. Optionally, one or more dsRNAs of the multiple target molecule comprise 2'-O-methyl modified nucleotides at all nucleobase positions not modified by 2'-fluoro, 2'-alkyl, or glycol nucleic acid (GNA). Optionally, the two or more 2'-O-methyl modified nucleotides are located at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5' end of the sense strand and positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5' end of the antisense strand. Alternatively, the two or more 2'-O-methyl modified nucleotides are located at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5' end of the sense strand and positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5' end of the antisense strand.

[0148] Another aspect of the invention provides a cell of a CNS tissue of a subject containing a nucleic acid composition comprising at least a first dsRNA molecule and a single-stranded nucleic acid agent or a second dsRNA molecule, wherein the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule are connected together by a linker and do not overlap with each other, and wherein the first dsRNA and the single-stranded nucleic acid molecule or the second dsRNA molecule each comprise at least one conjugated lipophilic moiety, and the nucleic acid composition inhibits the activity or expression of one or more distinct target RNAs in a cell or tissue of the CNS of the subject by at least 15% relative to a suitable control.

[0149] In some embodiments, the cell is a cell of a cell type selected from the group consisting of a neuron, an oligodendrocyte, a microglia, and an astrocyte.

[0150] Another aspect of the present invention provides a multi-target molecule for modulating two or more distinct target RNAs in the central nervous system (CNS) of a subject according to the formula: [ka] wherein A is a first double-stranded oligonucleotide (e.g., dsRNA (dsRNA)) molecule, B is a second double-stranded oligonucleotide (e.g., dsRNA) molecule, and L is a linker, A and B do not overlap with each other, each of A and B independently comprises at least one conjugated lipophilic moiety, and the multi-targeting molecule is capable of inhibiting the activity or expression of two or more distinct target RNAs in a CNS tissue of a subject by at least 15% relative to a suitable control.

[0151] In certain embodiments, A and B are first and second double-stranded RNA molecules (dsRNA), respectively.

[0152] In one embodiment, A is according to the formula: [ka] wherein ss1 is the sense strand of the first dsRNA molecule, as1 is the antisense strand of the first dsRNA molecule, * represents the bond between ss1 and L, and the dotted box indicates the optional 3' overhang region of as1.

[0153] In another embodiment, A is according to the formula: [ka] wherein ss2 is the sense strand of the second dsRNA molecule, as2 is the antisense strand of the second dsRNA molecule, ** represents the bond between ss2 and L, and the dotted box indicates the optional 3' overhang region of as2.

[0154] In a related embodiment, L is represented by -(nt1)(nt2)(nt3)-, where nt1, nt2, and nt3 are each independently a nucleotide or a modified nucleotide, and nt1 is attached to a first dsRNA molecule and nt3 is attached to a second dsRNA molecule.

[0155] In another embodiment, the multi-target molecule has the formula: [ka] wherein ss1 is the sense strand of the first dsRNA molecule, as1 is the antisense strand of the first dsRNA molecule, ss2 is the sense strand of the second dsRNA molecule, as2 is the antisense strand of the second dsRNA molecule, and the dotted boxes indicate the optional 3' overhang regions of as1 and as2, respectively.

[0156] In some embodiments, L is represented by -(nt1)(nt2)(nt3)-, where nt1, nt2, and nt3 are each independently a nucleotide or a modified nucleotide, and nt1 is attached to a first dsRNA molecule and nt3 is attached to a second dsRNA molecule.

[0157] In some embodiments, L is represented by -(nt1)(nt2)(nt3)-, where each of nt1, nt2, and nt3 is independently a nucleotide or a modified nucleotide, nt1 is attached to ss1, and nt3 is attached to ss2. In related embodiments, nt1, nt2, and nt3 are each independently selected from A, T, U, G, C, dA, dT, dU, dG, dC, a, t, u, g, c, Af, Tf, Uf, Gf, and Cf, as defined in Table 1.

[0158] In another embodiment, nt1, nt2, and nt3 are each independently A, T, U, G, or C, as defined in Table 1.

[0159] In further embodiments, nt1, nt2, and nt3 are each independently dA, dT, dU, dG, or dC, as defined in Table 1.

[0160] In some embodiments, nt1, nt2, and nt3 are each independently a, t, u, g, or c, as defined in Table 1.

[0161] In certain embodiments, nt1, nt2, and nt3 are each independently Af, Tf, Uf, Gf, or Cf, as defined in Table 1.

[0162] In some embodiments, nt1, nt2, and nt3, taken together, are one of the following: dTdTdT, UUU, uuu, and UfUfUf, as defined in Table 1.

[0163] In some embodiments, as1 and as2 each comprise an independent dinucleotide 3' overhang. In related embodiments, the dinucleotide 3' overhang of as2 is complementary to the linker. In certain embodiments, the dinucleotide 3' overhang of as2 has one mismatch with the linker. In other embodiments, the dinucleotide 3' overhang of as2 has two mismatches with the linker.

[0164] In some embodiments, each lipophilic moiety is a hexadecyl group.

[0165] In certain embodiments, one or more non-terminal nucleotide positions (eg, position 6) of the first sense strand and one or more non-terminal nucleotide positions of the second sense strand independently have the structure: [ka] wherein B is a nucleotide base or nucleotide base analog, optionally B is adenine, guanine, cytosine, thymine, or uracil, and the n-hexadecyl chain is a lipophilic moiety.

[0166] In some embodiments, the one or more non-terminal nucleotide positions of the first sense strand are selected from the group consisting of positions 2-8 and 13-20 of the first sense strand, optionally selected from the group consisting of positions 4-8 and 13-18; optionally the non-terminal nucleotide positions are positions 4, 6, 7 and 8, or positions 5, 6, 7, 15 and 17 of the first sense strand; and the one or more non-terminal nucleotide positions of the second sense strand are selected from the group consisting of positions 2-8 and 13-20 of the first sense strand, optionally selected from the group consisting of positions 4-8 and 13-18; optionally the non-terminal nucleotide positions are positions 4, 6, 7 and 8, or positions 5, 6, 7, 15 and 17 of the second sense strand, independently counting from the 5' end of the first and second sense strands, respectively.

[0167] In certain embodiments, only one of the non-terminal nucleotide positions of the first sense strand and only one of the non-terminal nucleotide positions of the second sense strand independently has the structure: [ka] wherein B is a nucleotide base or nucleotide base analog, optionally B is adenine, guanine, cytosine, thymine, or uracil, and the n-hexadecyl chain is a lipophilic moiety.

[0168] In some embodiments, the one non-terminal nucleotide position of the first sense strand is selected from the group consisting of positions 2-8 and 13-20 of the first sense strand, optionally selected from the group consisting of positions 4-8 and 13-18, and optionally selected from the group consisting of positions 4-8, 15, and 17 of the first sense strand, and the one non-terminal nucleotide position of the second sense strand is selected from the group consisting of positions 2-8 and 13-20 of the first sense strand, optionally selected from the group consisting of positions 4-8 and 13-18, and optionally selected from the group consisting of positions 4-8, 15, and 17 of the second sense strand, independently counting from the 5' end of the first and second sense strands, respectively.

[0169] In certain embodiments, L is a biocleavable linker. All statements regarding biocleavable linkers in the above aspects or embodiments may apply to L herein. In certain embodiments, L is [ka] [ka] (Y195), and [ka] (Y254).

[0170] In certain embodiments, L is a redox-cleavable linking group. Optionally, L is or includes -SS- or -C(R)2-SS-, where R is H or C1-C6 alkyl and at least one R is C1-C6 alkyl, optionally CH3 or CH2CH3.

[0171] In some embodiments, L is a phosphate-based cleavable linking group. Optionally, L is selected from the group consisting of -OP(O)(OR)-O-, -OP(S)(OR)-O-, -OP(S)(SR)-O-, -SP(O)(OR)-O-, -OP(O)(OR)-S-, -SP(O)(OR)-S-, -OP(S)(ORk)-S-, -SP(S)(OR)-O-, -OP(O)(R)-O-, -OP(S)(R)-O-, -SP(O)(R)-O-, -SP(O)(R)-S-, -OP(S)(R)-S-, -OP(O)(OH)-O-, -OP wherein R is or comprises an optionally substituted straight or branched chain C-C 10 It is alkyl.

[0172] In certain embodiments, L is an acid-cleavable linking group. Optionally, L is or includes a hydrazone, an ester, an ester of an amino acid, -C=NN-, or -OC(O)-.

[0173] In some embodiments, L is an ester-based cleavable linking group. Optionally, L is or includes -C(O)O-.

[0174] In certain embodiments, L is a peptide-based cleavable linking group. Optionally, L is or includes a linking group that is cleaved by a cellular enzyme. Optionally, the cellular enzyme is a peptidase or protease. Optionally, L is -NHCHR A C(O)NHCHR B is or includes C(O)—, wherein R A and R B are the R groups of two adjacent amino acids.

[0175] In some embodiments, the nucleic acid compositions or pharmaceutical compositions of the present disclosure target one or more target RNAs, including two or more distinct target RNA sequences.

[0176] For the multi-targeting molecules of the present disclosure, which include at least one lipophilic moiety conjugated to each dsRNA molecule, surprising stability of delivery and inhibitory efficacy of such molecules, particularly in CNS tissues, has been observed, which distinguishes the molecules of the present disclosure from the multi-targeting single conjugates described in PCT application PCT / US2016 / 042498.

[0177] Another aspect of the present invention relates to a small circular interfering RNA (sciRNA) for regulating one or more target mRNAs in a subject's central nervous system (CNS), the sciRNA comprising a first strand having a length of at least 40 nucleotides and at least two first strand nucleotide sequences (each nucleotide sequence having a length of about 18 to about 28 nucleotides) connected together by a bis-linker, and at least one second strand nucleotide sequence having a length of about 19 to 23 nucleotides and annealing to at least one of the first strand nucleotide sequences. The first strand has a circular or substantially circular structure. The first strand nucleotide sequence and the second strand nucleotide sequence each contain at least one nucleic acid modification. The first strand nucleotide sequence or the second strand nucleotide sequence contains one or more ligands.

[0178] In some embodiments, each of the first and second strand nucleotide sequences is at least 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, or 40 nucleotides in length. Each of the first strand nucleotide sequences may be about 18 to about 28 nucleotides in length, e.g., about 19 to 25 nucleotides in length, about 19 to 23 nucleotides in length, or about 20 to 21 nucleotides in length. Each of the second strand nucleotide sequences may be about 19 to about 25 nucleotides in length, about 19 to 23 nucleotides in length, or about 21 to 23 nucleotides in length.

[0179] In some embodiments, the first strand is an antisense strand, and the second strand nucleotide sequence is a sense strand nucleotide sequence. Thus, the antisense strand has a circular or substantially circular structure, and at least two antisense strand nucleotide sequences are connected together by a bis-linker. At least one of the antisense strand nucleotide sequences is annealed with a sense strand nucleotide sequence. In one embodiment, each of the antisense strand nucleotide sequences is annealed with the same or different sense strand nucleotide sequence.

[0180] In some embodiments, the first strand is a sense strand, and the second strand nucleotide sequence is an antisense strand nucleotide sequence. Thus, the sense strand has a circular or substantially circular structure, and at least two sense strand nucleotide sequences are connected together by a bis-linker. At least one of the sense strand nucleotide sequences is annealed with an antisense strand nucleotide sequence. In one embodiment, each of the sense strand nucleotide sequences is annealed with the same or different antisense strand nucleotide sequence.

[0181] In some embodiments, each of the circular or substantially circular sense strand nucleotide sequences is at least 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, or 40 nucleotides in length. In one embodiment, each of the sense strand nucleotide sequences is about 19-23 nucleotides in length, or about 20-21 nucleotides in length.

[0182] In some embodiments, each of the antisense strand nucleotide sequences is at least 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, or 40 nucleotides in length. In one embodiment, each of the antisense strand nucleotide sequences is about 21-23 nucleotides in length, or 23 nucleotides in length.

[0183] The antisense strand nucleotide sequence is annealed to a circular or substantially circular sense strand nucleotide sequence. In some embodiments, one or more sense nucleotide sequences are annealed to the antisense strand nucleotide sequence. In some embodiments, each of the sense nucleotide sequences is annealed to an antisense strand nucleotide sequence. In some embodiments, at least one sense nucleotide sequence is not annealed to an antisense strand nucleotide sequence.

[0184] In some embodiments, the sense nucleotide sequence that is not annealed to the antisense strand nucleotide sequence may be, for example, an inhibitory single-stranded oligonucleotide such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide.

[0185] In some embodiments, a double-stranded region is formed between the sense strand nucleotide sequence and the antisense strand nucleotides in at least a seed region of the antisense strand nucleotide sequence.

[0186] In certain embodiments, the circular or substantially circular sense strand comprises at least two symmetrical sense nucleotide sequences, each having a length of about 19 to about 23 nucleotides. In one embodiment, the circular or substantially circular sense strand comprises at least two symmetrical sense nucleotide sequences, each having a length of about 20 to about 21 nucleotides. "Symmetrical" means that the same antisense nucleotide sequence can anneal with either of the two sense nucleotide sequences. In one embodiment, the sense strand nucleotide sequence anneals with at least two identical antisense nucleotide sequences, each having a length of 23 nucleotides and targeting the same mRNA transcript nucleotide sequence. Thus, the sciRNA (bis-sciRNA) can inhibit the activity or expression of at least one target mRNA transcript in a CNS tissue of a subject.

[0187] In certain embodiments, the circular or substantially circular sense strand comprises at least two asymmetric sense nucleotide sequences, each having a length of about 19 to about 23 nucleotides. In one embodiment, the circular or substantially circular sense strand comprises at least two asymmetric sense nucleotide sequences, each having a length of about 20 to about 21 nucleotides. "Asymmetric" means that the antisense nucleotide sequences that can anneal to the at least two sense nucleotide sequences are different. In one embodiment, the sense strand nucleotide sequence anneals to at least two different antisense nucleotide sequences, each having a length of 23 nucleotides, and targeting at least two different mRNA transcript nucleotide sequences. Thus, sciRNAs (bis-sciRNAs) can inhibit the activity or expression of two or more distinct target mRNA transcripts in a subject's CNS tissue. In one embodiment, the two or more distinct target mRNAs are located within the same nucleic acid.

[0188] In some embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) is an organic polymer linker. The organic polymer linker may be a biocleavable linker.

[0189] In some embodiments, the circular or substantially circular sense strand contains a nucleotide-based linker (tether). In some embodiments, the circular or substantially circular sense strand contains a non-nucleotide-based linker (tether). In one embodiment, the bis-linker connecting the sense nucleotide sequences is a nucleotide-based or non-nucleotide-based linker (tether).

[0190] In some embodiments, the circular or substantially circular antisense strand contains a nucleotide-based linker (tether). In some embodiments, the circular or substantially circular antisense strand contains a non-nucleotide-based linker (tether). In one embodiment, the bis-linker connecting the antisense nucleotide sequences is a nucleotide-based or non-nucleotide-based linker (tether).

[0191] In certain embodiments, the nucleotide or non-nucleotide based linker (tether) is a stable linker (tether) that is stable in biological fluids, for example, the nucleotide or non-nucleotide based stable linker (tether) is stable in plasma or artificial cerebrospinal fluid.

[0192] In certain embodiments, the nucleotide or non-nucleotide cleavable linker (tether) may be cleavable in liver homogenate, liver tritosomes, liver lysosomes, liver cytosol, brain homogenate, brain tritosomes, brain lysosomes, or brain cytosol.

[0193] In certain embodiments, the cleavable linker (tether) is a redox-cleavable linker (e.g., a reductively cleavable linker, such as a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate group), or a peptidase-cleavable linker (e.g., a peptide bond).

[0194] In certain embodiments, the cleavable linker comprises at least one modified internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester, hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate, phosphorothioate, methylenemethylimino, thiodiester, thionocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, boranophosphate, boranophosphate ester, amide, hydroxylamino, siloxane, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonic acid ester, thioformacetal, formacetal, oxime, methyleneimino, methylenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether, thioacetamide, and combinations thereof.

[0195] In some embodiments, the bis-linker in the circular or substantially circular sense strand contains a nucleotide-based cleavable linker (tether) that is cleavable by DICER. In some embodiments, the circular or substantially circular sense strand comprises a substrate that is cleavable by DICER.

[0196] In some embodiments, the bis-linker in the circular or substantially circular antisense strand contains a nucleotide-based cleavable linker that is cleavable by DICER. In some embodiments, the circular or substantially circular antisense strand comprises a substrate that is cleavable by DICER.

[0197] In certain embodiments, the antisense strand contains a cleavable linker (nucleotide or non-nucleotide) that can form a circular or substantially circular structure and generate a 5'-monophosphate metabolite at the antisense nucleotide sequence of the antisense strand. The circular or substantially circular antisense strand can be cleaved to generate a linear structure containing a 5'-monophosphate metabolite at the antisense nucleotide sequence of the antisense strand.

[0198] In some embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) contains a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0199] In some embodiments, the bis-linker is an endosomally cleavable linker or a protease-cleavable linker, e.g., a carbohydrate linker, in which case the linker is cleaved at least 1.25 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0200] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (either the circular or substantially circular sense strand or the circular or substantially circular antisense strand) contains a moiety selected from the group consisting of: aliphatic saturated or unsaturated alkyl chains; phosphorus-containing linkages, including phosphate, phosphonate, phosphoramidate, phosphodiester, phosphotriester, and phosphorothioate; (poly)ethylene glycol chains, including diethylene glycol, triethylene glycol, tetra-, penta-, hexa-, hepta-, octa-, nona-, or decaethylene glycol; glycerol or glycerol esters; aminoalkyl; and combinations thereof.

[0201] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) comprises a moiety selected from the group consisting of: [ka]

[0202] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) contains a moiety selected from: -(CH2) 12 -(C12 linker or Q50), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker or Q51), [ka] -CH2CH2O-(CH2CH2) n -CH2CH2O-CH2CH2O-, where n is 0 or 1 to 20; -(CH2)9-(CH2) n -CH2-, where n is 0 or 1 to 20; mono-, di-, tri-, tetra-, penta-, or poly-prolinol, optionally conjugated to a ligand; Mono-, di-, tri-, tetra-, penta-, or poly-hydroxyprolinol, optionally conjugated to a ligand.

[0203] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) comprises a nucleic acid linker 1 to 15 nucleotides in length. For example, the nucleic acid linker may be 2 to 5 nucleotides, 3 to 4 nucleotides, or 3 nucleotides in length.

[0204] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand, or the circular or substantially circular antisense strand) comprises one or more sequences selected from the group consisting of UUU, 2'-O-methyl-UUU (uuu), 2'-fluoro-UUU (UfUfUf), and (dT)n, where n is 1 to 20 (e.g., dTdTdT).

[0205] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand, or the circular or substantially circular antisense strand) comprises a nucleic acid linker comprising one or more nucleotides selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, deoxyribonucleotides, and ribonucleotides. In one embodiment, all of the nucleic acid linker nucleotides are the same type of nucleotide. In one embodiment, the nucleic acid linker comprises entirely 2'-O-methyl nucleotides, entirely 2'-fluoro nucleotides, or entirely deoxyribonucleotides.

[0206] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand or the circular or substantially circular antisense strand) comprises a polynucleotide comprising a modified ribonucleotide sequence, optionally a polynucleotide comprising one or more modifications selected from the group consisting of a 2'-O-methyl ribonucleotide modification, a 2'-fluoro-ribonucleotide modification, a 2'-5'-linked nucleotide including different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), a glycol nucleic acid (GNA) modification, a locked nucleic acid (LNA) modification, a hexanol nucleic acid (HNA) modification, an abasic ribose modification, an abasic deoxyribose modification, and an abasic hydroxyprolinol modification.

[0207] In certain embodiments, the bis-linker connecting the nucleotide sequences of the first strand (the circular or substantially circular sense strand, or the circular or substantially circular antisense strand) comprises one or more moieties selected from the group consisting of a phosphodiester bond, a phosphotriester bond (optionally containing a linking phosphorus atom in either the Rp or Sp configuration), a phosphorothioate diester bond (optionally containing a linking phosphorus atom in either the Rp or Sp configuration), a phosphoramidate diester bond (optionally containing a linking phosphorus atom in either the Rp or Sp configuration), and a disulfide bond.

[0208] In certain embodiments, the circular or substantially circular sense strand has two nucleotide sequences, ss1 and ss2, where the 3' end of ss1 is connected to the 5' end of ss2 by a bis-linker. [ka]

[0209] In another embodiment, the circular or substantially circular sense strand has two nucleotide sequences, ss1 and ss2, where the 3' end of ss1 is connected to the 3' end of ss2 by a bis-linker. [ka]

[0210] In another embodiment, the circular or substantially circular sense strand has two nucleotide sequences, ss1 and ss2, where the 5' end of ss1 is connected to the 5' end of ss2 by a bis-linker. [ka]

[0211] In one embodiment, ss1 is annealed to as1 of the antisense strand nucleotide sequence. The 3' end of as1 may form a 3' overhang of 1 to 2 nucleotides relative to the 5' end of ss1. The 5' end of as1 may form a 5' overhang of 1 to 2 nucleotides relative to the 3' end of ss1.

[0212] In one embodiment, ss2 is annealed to as2 of the antisense strand nucleotide sequence. The 3' end of as2 may form a 3' overhang of 1 to 2 nucleotides relative to the 5' end of ss2. The 5' end of as2 may form a 5' overhang of 1 to 2 nucleotides relative to the 3' end of ss2.

[0213] In certain embodiments, the bis-linker between ss1 and ss2 is represented by -(nt1)(nt2)(nt3)-, where nt1, nt2, and nt3 are each independently a nucleotide or a modified nucleotide. In some embodiments, nt1, nt2, and nt3 are each independently selected from the group consisting of A, T, U, G, C, and various modifications thereof. Each of A, T, U, G, and C may be in the form of a nucleotide selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, deoxyribonucleotides, and ribonucleotides. In one embodiment, nt1, nt2, and nt3 are one of the following: UUU, 2'-O-methyl-UUU(uuu), 2'-fluoro-UUU(UfUfUf), or dTdTdTdT.

[0214] In one embodiment, ss1 is annealed to as1 of the antisense strand nucleotide sequence, and ss2 is annealed to as2 of the antisense strand nucleotide sequence. [ka] The antisense strand nucleotide sequences as1 and as2 may each include a 3' overhang of 2 nucleotides in length. The antisense strand nucleotide sequences as1 and as2 may each include a 5' overhang of 1 to 2 nucleotides in length.

[0215] In some embodiments, the bis-linker between ss1 and ss2 is a nucleic acid linker three nucleotides in length, and the antisense strand nucleotide sequences as1 and as2 each comprise a two-nucleotide 3' overhang. In one embodiment, the two nucleotides in the 3' overhang of as2 are complementary to the nucleotides in the bis-linker. In one embodiment, the two nucleotides in the 3' overhang of as2 have one mismatch with the nucleotides in the bis-linker. In one embodiment, the two nucleotides in the 3' overhang of as2 have two mismatches with the nucleotides in the bis-linker.

[0216] In another embodiment, ss1 is annealed to as1 of the antisense strand nucleotide sequence, and ss2 is annealed to as2 of the antisense strand nucleotide sequence. [ka] The antisense strand nucleotide sequences as1 and as2 may each include a 3' overhang of 2 nucleotides in length. The antisense strand nucleotide sequences as1 and as2 may each include a 5' overhang of 1 to 2 nucleotides in length.

[0217] In some embodiments, the bis-linker between ss1 and ss2 is a nucleic acid linker three nucleotides in length, and the antisense strand nucleotide sequences as1 and as2 each comprise a 5' overhang one to two nucleotides in length. In one embodiment, one or two nucleotides in the 5' overhang of as2 or as1 are complementary to nucleotides in the bis-linker. In one embodiment, one or two nucleotides in the 5' overhang of as2 or as1 have one mismatch with nucleotides in the bis-linker. In one embodiment, two nucleotides in the 5' overhang of as2 or as1 have two mismatches with nucleotides in the bis-linker.

[0218] In another embodiment, ss1 is annealed to as1 of the antisense strand nucleotide sequence, and ss2 is annealed to as2 of the antisense strand nucleotide sequence. [ka] The antisense strand nucleotide sequences as1 and as2 may each include a 3' overhang of 1 to 2 nucleotides in length. The antisense strand nucleotide sequences as1 and as2 may each include a 5' overhang of 1 to 2 nucleotides in length.

[0219] In some embodiments, the bis-linker between ss1 and ss2 is a nucleic acid linker three nucleotides in length, and the antisense strand nucleotide sequences as1 and as2 each comprise a 1-2 nucleotide 3' overhang. In one embodiment, one or two nucleotides in the 3' overhang of as2 or as1 are complementary to nucleotides in the bis-linker. In one embodiment, one or two nucleotides in the 3' overhang of as2 or as1 have one mismatch with nucleotides in the bis-linker. In one embodiment, two nucleotides in the 3' overhang of as2 or as1 have two mismatches with nucleotides in the bis-linker.

[0220] In some embodiments, the sciRNA comprises at least one chemical modification, which may comprise an internucleoside linkage modification, a nucleobase modification, a sugar modification, or a combination thereof.

[0221] In certain embodiments, the chemical modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (e.g., 2'-modified L-nucleosides, e.g., 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic and open-chain alkyls, and combinations thereof.

[0222] In certain embodiments, the chemical modifications are 2'-O-methyl, 2'-deoxy, 2'-fluoro, 2'-C6-C 18 The 2'-modification is selected from the group consisting of: a hydrocarbon chain; and combinations thereof.

[0223] In certain embodiments, the chemical modification is a 2'-modification selected from the group consisting of 2'-O-methyl, 2'-deoxy, 2'-fluoro, and combinations thereof.

[0224] In some embodiments, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of all nucleotides are modified.For example, if 50% of all nucleotides are modified, 50% of all nucleotides present in sciRNA contain the modifications described herein.

[0225] In some embodiments, every nucleotide in the first strand (eg, sense strand) nucleotide sequence is modified.

[0226] In some embodiments, every nucleotide in the second strand (eg, antisense strand) nucleotide sequence is modified.

[0227] In one embodiment, at least 50% of the nucleotides of the sciRNA are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-deoxy, or 2'-fluoro.

[0228] The sciRNA comprises one or more ligands.

[0229] In some embodiments, the circular or substantially circular sense strand may comprise one or more ligands. In one embodiment, each sense nucleotide sequence of the circular or substantially circular sense strand comprises at least one ligand, which may be particularly effective in regulating gene expression. Thus, at least two ligands (e.g., lipophilic ligands) are conjugated to the multi-target bis-sciRNA molecule.

[0230] In some embodiments, the antisense strand nucleotide sequence comprises one or more ligands. In one embodiment, each antisense nucleotide sequence comprises at least one ligand, which may be particularly effective in regulating gene expression. Thus, at least two ligands (e.g., lipophilic ligands) are conjugated to the multi-target bis-sciRNA molecule.

[0231] In certain embodiments, at least one of the ligands is conjugated to a chain having a cyclic or substantially cyclic structure. In certain embodiments, at least one of the ligands is conjugated to a chain that does not have a cyclic or substantially cyclic structure. In one embodiment, at least one of the ligands is conjugated to a chain having a cyclic or substantially cyclic structure and at least one of the ligands is conjugated to a chain that does not have a cyclic or substantially cyclic structure.

[0232] In certain embodiments, at least one of the ligands is conjugated to the sense nucleotide sequence of the sense strand. At least one of the ligands may be conjugated to the 3' end, 5' end, or an internal position of the sense nucleotide sequence. In one embodiment, the conjugated sense strand has a cyclic or substantially cyclic structure. In one embodiment, the conjugated sense strand does not have a cyclic or substantially cyclic structure.

[0233] In certain embodiments, at least one of the ligands is conjugated to the antisense nucleotide sequence of the antisense strand. At least one of the ligands may be conjugated at the 3' end, 5' end, or internal position of the antisense nucleotide sequence. In one embodiment, the conjugated antisense strand has a cyclic or substantially cyclic structure. In one embodiment, the conjugated antisense strand does not have a cyclic or substantially cyclic structure.

[0234] In some embodiments, the ligand may be conjugated to the sciRNA by direct attachment to the ribosugars of the sciRNA, or the ligand may be conjugated to the sciRNA via one or more linkers (tethers) and / or carriers.

[0235] In some embodiments, the ligand may be conjugated to the sciRNA molecule via a monovalent, or branched bivalent or trivalent linker.

[0236] In some embodiments, the ligand may be conjugated to the sciRNA via a carrier that replaces one or more nucleotides. The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl. In one embodiment, the acyclic group is a moiety based on a serinol or diethanolamine backbone.

[0237] In certain embodiments, at least one of the ligands is a lipophilic moiety.

[0238] In one embodiment, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0239] In some embodiments, the lipophilic moiety is a saturated or unsaturated C4-C 30 Hydrocarbon chains (e.g., C4-C 30 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6-C 18 Hydrocarbon chains (e.g., linear C6-C 18alkyl or alkenyl), for example, saturated or unsaturated C 16 or C 22 Hydrocarbon chains (e.g., linear C 16 Or C 22 For example, one or more non-terminal positions of the sense strand nucleotide sequence may have the following structure: [ka] where B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine, or uracil, or modified derivatives thereof), and the n-hexadecyl chain is a lipophilic moiety. The modification shown in formula (1) is referred to herein as "2'-C16." Similar modifications can be made by modifying the n-hexadecyl chain from C4-C 30 The modification of the hydrocarbon chain is "2'-C4-C 30 The hydrocarbon chain (or C6-C 18 The substitution with the hydrocarbon chain is "2'-C6-C 18 (These are called "hydrocarbon chains").

[0240] In a related embodiment, one or more non-terminal nucleotide positions of the sense strand of the sense strand nucleotide sequence or the antisense strand nucleotide sequence is a 2'-C4-C 30 Hydrocarbon chain structure, 2'-C6-C 18 It has a hydrocarbon chain structure or a 2'-C16 structure.

[0241] In one embodiment, one or more non-terminal nucleotide positions of every sense strand nucleotide sequence is 2'-C4-C of Formula (1). 30 Hydrocarbon chain structure, 2'-C6-C 18 It has a hydrocarbon chain structure or a 2'-C16 structure.

[0242] In one embodiment, one or more non-terminal nucleotide positions of every antisense strand nucleotide sequence is a 2'-C4-C 30 Hydrocarbon chain structure, 2'-C6-C 18It has a hydrocarbon chain structure or a 2'-C16 structure.

[0243] In some embodiments, one or more of the circular or substantially circular sense strand nucleotide sequences comprise one or more lipophilic moieties independently conjugated to one or more non-terminal positions other than positions 9-12 of the sense strand nucleotide sequence, for example, positions 4-8 and 13-18 of the sense strand nucleotide sequence, positions 5, 6, 7, 15 and 17 of the sense strand nucleotide sequence, or positions 4, 6, 7 and 8 of the sense strand nucleotide sequence, counting the 5' end of the sense strand nucleotide sequence as position 1.

[0244] In some embodiments, one or more of the circular or substantially circular sense strand nucleotide sequences comprise one or more lipophilic moieties independently conjugated to position 6 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence. In one embodiment, each sense strand nucleotide sequence comprises a lipophilic moiety conjugated to position 6 of the nucleotide sequence, and optionally the lipophilic moiety is a saturated or unsaturated C6-C 18 The lipophilic moiety comprises a hydrocarbon chain, and optionally a saturated or unsaturated C 16 Contains a hydrocarbon chain.

[0245] In some embodiments, one or more of the antisense strand nucleotide sequences comprise one or more lipophilic moieties independently conjugated to one or more of the non-terminal positions of the antisense strand nucleotide sequence, e.g., positions 4-8 and 13-18 of the sense strand nucleotide sequence, positions 6-10 and 15-18 of the antisense strand nucleotide sequence, or positions 15 and 17 of the antisense strand nucleotide sequence, counting the 5' end of the antisense strand nucleotide sequence as position 1.

[0246] In certain embodiments, at least one of the ligands is a carbohydrate-based ligand, which may be D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, a glycosylated polyamino acid, or a lectin.

[0247] In certain embodiments, the carbohydrate-based ligand is an ASGPR ligand. For example, the ASGPR ligand may be linked to a bivalent or trivalent branched linker, such as [ka] and one or more GalNAc derivatives attached via

[0248] In some embodiments, the antisense strand nucleotide sequence comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand nucleotide sequence, hi one embodiment, at least one phosphate mimetic is at the 5' end of each antisense nucleotide sequence.

[0249] The phosphate mimic may be a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl. In one embodiment, the phosphate mimic is a 5'-vinylphosphonate (VP). The 5'-VP may be either the 5'-E-VP isomer (i.e., trans-vinyl phosphate), the 5'-Z-VP isomer (i.e., cis-vinyl phosphate), or a mixture thereof.

[0250] In one embodiment, the phosphate mimetic is a 5'-vinylphosphonate (VP).

[0251] In some embodiments, the sciRNA further comprises at least one terminal chiral phosphorus atom.

[0252] The site-specific chiral modification of the internucleotide bond may be at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the sense or antisense nucleotide sequence. This is referred to herein as a "terminal" chiral modification. The terminal modification may be at the 3'-end or 5'-end position within the terminal region, for example, at the terminal nucleotide position of the sense or antisense nucleotide sequence, or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. The chiral modification may be in the sense strand nucleotide sequence, the antisense strand nucleotide sequence, or both the sense and antisense strand nucleotide sequences. Each chirally pure phosphorus atom may be in either the Rp or Sp configuration, and combinations thereof. Details regarding chiral modifications and chiral modified dsRNA agents can be found in WO2019 / 126651A1, which is incorporated herein by reference in its entirety.

[0253] In some embodiments, the sciRNA contains at least two blocks of two consecutive phosphorothioate or methylphosphonate internucleotide linkage modifications.

[0254] In some embodiments, the sciRNA has at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end) of the antisense strand nucleotide sequence.

[0255] In some embodiments, the antisense nucleotide sequence contains two blocks of one, two, or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0256] In one embodiment, the antisense strand nucleotide sequence comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense nucleotide sequence, counting from the 5' end of the antisense nucleotide sequence, and the sense strand nucleotide sequence comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense nucleotide sequence, counting from the 5' end of the sense nucleotide sequence.

[0257] In some embodiments, each of the nucleotide sequences of the sciRNA contains at least two blocks of two consecutive phosphorothioate internucleotide linkage modifications. In one embodiment, each of the nucleotide sequences of the sciRNA includes, counting from the 5' end of the nucleotide sequence, at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18-23 of the nucleotide sequence and at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1-5 of the nucleotide sequence.

[0258] In some embodiments, the sciRNA comprises the following characteristics: The circular or substantially circular sense strand has two nucleotide sequences, ss1 and ss2, the 3' end of ss1 is connected to the 5' end of ss2 by a bis-linker, ss1 is annealed to as1 of the antisense strand nucleotide sequence, and ss2 is annealed to as2 of the antisense strand nucleotide sequence. [ka] as1 and as2 each contain a 3' overhang that is 2 nucleotides in length, and / or as1 and as2 each contain a 5' overhang of 1 nucleotide in length, All nucleotides of ss1, ss2, as1, and as2 are modified, the sciRNA contains at least two blocks of two consecutive phosphorothioate or methylphosphonate internucleotide linkage modifications; One or both of as1 and as2 comprises a 5'-terminal phosphate mimetic selected from the group consisting of 5'-phosphorothioate (5'-PS), 5'-phosphorodithioate (5'-PS2), 5'-vinylphosphonate (5'-VP), 5'-methylphosphonate (5'-MePhos), and 5'-deoxy-5'-C-malonyl; and One or both of ss1 and ss2 contain one or more ligands.

[0259] In one embodiment, the sciRNA has the following characteristics: the bis-linker between ss1 and ss2 is a nucleic acid linker three nucleotides in length; all nucleotides of ss1, ss2, as1 and as2 are modified with 2'-O-methyl or 2'-fluoro modifications; each of as1 and as2 comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence; each of ss1 and ss2 comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence; and One or both of ss1 and ss2 are one or more lipophilic moieties conjugated to position 6 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence; or at least one carbohydrate-based ligand conjugated to the 3' end of the nucleotide sequence.

[0260] In some embodiments, the sense strand forms a circular or substantially circular structure via a cycling linking moiety that connects one end of the sense strand to the other end of the sense strand.

[0261] In some embodiments, the antisense strand forms a circular or substantially circular structure via a cyclization linkage that connects one end of the antisense strand to the other end of the antisense strand.

[0262] In certain embodiments, the cyclization linkage moiety is a triazole bond, an amide bond, a sulfide or disulfide bond, a phosphate bond, an oxime bond, a hydrazo bond, an N,N'-dialkylenehydrazo bond, a methyleneimino bond, a methylenecarbonylamino bond, a methylenemethylimino bond, a methylenehydrazo bond, a methylenedimethylhydrazo bond, a methyleneoxymethylimino bond, a hydroxylamino bond, a formacetal bond, an alkyl or aryl bond, a PEG bond, an ether bond, a thioether bond, a thiodiester bond, a thionocarbamate bond, a thioacetamide bond, a sulfonic acid bond, a sulfonamide bond, a sulfonate ester ... The compound may contain one or more bonds selected from the group consisting of a tert-butyl bond, a thioformacetal bond, a urea bond, a carbonate bond, an amine bond, a maleimide-thioether bond, a phosphodiester bond, a phosphotriester bond, a phosphonate hydrogen bond, a phosphonate alkyl bond or a phosphonate aryl bond, a phosphoramidate bond, a phosphorothioate bond, a phosphoroselenate bond, a boranophosphate bond, a boranophosphate ester bond, a sulfonamide bond, a carbamate bond, a carboxamide bond, a carboxymethyl bond, a carboxylic acid ester bond, a siloxane bond, a dialkylsiloxane bond, an ethylene oxide bond, and combinations thereof.

[0263] In certain embodiments, the cyclized linking moiety may contain one or more cyclic groups selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0264] In certain embodiments, the cyclization binding moiety also serves as a carrier, carrying the ligand and connecting the ligand to the sciRNA.

[0265] Another aspect of the present invention relates to a pharmaceutical composition comprising a sciRNA for regulating one or more target mRNAs in a subject's central nervous system (CNS) and a pharmaceutically acceptable excipient. The sciRNA comprises a first strand having a length of at least 40 nucleotides and at least two first-strand nucleotide sequences (each nucleotide sequence having a length of about 18 to about 28 nucleotides) connected together by a bis-linker, and at least one second-strand nucleotide sequence having a length of about 19 to 23 nucleotides and annealing to at least one of the first-strand nucleotide sequences. The first strand has a circular or substantially circular structure. The first-strand nucleotide sequence and the second-strand nucleotide sequence each contain at least one nucleic acid modification. The first-strand nucleotide sequence or the second-strand nucleotide sequence contains one or more ligands.

[0266] All of the above embodiments relating to the first strand, first strand nucleotide sequence, second strand nucleotide sequence, sense strand, sense strand nucleotide sequence, antisense strand, antisense strand nucleotide sequence, chemical modifications of the sense strand nucleotide sequence and antisense strand nucleotide sequence, bis-linkers, nucleotide-based linkers and non-nucleotide-based linkers, ligands and ligand conjugations, and cyclization linking moieties disclosed in the first aspect of the invention relating to sciRNA for modulating one or more target mRNAs in the central nervous system (CNS) of a subject are suitable for this aspect of the invention relating to pharmaceutical compositions.

[0267] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target mRNAs in a subject's central nervous system (CNS), comprising contacting a CNS cell of the subject with a sciRNA for regulating one or more target mRNAs in the CNS cell of the subject, in an amount sufficient to inhibit the activity or expression of the one or more target mRNAs in the CNS cell of the subject. The sciRNA comprises a first strand having a length of at least 40 nucleotides and having at least two first-strand nucleotide sequences (each nucleotide sequence having a length of about 18 to about 28 nucleotides) connected together by a bis-linker, and at least one second-strand nucleotide sequence having a length of about 19 to 23 nucleotides and annealing to at least one of the first-strand nucleotide sequences. The first strand has a circular or substantially circular structure. The first-strand nucleotide sequence and the second-strand nucleotide sequence each contain at least one nucleic acid modification. The first-strand nucleotide sequence or the second-strand nucleotide sequence comprises one or more ligands.

[0268] All of the above-described embodiments of the first strand, first strand nucleotide sequence, second strand nucleotide sequence, sense strand, sense strand nucleotide sequence, antisense strand, antisense strand nucleotide sequence, chemical modifications of the sense strand nucleotide sequence and antisense strand nucleotide sequence, bis-linkers, nucleotide-based linkers and non-nucleotide-based linkers, ligands and ligand conjugations, and cyclization linking moieties disclosed in the first aspect of the invention relating to sciRNA for modulating one or more target mRNAs in the central nervous system (CNS) of a subject are suitable for this aspect of the invention relating to a method for inhibiting expression of one or more target mRNAs in the central nervous system (CNS) of a subject.

[0269] In some embodiments, the cell is in a subject. In one embodiment, the subject is a human. In one embodiment, the subject is a non-human mammal, such as a rhesus monkey, a cynomolgus monkey, a mouse, or a rat.

[0270] Another aspect of the present invention relates to a method for treating or preventing a CNS disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a sciRNA for regulating one or more target mRNAs in the subject's central nervous system (CNS), thereby treating or preventing the CNS disease or disorder in the subject. The sciRNA comprises a first strand having a length of at least 40 nucleotides and having at least two first-strand nucleotide sequences (each nucleotide sequence having a length of about 18 to about 28 nucleotides) connected together by a bis-linker, and at least one second-strand nucleotide sequence having a length of about 19 to 23 nucleotides and annealing to at least one of the first-strand nucleotide sequences. The first strand has a circular or substantially circular structure. The first-strand nucleotide sequence and the second-strand nucleotide sequence each contain at least one nucleic acid modification. The first-strand nucleotide sequence or the second-strand nucleotide sequence contains one or more ligands.

[0271] All of the above embodiments relating to the first strand, first strand nucleotide sequence, second strand nucleotide sequence, sense strand, sense strand nucleotide sequence, antisense strand, antisense strand nucleotide sequence, chemical modifications of the sense strand nucleotide sequence and antisense strand nucleotide sequence, bis-linkers, nucleotide-based linkers and non-nucleotide-based linkers, ligands and ligand conjugations, and cyclization linking moieties disclosed in the first aspect of the invention relating to sciRNA for modulating one or more target mRNAs in the central nervous system (CNS) of a subject are suitable for this aspect of the invention relating to a method for treating or preventing a disease or disorder of the CNS in a subject.

[0272] In all of the above aspects of the invention, the sciRNA can inhibit the activity or expression of one or more target mRNAs in a subject's CNS tissue by at least 15%, optionally by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, respectively, compared to a suitable control (e.g., compared to an untreated subject or a placebo-treated subject, or compared to a baseline value, including, for example, target mRNA or protein levels in a treated subject measured before treatment with the sciRNA is administered). In one embodiment, a suitable control is an untreated subject. In one embodiment, a suitable control is a baseline value, such as, for example, a value obtained for a subject before administering the sciRNA to the subject.

[0273] In related embodiments, the sciRNA can inhibit expression of a target mRNA throughout a subject's CNS or within a location within the subject's CNS. In certain embodiments, the sciRNA can inhibit expression of a target mRNA in one or more of the following CNS locations of a subject: right hemisphere, left hemisphere, cerebellum, striatum, brainstem, and spinal cord. Targeted CNS cell types include, but are not limited to, neurons, oligodendrocytes, microglia, and astrocytes, among others.

[0274] In certain embodiments, the sciRNA may be formulated for intrathecal or intracerebroventricular (ICV) administration. In certain embodiments, in the methods described herein, the contacting or administering step comprises administering to the subject an intrathecal injection or an intracerebroventricular (ICV) injection.

[0275] In certain embodiments, the two or more distinct target RNAs are transcripts of genes associated with a disease or disorder of the CNS.

[0276] Examples of diseases or disorders of the CNS include neurodegenerative disorders (e.g., Parkinson's disease (PD), Alzheimer's disease, early-onset familial Alzheimer's disease (EOFAD), cerebral amyloid angiopathy (CAA), spinal muscular atrophy (SMA), Angelman syndrome, ataxic / neurodegenerative disorders of the nervous system (e.g., Friedreich's ataxia), Huntington's disease (Huntington's chorea), multiple sclerosis, amyotrophic lateral sclerosis (ALS)), depression, Down's syndrome, psychosis, schizophrenia, Creutzfeldt-Jakob disease, multiple system atrophy, dementia with Lewy bodies (LBD), pure autonomic dysfunction (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig disease, and tangle predominant dementia. dementia, argyrophilic granule disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, ribofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, vascular disorders (e.g., stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage), infections (e.g., meningitis, encephalitis, polio, epidural abscess), structural disorders (e.g., brain or spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, Guillain-Barré syndrome), and functional disorders (e.g., headache, epilepsy, dizziness, neuralgia). [Brief explanation of the drawings]

[0277] [Figure 1]Figures 1A and 1B show the structures of two siRNA molecules linked to form a bis-siRNA complex exemplified herein, and their respective CNS-directed inhibitory efficiencies (when administered as a mixed siRNA to mice via ICV injection). Figure 1A shows the sequences, structures, and modification patterns of an SOD-targeting siRNA (top, containing the sense strand sequence 5'-CAUUUUAAUCCUCACUCUAAA-3' (SEQ ID NO: 1) and the antisense strand sequence 5'-UUUAGAGUGAGGAUUAAAAUGAG-3' (SEQ ID NO: 2)) and a CTNNB1-targeting siRNA (bottom, containing the sense strand sequence 5'-UACUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 3) and the antisense strand sequence 5'-UUUCGAAUCAAUCCAACAGUAGC-3' (SEQ ID NO: 4)). Figure 1B shows the respective inhibitory efficiencies of SOD1 and CTNNB1 observed when the two siRNA molecules were administered as a mixture of 100 μg to mice via ICV injection. Inhibition levels measured in the right hemisphere, cerebellum, and brainstem in the brain, and in the liver on day 21. Mouse number 8 was identified as an injection failure.

[0278] [Figure 2-1]Figures 2A-2D show examples of bis-siRNA complexes created and tested herein for tandem inhibition of mCTNNB1 and mSOD1. Figure 2A summarizes the duplex identifiers, sense strand identifiers, linkers, and configuration patterns for the exemplary bis-siRNA complexes and control mixed duplexes. All bis-siRNA complexes contain two sets of 21-mer sense strands and 23-mer antisense strands. The sense strands of both siRNAs are contiguous by including a trinucleotide single-stranded linker, while each antisense strand of the siRNA duplex is a noncontiguous, independent strand. The duplex configuration patterns shown include the order of the RNAi target duplexes and the number of C16 modifications in each bis-complex. The number of mice in the cohort tested, the day of target inhibition assessment, the dose employed, and the position of the resulting readout are also shown. Figure 2B shows the complete sense strands of various different bis siRNA complexes, including the linkers for each bis complex, as indicated (from top to bottom: SEQ ID NOS: 5-12, as shown in Table 3 herein; and SEQ ID NOS: 17-24, as listed in Table 2 herein, along with modified versions of these sequences). The modifications present on each presented oligonucleotide sequence are indicated with reference to the legend to the right of each strand, including "DNA" for DNA nucleotides, "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. Figure 2C summarizes the construction patterns and linkers used for each bis siRNA duplex of the present disclosure. Figure 2D shows independent antisense strands (SEQ ID NO: 4 on top and SEQ ID NO: 2 on bottom, summarized in Table 3 herein), which are complementary to the bound CTNNB1 and SOD1 sense strands, respectively, and complexes of each of the two antisense strand sequences shown are hybridized to the fused sense strand sequence in Figure 2B to form the various bis-siRNA complex types tested herein. [Figure 2-2] Same as above.

[0279] [Figure 3A]Figures 3A-3C show the structure of a CTNNB1(C16)-SOD1(C16)bis siRNA multi-target molecule in which each siRNA effector molecule sense strand is linked by a three-nucleotide DNA linker (dTdTdT), as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with the constructs. Figure 3A shows the sequence and modifications of the CTNNB1(C16)-SOD1(C16)bis siRNA complex with a DNA(dTdTdT) linker, including "DNA" for DNA nucleotides, "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The respective sequences shown are SEQ ID NO: 17 for the fusion sense strand sequence, SEQ ID NO: 16 for the CTNNB1 antisense strand sequence, and SEQ ID NO: 14 for the SOD1 antisense strand sequence. Figure 3B shows the percentage of SOD1 (top) and CTNNB1 (bottom) remaining at day 21 after ICV injection of 100 μg of CTNNB1(C16)-SOD1(C16) bis siRNA molecules in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) for each individual mouse treated. (Note: Three animals, numbers 9, 10, and 11, exhibited failed injections and were excluded from specific analyses.) Figure 3C shows the total percentage of SOD1 and CTNNB1 remaining at day 21 after ICV injection of 100 μg, measured in the right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver. Results are analyzed either for all five injected animals combined (top) or for only the two successfully injected animals combined (bottom). [Figure 3B] Same as above. [Figure 3C] Same as above.

[0280] [Figure 4-1]Figures 4A-4C show the structure of a CTNNB1(C16)-SOD1(C16)bis siRNA multi-target molecule in which each siRNA effector molecule sense strand is linked by a trinucleotide 2'-O-methyl linker (uuu), as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with the constructs. Figure 4A shows the sequence and modifications of the CTNNB1(C16)-SOD1(C16)bis siRNA complex with a 2'O-methyl linker (uuu), including "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The respective sequences shown are SEQ ID NO: 18 for the fusion sense strand sequence, SEQ ID NO: 16 for the CTNNB1 antisense strand sequence, and SEQ ID NO: 14 for the SOD1 antisense strand sequence. Figure 4B shows the percentage of SOD1 (top) and CTNNB1 (bottom) remaining at day 21 after ICV injection of 100 μg of CTNNB1(C16)-SOD1(C16) bis siRNA molecules in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) for each individual mouse treated, for a cohort of four animals. Figure 4C shows the total percentage of SOD1 and CTNNB1 remaining at day 21 after ICV injection of 100 μg, measured in the right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver. Results were analyzed pooled across all four injected animals. [Figure 4-2] Same as above.

[0281] [Figure 5-1]Figures 5A-5C show the structure of a CTNNB1(C16)-SOD1(C16)bis siRNA multi-target molecule in which each siRNA effector molecule sense strand is linked by a trinucleotide RNA linker (UUU), as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with the constructs. Figure 5A shows the sequence and modifications of the CTNNB1(C16)-SOD1(C16)bis siRNA complex with an RNA linker (UUU), including "RNA" for unmodified ribonucleotides, "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The respective sequences shown are SEQ ID NO: 19 for the fusion sense strand sequence, SEQ ID NO: 16 for the CTNNB1 antisense strand sequence, and SEQ ID NO: 14 for the SOD1 antisense strand sequence. Figure 5B shows the percentage of SOD1 (top) and CTNNB1 (bottom) remaining at day 21 after ICV injection of 100 μg of CTNNB1(C16)-SOD1(C16) bis siRNA molecules in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) for each individual mouse treated, for a cohort of four animals. Figure 5C shows the total percentage of SOD1 and CTNNB1 remaining at day 21 after ICV injection of 100 μg, as measured in the right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver. Results were analyzed pooled across all four injected animals. [Figure 5-2] Same as above.

[0282] [Figure 6-1]Figures 6A-6C show the structure of a CTNNB1(C16)-SOD1(C16)bis siRNA multi-target molecule in which each siRNA effector molecule sense strand is linked by a trinucleotide 2'-fluoro linker (UfUfUf), as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with the constructs. Figure 6A shows the sequence and modifications of the CTNNB1(C16)-SOD1(C16)bis siRNA complex with a 2'-fluoro linker (UfUfUf), including "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The respective sequences shown are SEQ ID NO: 20 for the fusion sense strand sequence, SEQ ID NO: 16 for the CTNNB1 antisense strand sequence, and SEQ ID NO: 14 for the SOD1 antisense strand sequence. Figure 6B shows the percentage of SOD1 (top) and CTNNB1 (bottom) remaining at day 21 after ICV injection of 100 μg of CTNNB1(C16)-SOD1(C16) bis siRNA molecules in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) for each individual mouse treated, for a cohort of four animals. Figure 6C shows the total percentage of SOD1 and CTNNB1 remaining at day 21 after ICV injection of 100 μg, as measured in the right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver. Results were analyzed for all four injected animals combined. [Figure 6-2] Same as above.

[0283] [Figure 7-1]Figures 7A-7C show the structure of SOD1(C16)-CTNNB1(C16)bis siRNA multi-target molecules in which each siRNA effector molecule sense strand is linked by a trinucleotide DNA linker (dTdTdT), as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with the constructs. Figure 7A shows the sequence and modifications of SOD1(C16)-CTNNB1(C16)bis siRNA complexes with DNA(dTdTdT) linkers, including "DNA" for DNA nucleotides, "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The respective sequences shown are SEQ ID NO: 22 for the fusion sense strand sequence, SEQ ID NO: 16 for the CTNNB1 antisense strand sequence, and SEQ ID NO: 14 for the SOD1 antisense strand sequence. Figure 7B shows the percentage of SOD1 (top) and CTNNB1 (bottom) remaining in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) at day 21 after ICV injection of 100 μg of SOD1(C16)-CTNNB1(C16) bis siRNA molecules for each individual mouse treated, for a cohort of four animals. (Note: Animal number 30 indicates an unsuccessful injection, and its data was excluded from subsequent analyses.) Figure 7C shows the total percentage of SOD1 and CTNNB1 remaining in the right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver at day 21 after ICV injection of 100 μg. Results are analyzed either for all four injected animals (top) or for only the three successfully injected animals (bottom). [Figure 7-2] Same as above.

[0284] [Figure 8-1]Figures 8A-8C show the structure of SOD1(C16)-CTNNB1(C16)bis siRNA multi-target molecules in which each siRNA effector molecule sense strand is linked by a trinucleotide 2'-O-methyl linker (uuu), as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with the constructs. Figure 8A shows the sequence and modifications of SOD1(C16)-CTNNB1(C16)bis siRNA complexes with 2'O-methyl linkers (uuu), including "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The respective sequences shown are SEQ ID NO: 23 for the fusion sense strand sequence, SEQ ID NO: 16 for the CTNNB1 antisense strand sequence, and SEQ ID NO: 14 for the SOD1 antisense strand sequence. Figure 8B shows the percentage of SOD1 (top) and CTNNB1 (bottom) remaining at day 21 after ICV injection of 100 μg of SOD1(C16)-CTNNB1(C16) bis siRNA molecules in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) for each individual mouse treated, for a cohort of four animals. Figure 8C shows the total percentage of SOD1 and CTNNB1 remaining at day 21 after ICV injection of 100 μg, measured in the right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver. Results were analyzed pooled across all four injected animals. [Figure 8-2] Same as above.

[0285] [Figure 9A]Figures 9A and 9B show the structures of the CTNNB1-SOD1(C16) and CTNNB1(C16)-SOD1 bis siRNA multi-target molecules, respectively, in which each siRNA effector molecule sense strand is connected by a trinucleotide DNA linker (dTdTdT), and only one effector molecule within each multi-target molecule possesses a C16-modified nucleotide (located within each effector molecule as shown). The levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with these constructs are also shown. 9A shows the sequences and modifications of CTNNB1-SOD1(C16) and CTNNB1(C16)-SOD1 bis siRNA complexes with DNA(dTdTdT) linkers, including "DNA" for DNA nucleotides, "2'OMe" for 2'-O-methyl modified nucleotides, "F" for 2'-fluoro modified nucleotides, "PS" for 3' phosphorothioate modified nucleotides, and "2-C16" for C16 modified nucleotides. The sequences shown are: (a) CTNNB1-SOD1(C16) bis siRNA complex with fusion sense strand sequence SEQ ID NO:21, CTNNB1 antisense strand sequence SEQ ID NO:16, and SOD1 antisense strand sequence SEQ ID NO:14; (b) CTNNB1(C16)-SOD1 bis siRNA complex with fusion sense strand sequence SEQ ID NO:24, CTNNB1 antisense strand sequence SEQ ID NO:16, and SOD1 antisense strand sequence SEQ ID NO:14. Figure 9B shows the measured levels of SOD1 and CTNNB1 remaining at day 21 after ICV injection of 100 μg of CTNNB1-SOD1(C16) bis siRNA molecules (left) and CTNNB1(C16)-SOD1 bis siRNA molecules (right) in each of the indicated tissues (right brain hemisphere, left brain hemisphere, cerebellum, brainstem, and liver) from each cohort of four animals. [Figure 9B] Same as above.

[0286] [Figure 10-1]Figures 10A-10E compare the target gene inhibition efficiencies observed for various bis-siRNA complexes disclosed herein targeting SOD1 and CTNNB1, including the duplexes shown in Figures 3A-3C, 4A-4C, 5A-5C, 6A-6C, 7A-7C, 8A-8C, 9A, and 9B, in all analyzed CNS tissues (right hemisphere, left hemisphere, cerebellum, and brainstem) compared to a mixed siRNA delivery format. Figure 10A shows the observed remaining levels of SOD1 (left) and CTNNB1 (right) after 21 days of treatment with each of the indicated bis-siRNA complexes (shown in Figures 3A, 4A, 5A, and 6A, respectively), measured in the right hemisphere of the brain, the left hemisphere of the brain, the cerebellum, and the brainstem, also compared to a mixed siRNA treatment format. Notably, the best inhibitory activity was observed with bis-siRNA complexes containing a DNA (dTdTdT) linker. Figure 10B shows the observed remaining levels of SOD1 (left) and CTNNB1 (right) 21 days after treatment with each of the indicated bis-siRNA complexes (shown in Figures 3A, 4A, 7A, and 8A, respectively) measured in the right hemisphere of the brain, the left hemisphere of the brain, the cerebellum, and the brainstem, also compared to the mixed siRNA treatment format. Notably, reversing the positions of the CTNNB1 and SOD1 effector molecules within the multi-targeting complex resulted in reduced activity for the bis-siRNA complex with the SOD1(C16)-CTNNB1(C16) configuration. Figure 10C shows the levels of SOD1 (left) and CTNNB1 (right) inhibition observed with a mixture of siRNAs, a stable and effective CTNNB1(C16)-SOD1(C16) bis siRNA duplex with a DNA linker (dTdTdT), and surprisingly, no inhibition was observed with two bis siRNA duplexes that each had a C16 modification on only one of the two effector molecules, i.e., CTNNB1-SOD1(C16) bis siRNA (having a C16 modification only on the SOD1-targeting siRNA effector molecule) and CTNNB1(C16)-SOD1 bis siRNA (having a C16 modification only on the CTNNB1-targeting siRNA effector molecule).The effects of all bis siRNA multi-target molecules tested, as well as the mixed siRNA control, on SOD1 (left) and CTNNB1 (right) levels were assessed in the right hemisphere, left hemisphere, cerebellum, and brainstem. Figure 10D shows a comparison of the effects of all bis siRNA multi-target molecules tested, as well as the mixed siRNA control, on SOD1 and CTNNB1 levels, broken down by target gene and measured in all brain tissues tested (right hemisphere, left hemisphere, cerebellum, and brainstem). Figure 10E shows a comparison of the effects of all bis siRNA multi-target molecules tested, as well as the mixed siRNA control, on SOD1 and CTNNB1 levels, broken down by location (upper left: right hemisphere, upper right: left hemisphere, lower right: cerebellum, and lower left: brainstem). A legend indicating the reference identifiers and associated structures used in Figures 10D and 10E is also shown. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0287] [Figure 11] FIG. 11 shows the degradation of bis-siRNA designs AM-183 to AM-190 in the CSF of rats after 0, 4, or 24 hours of incubation, or after 24 hours of incubation with PBS as a control.

[0288] [Figure 12] Figure 12 shows the nature of sense strand metabolites observed after 24 hours of incubation of bis-siRNA designs AM-183 to AM-190 in rat brain homogenates analyzed via MS, as described above.

[0289] [Figure 13] FIG. 13 shows a schematic diagram of the structure of the parent SOD1-targeting siRNA AD-401824, indicating the presence of SEQ ID NOs: 29 (top strand) and 31 (bottom strand).

[0290] [Figure 14]Figures 14A and 14B present study design information for testing a series of early bis-siRNA designs. Figure 14A shows the study design of three differently linked bis-siRNA designs compared with the parent siRNA and appropriate CSF control. Figure 14B shows the structure of the "Q315" linker used in the AM-182 bis-siRNA design.

[0291] [Figure 15A] Figures 15A and 15B show the results obtained with fluoro-conjugated bis-siRNA AM-178. Figure 15A shows a schematic of the fluoro-conjugated AM-178 bis-siRNA design. Figure 15B shows the tissue distribution after IT injection of AM-178 bis-siRNA compared to the parent siRNA AD-401824 at 7 and 28 days. [Figure 15B] Same as above.

[0292] [Figure 16A] Figures 16A and 16B show the results obtained with DNA-binding bis-siRNA AM-181. Figure 16A shows a schematic of the DNA-binding AM-181 bis-siRNA design. Figure 16B shows the tissue distribution after IT injection of AM-181 bis-siRNA compared to the parent siRNA AD-401824 at 7 and 28 days. [Figure 16B] Same as above.

[0293] [Figure 17A] Figures 17A and 17B show the results obtained with 3x Q315-conjugated bis-siRNA AM-182. Figure 17A shows a schematic of the 3x Q315-conjugated AM-182 bis-siRNA design. Figure 17B shows the tissue distribution after IT injection of AM-182 bis-siRNA compared to the parent siRNA AD-401824 at 7 and 28 days. [Figure 17B] Same as above.

[0294] [Figure 18]Figures 18A and 18B show the delivery levels of parental siRNA and bis-siRNA designs in the distal CSF, assessed on days 7 and 28. Figure 18A shows the results obtained for all tested animals. Figure 18B shows a chart in which, compared to Figure 18A above, two animals were removed: animal #11 (AM-181 on day 7) and #19 (AD-401824 parental siRNA on day 28). Notably, no CSF ​​was obtained from animal #30, the AM-182-treated animal on day 28, and no significant difference was observed in the CSF concentrations of the administered agents.

[0295] [Figure 19] Figures 19A and 19B show the actual values ​​of parental siRNA and bis-siRNA designs in plasma evaluated on day 7. Figure 19A shows the results obtained for all tested animals. Figure 19B shows a chart in which animal #11 (AM-181 on day 7) is removed, compared to Figure 19A above. Low levels of AM-182 were specifically observed on day 7.

[0296] [Figure 20] Figures 20A and 20B show the actual values ​​of the parental siRNA and bis-siRNA designs in plasma, evaluated on day 28. Specifically, no significant differences were observed in long-term pharmacokinetics up to day 28. Figure 20A shows the results obtained for all tested animals. Figure 20B shows a chart in which an animal (#19, AD-401824 on day 28) was removed, compared to Figure 20A above.

[0297] [Figure 21] Figures 21A and 21B show the results of bis-sense strand quantification. Figure 21A shows that after IT injection, intact AM-178 and AM-181 bis-siRNAs were detected in plasma 30 minutes after administration. Figure 21B also reveals that bis-sense strand quantification revealed that intact AM-178, but not AM-181, was detected in CSF on days 7 and 28.

[0298] [Figure 22-1]Figures 22A-22H show the structures of exemplary CTNNB1(C16)-SOD1(C16) bis siRNA multi-targeting molecules in which each siRNA effector molecule sense strand is linked by a trinucleotide DNA linker, as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with these constructs. All exemplary bis-siRNA complexes tested are shown in Figure 2A. Figure 22A shows the sequences, structures, and modification patterns of the SOD-targeting siRNA and CTNNB1-targeting siRNA. The sequences are the same as those shown in Figure 1A, and the modification patterns are the same as those shown in Figure 2B. Figure 22B summarizes the tRNA configuration patterns and linkers used for each bis-siRNA duplex in the exemplary bis-siRNA complexes used. Figure 22C shows the percentage of SOD1 and CTNNB1 remaining at day 21 for each individual mouse administered in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, and brainstem) after ICV injection of 100 μg of each of the CTNNB1(C16)-SOD1(C16)bis siRNA molecules for a cohort of four animals. Figure 22D shows the results for the percentage of SOD1 and CTNNB1 remaining in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, and brainstem) at 21 days after 100 μg ICV injection for each cohort of four animals, comparing mixed duplex delivery (a mixture of siRNAs targeting SOD1, AD-413709, and siRNAs targeting CTTNB1, shown in Figure 2A and Table 2) with several CTNNB1(C16)-SOD1(C16) bis siRNA molecules (AM-183, AM-184, AM-185, and AM-186, shown in Figure 2A and Table 2).Figure 22E shows the results for the percentage of SOD1 and CTNNB1 remaining in each indicated tissue (right brain hemisphere, left brain hemisphere, cerebellum, and brainstem) at 21 days after 100 μg ICV injection for each cohort of four animals, comparing bis-siRNA complexes with one C16 modification in the SOD1-targeting siRNA (SOD1-C16) or CTNNB1-targeting siRNA (CTNNB1-C16) with bis-siRNA complexes with two C16 modifications in both the SOD1-targeting siRNA and the CTNNB1-targeting siRNA (2C16 or CTNNB1(C16)-SOD1(C16)), and mixed duplex delivery (a mixture of siRNAs AD-413709 targeting SOD1 and AD-320650 targeting CTNNB1, as shown in Figure 2A and Table 2). Figure 22F shows the results for the percentage of SOD1 and CTNNB1 remaining in each designated tissue (right brain hemisphere, left brain hemisphere, cerebellum, and brainstem) at day 21 after 100 μg ICV injection in mice for each cohort of four animals, comparing several different bis siRNA molecules (AM-183, AM-184, AM-188, and AM-189, shown in Figure 2A and Table 2) by varying the position of each siRNA effector within the bis-siRNA complex. Figure 22G shows the percentage of SOD1 and CTNNB1 remaining in the liver at day 21 after 100 μg ICV injection of each of the CTNNB1(C16)-SOD1(C16) bis siRNA molecules for each individual mouse administered. Figure 22H shows the percentage of SOD1 remaining in the liver at day 21 after injection of parental SOD1-targeting siRNA AD-401824 (Table 4) at various doses (50 μg, 150 μg, or 300 μg) for cohorts of four animals. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 22-4] Same as above.

[0299] [Figure 23-1]Figures 23A-23E show the structure of exemplary CTNNB1(C16)-SOD1(C16) bis siRNA multi-target molecules, in which each siRNA effector molecule sense strand is linked by a carbohydrate-based linker, as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with these constructs. All exemplary bis siRNA complexes tested are shown in Figure 23A. An exemplary circular bis-sciRNA (AM-206) is also illustrated in Figure 23A. Figure 23A summarizes the duplex ID, sense strand ID, target, and linker, as well as the control mixed duplex. The number of mice in the cohort tested, the day of target inhibition assessment, the dose employed, and the position of the resulting readout are also shown. All bis siRNA complexes contain two sets of 21-mer sense strands and 23-mer antisense strands. The sense strands of both siRNAs are contiguous due to the inclusion of a linker, while each antisense strand of the siRNA duplex is a non-contiguous, independent strand. Figure 23B shows the structures of various carbohydrate-based linkers in the exemplary bis-siRNA complex used in Figure 23A. Figure 23C summarizes the sequences, structures, organization patterns, and linkers used in each bis siRNA duplex of the exemplary bis-siRNA complex, as well as the exemplary circular bis-sciRNA (AM-206) used in Figure 23A. Figure 23D shows the percentage of SOD1 and CTNNB1 remaining in the brain at 21 days after ICV injection of 100 μg each of the CTNNB1(C16)-SOD1(C16) bis siRNA molecule and an exemplary cyclic bis-siRNA (AM-206) for each individual mouse administered, for a cohort of four animals, comparing bis siRNA complexes bearing three-carbohydrate-based linkers (AM-203, AM204, AM205) with bis siRNA complexes bearing three-nucleotide linkers (AM183, AM202) and with mixed duplex delivery (a mixture of siRNAs AD-401824 targeting SOD1 and AD-503801 targeting CTTNB1, as shown in Figure 23C).Figure 23E shows the percentage of SOD1 and CTNNB1 remaining at 21 days in each designated tissue (liver, heart) for each individual mouse administered, following ICV injection of 100 μg of each of the CTNNB1(C16)-SOD1(C16) bis siRNA molecule and an exemplary cyclic bis-siRNA (AM-206), for a cohort of four animals, comparing bis siRNA complexes bearing three-carbohydrate-based linkers (AM-203, AM204, AM205) to bis siRNA complexes bearing three-nucleotide linkers (AM183, AM202) and to mixed duplex delivery (a mixture of siRNAs AD-401824 targeting SOD1 and AD-503801 targeting CTTNB1, as shown in Figure 23C). [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above.

[0300] [Figure 24-1]Figures 24A-24D show the structures of exemplary CTNNB1(C16)-SOD1(C16) bis siRNA multi-target molecules, in which each siRNA effector molecule sense strand is attached by various linkers and has various chemical modifications in the bis-siRNA, as well as the respective mSOD1 and mCTNNB1 inhibition levels observed in mice injected with these constructs. All exemplary bis-siRNA complexes tested are shown in Figure 24A. An exemplary circular bis-sciRNA (AM-206) is also illustrated in Figure 24A. Figure 24A summarizes the duplex IDs, linkers, and chemistries, as well as the control mixed duplex. Also shown are the number of rats in the cohort tested, the duration of target inhibition evaluation, the doses employed, and the position of the resulting readouts. All bis siRNA complexes contain two sets of 21mer sense strands and 23mer antisense strands, and the sense strands of both siRNAs are made continuous by including a single-stranded trinucleotide linker, while each antisense strand of the siRNA duplex is a discontinuous independent strand. Figure 24B shows the structure of an exemplary bis-siRNA complex used in Figure 24A and various linkers in an exemplary circular bis-siRNA. Figure 24C summarizes the sequence, structure, composition pattern, and linker used in each bis siRNA duplex of the exemplary bis-siRNA complex used in Figure 24A. Figure 24D shows the percentage of SOD1 and CTNNB1 remaining at days 15 and 29, respectively, for cohorts of four animals in each designated tissue (thoracic spinal cord, frontal lobe, hippocampus, and striatum) following intrathecal (IT) administration (0.3 mg) of each of the CTNNB1(C16)-SOD1(C16) bis siRNA molecules (various linkers connecting each sense strand of the individual effector molecules (siRNAs) and containing various chemical modifications in the bis-siRNA molecule, as shown in Figures 24A-24C) and an exemplary circular bis-sciRNA (AM-206) at t0, compared to mixed duplex delivery (a mixture of siRNAs, AD-401824 targeting SOD1 and AD-503801 targeting CTTNB1, as shown in Figure 23C). [Figure 24-2] Same as above. [Figure 24-3] Same as above.

[0301] [Figure 25A] Figures 25A-25D show the structures of exemplary CTNNB1(C16)-SOD1(C16) bis siRNA multi-target molecules, in which each siRNA effector molecule sense strand is linked by various linkers, as well as the levels of mSOD1 and mCTNNB1 inhibition observed in mice injected with these constructs. All exemplary bis siRNA complexes tested are shown in Figure 25A. Figure 25A summarizes the duplex IDs, linkers, chemistries, and control mixed duplexes. The number of rats in the cohort tested, the duration of target inhibition assessment, the doses employed, and the positions of the resulting readouts are also shown. All bis siRNA complexes contain two sets of 21-mer sense strands and 23-mer antisense strands. The sense strands of both siRNAs are contiguous by including a trinucleotide single-stranded linker, while each antisense strand of the siRNA duplex is a discontinuous, independent strand. Figure 25B shows the structures of various linkers in the exemplary bis-siRNA complexes used in Figure 25A. Figure 25C summarizes the sequence, structure, organization pattern, and linker used in each bis siRNA duplex of the exemplary bis-siRNA complex used in Figure 25A. Figure 25D shows the percentage of SOD1 and CTNNB1 remaining at day 15 for cohorts of four animals in each designated tissue (thoracic spinal cord, cerebellum, frontal lobe, hippocampus, and striatum) following intrathecal (IT) administration (0.6 mg) of each of the CTNNB1(C16)-SOD1(C16) bis siRNA molecules (containing various linkers connecting each sense strand of the individual effector molecules (siRNAs) as shown in Figures 25A-25C) at t0 for each individual mouse administered, compared to mixed duplex delivery (a mixture of siRNAs, AD-401824 targeting SOD1 and AD-503801 targeting CTTNB1, as shown in Figure 23C). [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above.

[0302] [Figure 26A] Figures 26A-26D are summaries showing the in vivo stability of exemplary bis-siRNAs and / or circular bis-siRNAs after incubation of the molecules in rat brain homogenate, as measured using LC-MS. Figure 26A shows the stability of exemplary bis-siRNAs with various linker chemistries (AM-183 to AM-186) in rat brain homogenate. Figure 26B shows the stability of exemplary bis-siRNAs of various orientations (AM-183, AM-184, AM-188, and AM-189) in rat brain homogenate. Figure 26C shows the stability of exemplary bis-siRNAs (AM-190 and AM-187) bearing one C16 modification in the SOD1- or CTNNB1-targeting siRNA, and the stability of a bis-siRNA complex (AM-183) bearing two C16 modifications in both the SOD1- and CTNNB1-targeting siRNA, compared to a duplex mixture (AD-320650 and AD-413709). Figure 26D shows the metabolic trends of exemplary bis-siRNAs and exemplary circular bis-siRNAs in rat brain homogenate for CNS targeting (AM-183, AM-202, AM-203, AM-204, AM-205, and AM-206) and liver targeting (AM-191, AM-207, AM-208, AM-209, AM-210, and AM-211). [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above.

[0303] [Figure 27]Figure 27A is a schematic diagram of an exemplary GalNAc-sciRNA duplex. Figure 27B shows the chemical modifications used in the exemplary GalNAc-sciRNA duplex.

[0304] [Figure 28-1] Figures 28A-28D are graphs of enzymatic digestion decay curves using single-stranded poly 2'-deoxy linear and cyclic oligonucleotides (ON-3 and ON-4, respectively) and single-stranded fully 2'-modified linear and cyclic oligonucleotides (ON-5 and ON-6, respectively) in in vitro assays using either 3'-exonucleases (Figures 28A and 28B) and 5'-exonucleases (Figures 28C and 28D). [Figure 28-2] Same as above.

[0305] [Figure 29] Figures 29A-29B are graphs showing the stability of the full-length sense strand in plasma and liver homogenate after incubation of the duplex, as measured using LC-MS. Figure 29A shows the mean natural logarithm of the fraction of sense strand remaining in rat plasma. Figure 29B shows the mean natural logarithm of the fraction of sense strand remaining in rat liver homogenate. Averages are plotted. Error bars are standard deviations from triplicates per time point.

[0306] [Figure 30] Figure 30 shows the imino region of the 1D 1H NMR spectra of GalNAc-siRNAs with linear structures (Table 9, si-1, si-2, si-3, and si-6) and GalNAc-sciRNA duplexes with circular structures (Table 9, si-4 and si-5). The imino protons engaged in Watson-Crick base pairing exhibit chemical shift values ​​in the δ range of 12–14 ppm.

[0307] [Figure 31]Figures 31A-31B are graphs of the pharmacological profiles in mice after a single subcutaneous administration of linear GalNAc-siRNA (Table 9, si-1, si-2, and si-6) and cyclic GalNAc-sciRNA (Table 9, si-4, si-5, and si-7) conjugates. A single dose of each conjugate (3 mg / kg) was administered to mice on day 0, and serum was collected on days 0 (pre-administration), 3, 7, and 14. Circulating serum protein levels were determined for TTR (Figure 31A) and C5 (Figure 31B) using appropriate ELISA kits compared to PBS controls. Error bars represent SD (n=3).

[0308] [Figure 32] Figures 32A-32B are graphs showing whole liver levels of antisense strands of linear GalNAc-siRNA (Table 9, si-1, si-2, and si-3) and cyclic GalNAc-sciRNA (Table 9, si-4 and si-5) conjugates and Ago2 levels in mice. Figure 32A shows liver levels of antisense strands isolated and measured from whole mouse liver. Figure 32B shows levels of antisense strands isolated and measured from immunoprecipitated Ago2 from whole mouse liver. Mice were administered a single dose of each conjugate (3 mg / kg) on ​​day 0, and livers were harvested on day 7. Levels were determined using SL-RT QPCR compared to PBS controls. Error bars are SD (n=3).

[0309] [Figure 33] Figure 33 shows a model of the sciRNA:Ago2 complex based on the crystal structure of Ago2 bound to double-stranded RNA with seed region pairing. The Z linker carbons are highlighted. Selected side chains of the Ago2 PIWI and MID domains and the L2 linker region are labeled. The diagram is a cross-section of the major groove (top) and minor groove (bottom) of the seed region duplex. DETAILED DESCRIPTION OF THE INVENTION

[0310] The present disclosure is based, at least in part, on the discovery of molecules that target two or more target nucleic acids and exhibit stable and surprising efficacy in CNS tissue of a subject following CNS-directed administration of the multi-targeted molecule. CNS-directed delivery and efficacy of the multi-targeted molecule are herein identified as stable when each effector molecule of the multi-targeted molecule contains at least one lipophilic moiety, and delivery and efficacy have been observed to be significantly reduced in CNS tissue for multi-targeted molecules that do not carry at least one lipophilic moiety conjugated to each effector molecule. Pharmaceutical compositions, injectables, methods (including therapeutic methods), and other related aspects are also described herein.

[0311] Bis siRNA compounds (two effector molecules connected by a bis-linker) In one embodiment, provided herein are multi-targeting molecules based on bis siRNA compounds.

[0312] Generally, a multi-target molecule comprises at least two nucleic acid-based effector molecules, which are covalently or non-covalently linked to each other. Without being limited thereto, any nucleic acid-based effector molecule capable of regulating gene expression of a target can be included in the multi-target molecule disclosed herein.

[0313] A multi-targeting molecule comprises at least two nucleic acid-based effector molecules linked to each other by a linker moiety (e.g., a nucleic acid sequence, one or more carbohydrate moieties, or other organic polymer, optionally comprising a cleavable form of the linker moiety) described herein. Each of the at least two nucleic acid-based effector molecules of a multi-targeting molecule comprises a lipophilic moiety (e.g., a saturated or unsaturated C 16 The multi-targeting molecules may carry a hydrocarbon chain, thereby facilitating effective CNS targeting of each molecular target of the multi-targeting molecule. Any nucleic acid-based effector molecule capable of regulating gene expression of a target may be included in the multi-targeting molecules disclosed herein, without limitation.

[0314] Thus, in certain embodiments, the present disclosure provides a multi-targeting molecule for modulating one or more distinct target RNA sequences in one or more target RNAs in a subject's central nervous system (CNS), the multi-targeting molecule comprising at least two nucleic acid-based effector molecules, the effector molecules connected together by a linker and not overlapping with one another, the at least two effector molecules each having at least one conjugated lipophilic moiety, when delivered to the subject's central nervous system (CNS) and capable of inhibiting the activity or expression of one or more target RNAs in the subject's CNS tissue by at least 15% relative to a suitable control.

[0315] Two target RNA sequences within a single target RNA are considered "distinct" if the target RNA sequences do not overlap with each other.

[0316] In certain embodiments, two nucleic acid-based effector molecules both target the same target RNA sequence. In such embodiments, the multi-targeting molecule may have a "symmetric" design (e.g., a linker may connect the 3'-end or 5'-end of the sense strands of two identical siRNAs). In certain embodiments, two nucleic acid-based effector molecules target different target RNA sequences. In such embodiments, the multi-targeting molecule has an "asymmetric" design. In the latter case, both nucleic acid-based effector molecules may also be "asymmetric" if they target the same target RNA but "distinct" target RNA sequences.

[0317] As used herein, a "suitable control" refers to either a composition that is otherwise identical to the composition containing, but lacking, the relevant active agent, or a composition that contains an active agent (e.g., an oligonucleotide) that does not target the relevant target nucleic acid. An otherwise identical composition lacking the active agent may, for example, contain a buffer used for parenteral administration, such as phosphate-buffered saline (PBS) or artificial cerebrospinal fluid (aCSF). aCSF may include a sterile aqueous composition having a pH of about 7.2 and the following ion concentrations (mM): Na + 150, K + 3.0, Ca 2+ 1.4, Mg 2+ 0.8, P 1.0, and Cl - 155. Oligonucleotides that do not target a related target nucleic acid include, for example, polyadenoside-based oligonucleotides such as AD-77748 (see Tables 2 and 3).

[0318] "Nucleic acid-based effector molecule" refers to a modified or unmodified single-stranded or double-stranded nucleic acid molecule capable of regulating the expression activity of a target nucleic acid. In some embodiments, the nucleic acid-based effector molecule is a modified or unmodified single-stranded or double-stranded nucleic acid molecule capable of regulating the gene expression of a target nucleic acid. Examples of 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 (e.g., siRNAs and shRNAs, also referred to herein as dsRNA agents), ribozymes, triplex-forming oligonucleotides, decoy oligonucleotides, immunostimulatory oligonucleotides, RNA activators, U1 adaptors, guide RNAs (gRNAs) for CRISPR Cas, combinations thereof, and the like. In certain embodiments, the single-stranded or double-stranded nucleic acid molecules of the effector molecule each contain at least one modified nucleotide or at least one modified internucleotide linkage.

[0319] Please 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, it is different from a double-stranded effector molecule in which each strand is directed to a different target, or it is different from an effector molecule in which at least a portion of the sequence is complementary to or can hybridize with two different target sequences.

[0320] In some embodiments, the multi-target molecule is assembled from two different siRNA molecules, at least one of which has at least one ligand that binds to it, while in some other embodiments, the multi-target molecule is assembled from two different siRNA molecules, each of which has at least one ligand that binds to it.

[0321] In various embodiments of the multi-target molecule, at least two siRNAs each have at least one ligand and are conjugated to each other, and the at least two ligands may be the same or different. Furthermore, the at least two ligands may be independently conjugated to any position of each siRNA. For example, one ligand may be conjugated to the sense strand of the first siRNA, and the other ligand may be conjugated to the sense strand of the second siRNA. Alternatively, one ligand may be conjugated to the sense strand of the first siRNA, and the other ligand may be conjugated to the antisense strand of the second siRNA. Alternatively, one ligand may be conjugated to the antisense strand of the first siRNA, and the other ligand may be conjugated to the antisense strand of the second siRNA. Without limitation, the first ligand may be independently conjugated to the 5'-end, 3'-end, or internal (non-terminal) position of one strand (sense or antisense) of the first siRNA. Similarly, the second ligand may be independently conjugated to the 5'-end, 3'-end, or internal (non-terminal) position of one strand (sense or antisense) of the second siRNA.

[0322] In some embodiments, one ligand is conjugated to the 3' end of the sense strand of a first siRNA, and the other ligand is conjugated to the 3' end of the antisense strand of a second siRNA.

[0323] In some embodiments, one ligand is conjugated to the 5'-end of the sense strand of the first siRNA, and the other 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 the other 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 the other 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 the other ligand is conjugated to an internal (non-terminal) 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 the other ligand is conjugated to an internal (non-terminal) 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 the other ligand is conjugated to the internal (non-terminal) 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 the other ligand is conjugated to the internal (non-terminal) position of the sense strand of the second siRNA.In some embodiments, one ligand is conjugated to the internal (non-terminal) position of the antisense strand of the first siRNA, and the other ligand is conjugated to the internal (non-terminal) position of the sense strand of the second siRNA.

[0324] In some embodiments, one ligand is conjugated to the 3'-end of the first sense strand, and the other ligand is conjugated to the 3'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first sense strand, and the other ligand is conjugated to the 5'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first sense strand, and the other ligand is conjugated to the 3'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first sense strand, and the other ligand is conjugated to the 5'-end of the second sense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first sense strand, and the other ligand is conjugated to an internal (non-terminal) position of the second sense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first sense strand, and the other ligand is conjugated to an internal (non-terminal) position of the second sense strand. In some embodiments, one ligand is conjugated to an internal (non-terminal) position of the first sense strand, and the other ligand is conjugated to an internal (non-terminal) position of the second sense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to the 3'-end of the second antisense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to the 5'-end of the second antisense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first antisense strand, and the other ligand is conjugated to the 3'-end of the second antisense strand. In some embodiments, one ligand is conjugated to the 5'-end of the first antisense strand, and the other ligand is conjugated to the 5'-end of the second antisense strand. In some embodiments, one ligand is conjugated to the 3'-end of the first antisense strand, and the other ligand is conjugated to an internal (non-terminal) position of the second antisense strand.In some embodiments, one ligand is conjugated to the 5'-end of the first antisense strand, and the other ligand is conjugated to an internal (non-terminal) position of the second antisense strand. In some embodiments, one ligand is conjugated to an internal (non-terminal) position of the first antisense strand, and the other ligand is conjugated to an internal (non-terminal) position of the second antisense strand.

[0325] In some embodiments, the multi-target molecule is assembled from two siRNAs, in which 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 linked to each other in any direction.For example, the 3' end of the first sense strand can be linked to the 5' end of the second sense strand.The 3' end of the first sense strand can be linked to the 3' end of the second sense strand.Or the 5' end of the first sense strand can be linked to the 5' end of the second sense strand.

[0326] In some embodiments, the multi-target molecule is assembled from two siRNAs, in which 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 linked to each other in any direction.For example, the 3' end of the first antisense strand can be linked to the 5' end of the second antisense strand.The 3' end of the first antisense strand can be linked to the 3' end of the second antisense strand.Or the 5' end of the first antisense strand can be linked to the 5' end of the second antisense strand.

[0327] In some embodiments, the multi-target molecule is assembled from two siRNAs, in which 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 direction.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.

[0328] In some embodiments, a multi-targeting molecule regulates two or more distinct target RNAs in the central nervous system (CNS), and the multi-targeting molecule is assembled from two double-stranded RNAs (dsRNAs) that target two or more distinct target RNAs, and the orientation of the two dsRNAs with respect to the linker connecting them may be varied.

[0329] In some embodiments, the multi-target molecule is assembled from two dsRNAs according to the following formula: dsRNA1-L-dsRNA2, wherein dsRNA1 is a first dsRNA targeting a first target RNA sequence, dsRNA2 is a second dsRNA targeting a second, different target RNA sequence, and L is a linker connecting dsRNA1 to dsRNA2. L connects the 3' end of the sense strand of dsRNA1 to dsRNA2 and / or the 5' end of the antisense strand of dsRNA1 to dsRNA2. In one embodiment, the multi-target molecule is represented by: 5' ss1-L- ss2 3' 3' as1 as2 5', In the formula, ss1 is the sense strand of dsRNA1, as1 is the antisense strand of dsRNA1, ss2 is the sense strand of dsRNA2, as2 is the antisense strand of dsRNA2, and L connects the 3'-end of ss1 to the 5'-end of ss2.

[0330] In some embodiments, the multi-target molecule is assembled from two dsRNAs according to the following formula: dsRNA2-L-dsRNA1, wherein dsRNA1 is a first dsRNA targeting a first target RNA sequence, dsRNA2 is a second dsRNA targeting a second, different target RNA sequence, and L is a linker connecting dsRNA2 to dsRNA1. L connects the 3' end of the sense strand of dsRNA2 to dsRNA1 and / or the 5' end of the antisense strand of dsRNA2 to dsRNA1. In one embodiment, the multi-target molecule is represented by: 5' ss2-L-ss1 3' 3' as2 as1 5', In the formula, ss2 is the sense strand of dsRNA2, as2 is the antisense strand of dsRNA2, ss1 is the sense strand of dsRNA1, as1 is the antisense strand of dsRNA1, and L connects the 3'-end of ss2 to the 5'-end of ss1.

[0331] In some embodiments, the multi-targeting molecule is assembled from two siRNAs, in which the sense strand of a first siRNA is covalently linked to the sense strand of a second siRNA, and the antisense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA.

[0332] In some embodiments, the multi-targeting molecule is assembled from two siRNAs, in which the antisense strand of a first siRNA is covalently linked to the sense strand of a second siRNA, and the sense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA.

[0333] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is a ribozyme. In some embodiments, the multi-targeting molecule comprises at least two ribozymes. Without limitation, the ribozymes may be the same or different.

[0334] In some embodiments, at least one of the effector molecules in a multi-targeting molecule disclosed herein is an siRNA and at least one of the effector molecules is a ribozyme.

[0335] 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. Without limitation, the aptamers may be the same or different.

[0336] In some embodiments, at least one of the effector molecules in a multi-target molecule disclosed herein is an siRNA and at least one of the effector molecules is an aptamer.

[0337] In some embodiments, at least one of the effector molecules in the multi-target molecule disclosed herein is a decoy oligonucleotide. In some embodiments, the multi-target molecule comprises at least two decoy oligonucleotides. Without limitation, the decoy oligonucleotides may be the same or different.

[0338] In some embodiments, at least one of the effector molecules in the multi-targeting molecules disclosed herein is an siRNA and at least one of the effector molecules is a decoy oligonucleotide.

[0339] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is a U1 aptamer. In some embodiments, the multi-target molecule comprises at least two U1 aptamers. Without limitation, the U1 aptamers may be the same or different.

[0340] In some embodiments, at least one of the effector molecules in a multi-target molecule disclosed herein is an siRNA and at least one of the effector molecules is a U1 aptamer.

[0341] In some embodiments, at least one of the effector molecules in the multi-target molecules disclosed herein is an activating aptamer. In some embodiments, the multi-target molecule includes at least two activator RNAs. Without limitation, the activator RNAs may be the same or different.

[0342] In some embodiments, at least one of the effector molecules in a multi-target molecule disclosed herein is an siRNA and at least one of the effector molecules is an activator RNA.

[0343] In some embodiments, at least one of the effector molecules in the multi-target molecule disclosed herein is a triplex-forming oligonucleotide. In some embodiments, the multi-target molecule comprises at least two triplex-forming oligonucleotides. Without limitation, the triplex-forming oligonucleotides may be the same or different.

[0344] In some embodiments, at least one of the effector molecules in a multi-targeting molecule disclosed herein is an siRNA and at least one of the effector molecules is a triplex-forming oligonucleotide.

[0345] lipophilic part Certain embodiments of the present disclosure feature lipophilic moieties conjugated to multiple targeting molecules. Lipophilic moieties / ligands have been identified as particularly useful for achieving CNS delivery and effective knockdown of target RNA in the CNS in the context of nucleic acid therapeutics. Examples of lipophilic moieties for use herein include, but are not limited to, lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, and phenoxazine.

[0346] The lipophilic moiety can be saturated or unsaturated C4-C 30 Optionally, the lipophilic moiety of the present disclosure contains a saturated or unsaturated C6-C 18 Optionally, the lipophilic moiety of the present disclosure may comprise a saturated or unsaturated C 16 It may also contain a hydrocarbon chain.

[0347] In certain embodiments, each lipophilic moiety is a saturated or unsaturated C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , and C 22 The hydrocarbon chains may be independently selected from:

[0348] In certain embodiments, each lipophilic moiety is linear and may be saturated or unsaturated C6, C8, C 10 , C 12 , C 14 , C 16, C 18 , C 20 , and C 22 The hydrocarbon chains may be independently selected from:

[0349] In certain embodiments, each lipophilic moiety is a linear saturated C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 , and C 22 The hydrocarbon chains may be independently selected from:

[0350] As described herein, lipophilic moieties can be conjugated to the multi-targeting molecules of the present disclosure via monovalent, or branched, bivalent, or trivalent linkers.

[0351] In some embodiments, at least one lipophilic moiety is conjugated to multiple targeting molecules via a monovalent linker, or a branched bivalent or trivalent linker.

[0352] Certain embodiments include the following C as specific exemplified forms of lipophilic molecules: 16 Characterized by hydrocarbon chain molecules: [ka] wherein B is a nucleotide base or a nucleotide base analog, and optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0353] Linker In certain embodiments, linkers are employed to connect the effector molecules of the multi-targeting molecules of the present disclosure. A range of specifically contemplated linkers are available for conjugating the effector molecules of the multi-targeting molecules of the present disclosure, including, but not limited to, DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, or other organic polymer linkers, and combinations thereof.

[0354] In some embodiments, at least two nucleic acid-based effector molecules in a multi-targeting molecule of the present disclosure can be covalently linked to one another via a nucleotide-based linker or a non-nucleotide-based linker generally known in the art (see, e.g., WO2017 / 05109 and WO2018 / 136620, each of which is incorporated by reference in its entirety) and described herein. Thus, in some embodiments, at least two effector molecules in a multi-targeting molecule are linked via a nucleotide-based linker. In some other embodiments, at least two effector molecules are linked via a non-nucleotide-based linker.

[0355] It should be noted that a nucleotide-based linker can form part of one or both of the effector molecules that are connected together. That is, at least a portion of the nucleotide sequence of the linker is required for the function of one of the effector molecules. In certain embodiments, the nucleotide sequence of the linker does not form part of the effector molecule. In other words, one of the effector molecules does not require any portion of the linker nucleotide sequence to regulate gene expression. For example, if the linker sequence is removed from the effector molecule, the effector molecule can still regulate gene expression at a level similar to that when the linker was present (e.g., within 95%). If the effector molecule requires complementarity with a target gene to be active, the linker may or may not be part of the effector molecule that is required for complementarity with the target sequence. In some embodiments, the linker does not have complementarity with the target sequence (e.g., less than 5% complementarity) or does not hybridize to the target sequence.

[0356] For nucleic acid linkers, oligonucleotides of any length and modification pattern can be employed, with examples of optional linker lengths including, but not limited to, lengths of 1 to 30 nucleotides. Optionally, the linker length is 2 to 20 nucleotides, optionally 2 to 15 nucleotides, optionally 2 to 10 nucleotides, optionally 2 to 5 nucleotides, and optionally 2, 3, or 4 nucleotides in length.

[0357] The nucleotide linker may be single-stranded or double-stranded. In some embodiments, the first strand of a double-stranded nucleotide-based linker connecting 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 connecting two effector molecules.

[0358] Without limitation, the nucleotide-based linker connecting the effector molecules can be all DNA, all RNA, or a mixture of DNA and RNA. In some embodiments, the nucleotide-based linker connecting two effector molecules is all DNA. RNA and DNA can be natural or modified. Thus, in some embodiments, the nucleotide-based linker connecting the effector molecules includes at least one modification selected from the following: a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. Examples of linker modifications include, but are not limited to, locked nucleic acids (e.g., LNA, ENA, and BNA), 2'-O-alkylnucleosides, 2'-halonucleosides (e.g., 2'-F nucleotides), 2'-aminonucleosides, 2'-S-alkylnucleosides, abasic nucleosides, 2'-cyanonucleosides, 2'-mercaptonucleosides; 2'-MOE nucleosides, acyclic nucleosides, (S)-cEt monomers, and modified internucleotide linkages (e.g., phosphodiester, phosphotriester, hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate, phosphorothioate, phosphorodithioate, methylenemethylimino, thio Examples of modifications include diesters, thionocarbamates, N,N'-dimethylhydrazine, phosphoroselenates, boranophosphates, boranophosphate esters, amides, hydroxylaminos, siloxanes, dialkylsiloxanes, carboxamides, carbonates, carboxymethyls, carbamates, carboxylic acid esters, thioethers, ethylene oxide linkers, sulfides, sulfonates, sulfonamides, sulfonate esters, thioformacetals, formacetals, oximes, methyleneiminos, methylenecarbonylaminos, methylenemethyliminos, methylenehydrazos, methylenedimethylhydrazos, methyleneoxymethyliminos, ethers, thioethers, and thioacetamides. Such modifications can also be present in non-linker elements of the multiple target molecules of the present disclosure. Nucleic acid modifications are described in more detail elsewhere in this disclosure.

[0359] In some embodiments, at least one of the internucleoside linkages between the linker and the effector molecule connecting the effector molecules is a modified internucleoside linkage. In some embodiments, the internucleoside linkage connecting the 5'-end of the linker to the 3'-end of one of the effector molecules is a modified internucleoside linkage. In some embodiments, the internucleoside linkage connecting the 3'-end of the linker to the 5'-end of one of the effector molecules is a modified internucleoside linkage.

[0360] In some embodiments, the first (e.g., first, second, third, fourth, or fifth) internucleoside linkage at the 5'-end and / or 3'-end of the linker connecting the effector molecule is a modified internucleoside linkage, hi some embodiments, one, two, three, four, five, or more internucleoside linkages from the 5'-end and / or 3'-end of the linker are modified internucleoside linkages.

[0361] In some embodiments, the linker connecting the effector molecules comprises at least one (e.g., one, two, three, four, five, six, or more) modified internucleoside linkage at an internal (non-terminal) position of the linker.

[0362] Without limitation, the nucleotide-based linker connecting the effector molecules can be of any desired length. For example, the nucleotide-based linker connecting 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 connecting the effector molecules can range in length from 1 nucleotide to 5 nucleotides. In certain embodiments, the nucleotide-based linker connecting two effector molecules is 4 nucleotides in length.

[0363] When a nucleotide-based linker connecting effector molecules contains a nucleic acid modification, the modification can be located at any position in the linker. For example, the modification can be at the 5'-nucleotide, the 3'-nucleotide, or an internal (non-terminal) nucleotide of the linker. In some embodiments, the first (e.g., the first, second, third, fourth, or fifth) nucleotide at the 5'-end 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'-end and / or 3'-end of the linker contain a nucleic acid modification. In some embodiments, one, two, three, four, five, or more internal (non-terminal) nucleotides of the linker contain a nucleic acid modification. In some embodiments, the internal (non-terminal) nucleotides of the linker comprise all DNA on the sense strand. In other embodiments, the internal (non-terminal) nucleotides of the linker comprise a mixture of DNA and 2'-O alkyl modifications on the antisense strand.

[0364] The nucleotide-based linker connecting the effector molecules may comprise one or two nucleic acid strands, and may be single-stranded, double-stranded, or may comprise a single-stranded and double-stranded region. In some embodiments, the nucleotide-based linker connecting the effector molecules comprises two nucleic acid strands that do not form a double-stranded structure. In other words, the nucleotide-based linker comprises two strands that do not hybridize to each other.

[0365] In some embodiments, a nucleotide-based linker connecting effector molecules comprises two nucleic acid strands, wherein the nucleotide sequence of a first strand of the linker comprises at least one (e.g., one, two, three, four, five, or more) nucleotide mismatch with the nucleotide sequence of a second strand of the linker. In some embodiments, at least one of the linker strands comprises a bulge or loop. For example, at least one of the linker strands comprises at least one (e.g., one, two, three, four, five, or more consecutive or non-consecutive) nucleobase that is non-complementary to the other linker strand.

[0366] Without limitation, the nucleotide-based linker connecting the effector molecules may contain one or more nucleic acid modifications disclosed herein. When the nucleotide-based linker connecting the effector molecules comprises two nucleic acid strands, each strand may independently be unmodified or may contain one or more nucleic acid modifications disclosed herein. Thus, in some embodiments, the nucleotide-based linker connecting the effector molecules comprises two nucleic acid strands, each of which is unmodified. In some embodiments, the nucleotide-based linker connecting the effector molecules comprises two nucleic acid strands, where one strand is unmodified and the other strand contains at least one modification selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. In some embodiments, the nucleotide-based linker connecting the effector molecules comprises two nucleic acid strands, where both strands contain at least one modification independently selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof.

[0367] In some embodiments, the nucleotide-based linker connecting the effector molecules comprises two nucleic acid strands, where one of the strands comprises all DNA and the other strand comprises a mixture of DNA and 2'-O alkyl modifications.

[0368] The nucleotide-based linker connecting the effector molecules can be resistant to degradation or cleavage by single-stranded or double-stranded nucleases. Alternatively, the nucleotide-based linker connecting the effector molecules can be a cleavable linker. For example, the linker connecting the effector molecules can be cleaved by single-stranded or double-stranded nucleases.

[0369] As described herein, the linker connecting the effector molecules in the multi-targeting molecule may be a non-nucleotide-based linker. In some embodiments, the non-nucleotide-based linker connecting the two oligonucleotides comprises a cleavable group.

[0370] In some embodiments, the non-nucleotide-based linker connecting the two oligonucleotides comprises at least one disulfide bond.

[0371] In certain 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 with at least one ligand. Without limitation, the ligand can be present anywhere in the multi-target molecule. For example, the ligand can be present at one end of one of the at least two effector molecules covalently linked by the linker, at an internal (non-terminal) position of one of the at least two effector molecules covalently linked by the linker, or at a position within the linker.

[0372] In some embodiments, a multi-target molecule comprising at least two covalently linked effector molecules is conjugated with at least one ligand. Without limitation, the ligands may be the same or different. The two ligands may be independently conjugated to any position on the multi-target molecule. For example, a first ligand may be present in a first effector molecule and a second ligand may be present in a linker connecting the first and second effector molecules, or a first ligand may be present in a first effector molecule and a second ligand may be present in a 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 a linker connecting the effector molecules.

[0373] In some embodiments, the linker connecting the effector molecule comprises a ligand. Without limitation, the ligand can be located at any position within the linker. For example, the ligand can be conjugated to the middle position of the linker, or within one, two, or three monomers or units within the middle of the linker.

[0374] In some embodiments, the multi-targeting molecule is assembled from two siRNAs, wherein the two siRNAs are covalently linked to each other via a nucleotide-based or non-nucleotide-based linker. In some embodiments, the linker connecting the two siRNAs comprises the nucleotide sequence UUU or (dT)n, where n is 1 to 20. In some embodiments, the linker connecting the effector molecules comprises a molecule selected from the group consisting of: -(CH2) 12 -("C12 linker" or "Q50"), -(CH2)6-SS-(CH2)6- ("C6-SS-C6 linker" or "Q51"), ·Q151, ·Q173, -CH2CH2O-(CH2CH2) n -CH2CH2O-CH2CH2O-, where n is 0 or 1 to 20; -(CH2)9-(CH2) n -CH2-, where n is 0 or 1 to 20; mono-, di-, tri-, tetra-, penta-, or poly-prolinol, optionally conjugated to a ligand; and Mono-, di-, tri-, tetra-, penta-, or poly-hydroxyprolinol (e.g., poly[4-hydroxyprolinol]), optionally conjugated to a ligand.

[0375] In some embodiments, the multi-target molecule is assembled from two siRNAs, and in this case, 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 linked to each other in any direction.For example, the 3' end of the first sense strand can be linked to the 5' end of the second sense strand, the 3' end of the first sense strand can be linked to the 3' end of the second sense strand, or the 5' end of the first sense strand can be linked to the 5' end of the second sense strand.

[0376] In some embodiments, the multi-target molecule is assembled from two siRNAs, and in this case, 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 linked to each other in any direction.For example, the 3' end of the first antisense strand can be linked to the 5' end of the second antisense strand, the 3' end of the first antisense strand can be linked to the 3' end of the second antisense strand, or the 5' end of the first antisense strand can be linked to the 5' end of the second antisense strand.

[0377] In some embodiments, multi-target molecule is assembled from two siRNAs, in this case, the sense strand of the first siRNA is covalently linked to the antisense strand of the second siRNA.Not limited to, the sense strand of the first siRNA can be linked to the antisense strand of the second siRNA in any direction.For example, the 3' end of sense strand can be linked to the 5' end of antisense strand, the 3' end of sense strand can be linked to the 3' end of antisense strand, or the 5' end of sense strand can be linked to the 5' end of antisense strand.

[0378] In some embodiments, the multi-targeting 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-targeting 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.

[0379] In some embodiments, the linker is -[(PQ"-R) q -X-(P'-Q'-R') q '] q "-T-, wherein P, R, T, P', R' and T are each independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, CHO; NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CH=NO, [ka] or heterocyclyl; [ka] and; Q" and Q"' are each independently for each occurrence absent, -(CH2) n -, -C(R 1 )(R 2 )(CH2) n -, -(CH2) n C(R 1 )(R 2 )-, -(CH2CH2O) m CH2CH2-, or -(CH2CH2O) m CH2CH2NH-; X is absent or a cleavable linking group; R a is H or an amino acid side chain; R 1 and R 2 is each independently for each occurrence H, CH3, OH, SH, or N(R N )2; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; q, q', and q'' are each independently for each occurrence 0 to 20, and the repeat units may be the same or different; n is, independently for each occurrence, 1 to 20; m is, independently for each occurrence, 0 to 50.

[0380] In some embodiments, the linker comprises at least one cleavable linking group.

[0381] In some embodiments, the linker is a branched linker. The branch point of a branched linker may be at least trivalent, but may also be a tetravalent, pentavalent, or hexavalent atom or group exhibiting such multiple valencies. In some embodiments, the branch point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC, where Q is, independently for each occurrence, H or an optionally substituted alkyl. In some embodiments, the branch point is glycerol or a derivative thereof.

[0382] A cleavable linking group is one that is sufficiently stable outside a cell but is cleaved once inside a target cell to release the two moieties held together by the linker. In certain embodiments, the cleavable linking group is cleaved at a rate at least 10 times faster, and preferably at least 100 times faster, in the target cell or under a first reference condition (e.g., which may be selected to mimic or represent intracellular conditions) than in the subject's blood or serum or under a second reference condition (e.g., which may be selected to mimic or represent conditions found in the blood or serum).

[0383] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction; esterases; amidases; endosomes; or agents that can create an acidic environment, such as agents that cause a pH of 5 or less; general acids, peptidases (which may be substrate-specific) and proteases, and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0384] The linker may contain a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, an iRNA agent targeting cells in the CNS may be conjugated to a tether containing sialic acid (SA). CNS cells are rich in neuraminidase enzymes (e.g., neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4), etc.). NEU3, in particular, is abundant in cells of the CNS and is localized to the inner membrane of the nuclear envelope. Meanwhile, NEU1 is localized to the outer membrane of the nuclear envelope as well as the plasma membrane (see, e.g., Ledeen et al. (2011) New findings on nuclear gangliosides: overview on metabolism and function. 116(5):714-720). NEU3 cleaves terminal 2,3 and 2,6 linked SA (see, eg, US Pat. No. 10,907,176).

[0385] In some embodiments, the cleavable linking group is cleaved at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). In some embodiments, the cleavable linking group is cleaved to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% faster in blood (or under in vitro conditions selected to mimic extracellular conditions) compared to cells (or under in vitro conditions selected to mimic intracellular conditions).

[0386] Examples of cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., -SS- and -C(R)2-SS-, where R is H or C1-C6 alkyl and at least one R is C1-C6 alkyl, e.g., CH3 or CH2CH3); phosphate-based cleavable linking groups (e.g., -OP(O)(OR)-O-, -OP(S)(OR)-O-, -OP(S)(SR)-O-, -SP(O)(OR)-O-, -OP(O)(OR)-S-, -SP(O)(OR)-S-, -OP(S)(ORk)-S-, -SP(S)(OR)-O-, -OP(O)(R)-O-, -OP(S)(R)- O-, -SP(O)(R)-O-, -SP(S)(R)-O-, -SP(O)(R)-S-, -OP(S)(R)-S-, -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-, wherein R is an optionally substituted straight or branched chain C-C 10alkyl); acid cleavable linking groups (e.g., hydrazones, esters, and esters of amino acids, -C=NN- and -OC(O)-); ester-based cleavable linking groups (e.g., -C(O)O-); peptide-based cleavable linking groups (e.g., linking groups that are cleaved in cells by enzymes such as peptidases and proteases, e.g., -NHCHR A C(O)NHCHR B C(O)-, where R A and R B are the R groups of two adjacent amino acids. Peptide-based cleavable linker groups include two or more amino acids. In some embodiments, the peptide-based cleavable bond includes an amino acid sequence that is a substrate for peptidases or proteases present in cells.

[0387] Additional exemplary cleavable linking groups include all of the exemplary endosomally cleavable linkers as well as phosphoramidites described herein below.

[0388] Cleavable Linker In certain aspects, cleavable linkers, such as endosomal and / or protease-cleavable linkers, are provided herein. In some embodiments, the cleavable linkers described herein can be included in a larger linker. In some embodiments, the cleavable linker is a carbohydrate linker that is cleaved at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions). In some embodiments, the cleavable linker is cleaved to 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) than in cells (or under in vitro conditions selected to mimic intracellular conditions). In some embodiments, the linker is cleaved at a rate that is at least 10 times faster, and preferably at least 100 times faster, in the target cell or under first reference conditions (e.g., which may be selected to mimic or represent intracellular conditions) than in the subject's blood or serum or under second reference conditions (e.g., which may be selected to mimic or represent conditions found in blood or serum).

[0389] The cleavable linkers described herein and known in the art may be used for any molecule that would be useful for cleavage in the endo-lysosomal compartment. The cleavable linkers described herein and known in the art may be particularly useful in prodrug methods, particularly for hydrophobic conjugates, endosomal cleavable agents, or any other drugs that may need to be activated or released in the endo-lysosomal compartment.

[0390] Specific examples of linkers for effector molecules of multi-targeting molecules include, but are not limited to, all endosomally cleavable linkers as well as phosphoramidites disclosed herein below.

[0391] Effector molecules Those skilled in the art are aware that the double-stranded oligonucleotide with about 20-23 base pairs, particularly 21 base pairs of double-stranded structure, is recognized to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001,20:6877-6888).However, it has also been found that shorter or longer double-stranded oligonucleotides can be equally effective.

[0392] As used herein, the term " siRNA " refers to the agent that mediates the targeted cleavage of RNA transcripts.These agents associate with a cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC).The agent that is effective in inducing RNA interference is also referred to herein as siRNA, RNAi agent or iRNA agent.As used herein, the terms " siRNA activity " and " RNAi activity " refer to the gene silencing caused by siRNA.

[0393] A double-stranded oligonucleotide comprises two oligonucleotide strands that are sufficiently complementary to hybridize to form a double-stranded structure. Typically, the double-stranded structure is 15-35, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded oligonucleotides, 25-30 base pairs in length, are preferred. In some embodiments, shorter double-stranded oligonucleotides, 10-15 base pairs in length, are preferred. In other embodiments, the double-stranded oligonucleotide is at least 21 nucleotides in length.

[0394] In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity to at least a portion of a target sequence, and the double-stranded region is 14 to 30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14 to 30, more commonly 18 to 25, even more commonly 19 to 24, and most commonly 19 to 21 nucleotides in length.

[0395] In some embodiments, the double-stranded region of the double-stranded oligonucleotide is at least 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 in length.

[0396] In some embodiments, the antisense strand of the double-stranded oligonucleotide is at least 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, or 40 or more nucleotides in length.

[0397] In some embodiments, the sense strand of the double-stranded oligonucleotide is at least 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 nucleotides in length.

[0398] In some embodiments, one strand has at least one single-stranded stretch of 1 to 10 nucleotides in the double-stranded region. A "single-stranded nucleotide stretch in a double-stranded region" means that there is at least one nucleotide in the double-stranded region that is not base-paired to another nucleotide. If the single-stranded nucleotide stretch is internal (non-terminal) in the double-stranded region, at least one nucleotide base pair can be present at each end of the single-stranded stretch. If it is present at the end of the double-stranded region, the single-stranded nucleotide stretch can be a single-stranded overhang. The single-stranded nucleotide stretch in the double-stranded region can be in the form of a bulge or can contain one or more mismatched nucleotides. In some embodiments, both strands have at least one single-stranded stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) nucleotides in the double-stranded region. In a double-stranded region, when both strands have a single-stranded stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) nucleotides, the single-stranded nucleotides can be opposite each other (e.g., a mismatch stretch), or can be positioned such that the second strand does not have a non-base-pairing nucleotide opposite the single-stranded oligonucleotide of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are located within 8 nucleotides of either end, e.g., within 8, 7, 6, 5, 4, 3, or 2 nucleotides of either the 5' or 3' end of the region of complementarity of the two strands.

[0399] Hairpin and dumbbell oligonucleotides have a double-stranded region of at least or equal to 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region can be 200, 100, or 50 nucleotide pairs or less in length. In some embodiments, the ranges for the double-stranded region are 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. In some embodiments, the nucleic acid effector molecule may have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3' end, in some embodiments on the antisense side of the hairpin; in some embodiments, the overhang is 1 to 4 nucleotides in length, more broadly 2 to 3 nucleotides in length. Hairpin oligonucleotides capable of inducing RNA interference are also referred to herein as "shRNAs."

[0400] In certain embodiments, two oligonucleotide strands specifically hybridize if there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, and conditions under which the assay is performed in the case of in vitro assays.

[0401] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound hybridizes to its target sequence but hybridizes to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances; the "stringent conditions" under which an antisense compound hybridizes to a target sequence are determined by the nature and composition of the antisense compound and the assay in which it is being tested.

[0402] It is understood in the art that incorporating modified nucleotide affinity can allow more mismatches compared to unmodified compounds.Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between oligonucleotides and target nucleic acids, for example, by determining melting temperature (Tm).Tm or ΔTm can be calculated by techniques known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes and RNA:RNA duplexes by the technique described in Freier et al. (Nucleic Acids Research, 1997,25,22:4429-4443).

[0403] Circular sciRNA structure design In the present disclosure, the inventors have also designed a new method for producing small circular interfering RNA (sciRNA), which uses chemically modified nucleotides to connect the ends of the nucleic acid of antisense strand to produce a circular sense construct with blocked 5'-end and 3'-end.For example, exemplary sciRNA is synthesized using the antisense strand annealed to the 5'-3' circularized sense strand that carries a trivalent GalNAc ligand, which is produced by "click" chemistry.These sciRNAs, especially the sciRNAs with phosphate mimetic modifications at the 5'-end of antisense nucleotide sequence, such as 5'-phosphorothioate (5'-PS), 5'-phosphorodithioate (5'-PS2), 5'-vinylphosphonate (5'-VP), 5'-methylphosphonate (5'-MePhos) and 5'-deoxy-5'-C-malonyl modification, have been observed to potently silence gene expression in vitro and in vivo.

[0404] Exemplary bis-sciRNAs have also been synthesized, each having two sense nucleotide sequences connected together by a bis-linker (e.g., a cleavable nucleotide or non-nucleotide linker). The 5' end of one sense nucleotide sequence is cyclized with the 3' end of the other sense nucleotide sequence using "click" chemistry to form a cyclized sense strand having bis-sense nucleotide sequences. One or two of the sense nucleotide sequences carry a lipophilic moiety (and / or a trivalent GalNAc ligand) at a non-terminal position of the sense nucleotide sequence. One or two antisense strand nucleotide sequences are annealed to the corresponding sense nucleotide sequence of the 5'-3' cyclized sense strand.

[0405] Thus, one embodiment of the present invention relates to a small circular interfering RNA (sciRNA) comprising a sense strand and an antisense strand, each of which comprises at least one nucleic acid modification.

[0406] In some embodiments, the sense strand has a cyclic or substantially cyclic structure. In some embodiments, the antisense strand has a cyclic or substantially cyclic structure.

[0407] The sense or antisense strand can form a cyclic or substantially cyclic structure via a cyclization linking moiety connecting one end of the sense (or antisense) strand to the other end of the sense (or antisense) strand. The cyclic or substantially cyclic structure of the sense or antisense strand can be formed by the cyclization procedure shown in Scheme 1. As shown in Scheme 1, a reactive linking moiety Q is attached to one end of the sense (or antisense) strand, and another reactive linking moiety Y is attached to the other end of the sense (or antisense) strand. Q and Y each may contain various linkers (tethers) and carriers that can carry ligands, and each contains terminal functional groups that are reactive with each other. Then, by activating the reaction between Q and Y via an addition reaction, a cyclization linking moiety Z is formed, and the ring is closed to form a cyclic or substantially cyclic structure. An example of a cyclization procedure via click chemistry (e.g., azide-alkyne cycloaddition to form a triazole) is shown in Scheme I of Example 12. [ka]

[0408] Depending on the reaction used to cyclize the sense strand (or antisense strand) and the linker / cyclizing groups contained in the reactive linking moieties Q and Y, the cyclization linking moiety Z in the cyclized sense strand (or antisense strand) may be a triazole bond, an amide bond, a sulfide or disulfide bond, a phosphate bond, an oxime bond, a hydrazo bond, an N,N'-dialkylenehydrazo bond, a methyleneimino bond, a methylenecarbonylamino bond, a methylenemethylimino bond, a methylenehydrazo bond, a methylenedimethylhydrazo bond, a methyleneoxymethylimino bond, a hydroxylamino bond, a formacetal bond, an alkyl or aryl bond, a PEG bond, an ether bond, a thioether bond, a thiodiester bond, a thionocarbamate bond, or a thiolamino bond. , thioacetamide bond, sulfonic acid bond, sulfonamide bond, sulfonate ester bond, thioformacetal bond, urea bond, carbonate bond, amine bond, maleimide-thioether bond, phosphodiester bond, phosphotriester bond, phosphonate hydrogen bond, phosphonate alkyl bond or phosphonate aryl bond, phosphoramidate bond, phosphorothioate bond, phosphoroselenic acid bond, boranophosphate bond, boranophosphate ester bond, sulfonamide bond, carbamate bond, carboxamide bond, carboxymethyl bond, carboxylic acid ester bond, siloxane bond, dialkylsiloxane bond, ethylene oxide bond, and combinations thereof.

[0409] In one embodiment, the cyclization linkage moiety Z in the cyclic sense strand (antisense strand) may comprise one or more bonds selected from the group consisting of a triazole bond, an amide bond, a disulfide bond, a phosphate bond, an oxime bond, an alkyl bond, a PEG bond, an ether bond, a thioether bond, a urea bond, a carbonate bond, an amine bond, a maleimide-thioether bond, a phosphodiester bond, a sulfonamide bond, a carbamate bond, and combinations thereof.

[0410] In certain embodiments, the cyclization linking moiety may further contain one or more carriers that can serve to connect the ligand to the sciRNA. The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl. In one embodiment, the acyclic group is a moiety based on a serinol or diethanolamine backbone.

[0411] One exemplary cyclization linking moiety contains a triazole bond formed by a click chemistry-mediated cyclization procedure (e.g., from an azide-alkyne cycloaddition). As described above in Scheme 1, the cyclization can be formed by adding a reactive linking moiety Q to one end of the sense strand (or antisense strand) and another reactive linking moiety Y to the other end of the sense strand (or antisense strand), and activating the reaction between Q and Y. In this case, the Q / Y pair is an azide / alkyne pair. Non-limiting examples of molecules containing reactive linking moieties Q / Y (in this case, azide / alkyne functional groups) are shown below. [Table 1] [Table 2] [Table 3]

[0412] As described above, these exemplary molecules may be attached to the end of an oligonucleotide chain, for example, via a phosphate. A cyclized oligonucleotide chain is formed by activating click chemistry between the reactive linking moieties between the Q / Y pair. For example, by attaching L123 and Q301 (shown in the table above) to each end of the oligonucleotide chain via a phosphate and activating click chemistry of the azide / alkyne pair of L123 and Q301, [ka] is formed (cyclization by activating the click reaction of Z49-3'-phosphate-Hyp-C9-1,4-triazole-C6-5'-phosphate)

[0413] Additional non-limiting examples of cyclized linking moieties Z formed by activating the Click reaction of the reactive linking moiety Q / Y pair described above are shown below. [Table 4]

[0414] One exemplary cyclization linking moiety contains a maleimide-thioether bond (or thiosuccinimide bond) formed by a cyclization procedure via a click chemistry reaction from a thiol-maleimide addition reaction. As described above in Scheme 1, the cyclization can be formed by adding a reactive linking moiety Q to one end of the sense strand (or antisense strand) and another reactive linking moiety Y to the other end of the sense strand (or antisense strand), and activating the reaction between Q and Y. In this case, the Q / Y pair is a thiol / maleimide pair. Non-limiting examples of molecules containing reactive linking moieties Q / Y (in this case, thiol / maleimide functional groups) are shown below. [Table 5] [Table 6]

[0415] The sense strand may be at least 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, or 40 nucleotides in length. In one embodiment, the sense strand is at least 20 nucleotides in length. In one embodiment, the sense strand is at least 40 nucleotides in length.

[0416] The antisense strand can be at least 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, or 40 nucleotides in length.

[0417] The antisense strand is annealed to the sense strand to form an at least partially double-stranded region. In some embodiments, one or more sense nucleotide sequences are annealed to the antisense strand. In some embodiments, at least one sense nucleotide sequence is not annealed to the antisense strand.

[0418] In some embodiments, the sense nucleotide sequence that is not annealed to the antisense strand can be a single-stranded oligonucleotide, such as, for example, an antisense oligonucleotide (ASO), an antimiR oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide.

[0419] In some embodiments, a double-stranded region is formed between the sense and antisense strands at least in the seed region of the antisense strand (ie, positions 2-8 of the 5' end of the antisense nucleotide sequence).

[0420] Increasing the length of the sense strand, i.e., increasing the length of the double-stranded region, can affect the melting temperature of the sciRNA and increase the thermal stability of the sciRNA duplex.

[0421] Increasing the length of the sense strand can be achieved by using a single sense nucleotide sequence or by having two or more sense nucleotide sequences in the sense strand.

[0422] In some embodiments, the sense strand may have a long circular sense nucleotide sequence having a length of at least 20 nucleotides, at least 25 nucleotides, or at least 30 nucleotides, e.g., a length of 20-45 nucleotides, or a length of 30-45 nucleotides.

[0423] In some embodiments, the antisense strand comprises at least one antisense nucleotide sequence, the at least one antisense nucleotide sequence having a length of about 20 to about 45 nucleotides.

[0424] In one embodiment, the long circular sense nucleotide sequence is annealed to an antisense strand having a length of about 19 to about 23 nucleotides that is complementary to the nucleotide sequence of a target mRNA transcript. In one embodiment, the long circular sense nucleotide sequence is annealed to two or more antisense nucleotide sequences having a length of about 19 to about 23 nucleotides that are complementary to the nucleotide sequences of two or more target mRNA transcripts.

[0425] The long circular sense nucleotide sequence may be a substrate cleavable by DICER.

[0426] Structural design of bis-sciRNA compounds A further aspect of the present invention relates to a small circular interfering RNA (sciRNA) for regulating one or more target mRNAs in a subject's central nervous system (CNS), the sciRNA comprising a first strand having a length of at least 40 nucleotides and at least two first strand nucleotide sequences (each nucleotide sequence having a length of about 18 to about 28 nucleotides) connected together by a bis-linker, and at least one second strand nucleotide sequence having a length of about 19 to 23 nucleotides and annealing to at least one of the first strand nucleotide sequences. The first strand has a circular or substantially circular structure. The first strand nucleotide sequence and the second strand nucleotide sequence each contain at least one nucleic acid modification. The first strand nucleotide sequence or the second strand nucleotide sequence contains one or more ligands.

[0427] The first strand of a bis-sciRNA comprises two or more nucleotide sequences connected together by a bis-linker and may be referred to herein as the "bis-strand" (e.g., the bis-sense strand or the bis-antisense strand).

[0428] Furthermore, the first strand of a bis-sciRNA has a circular or substantially circular structure and may be referred to herein as a "circular or substantially circular strand." The first strand comprises at least two first-strand nucleotide sequences and is formed by connecting the at least two first-strand nucleotide sequences with a bis-linker. Each of the at least two first-strand nucleotide sequences can anneal with the same or different second-strand nucleotide sequences. Each of the first and second-strand nucleotide sequences can target the same or different RNA molecules. Thus, a bis-sciRNA molecule can target one or more target mRNAs.

[0429] A bis-sciRNA molecule can be a multi-target molecule. A multi-target molecule comprises at least two nucleic acid-based effector molecules, which are linked to each other by a bis-linker moiety as described herein. A "nucleic acid-based effector molecule" refers to a modified or unmodified nucleic acid molecule capable of modulating the expression activity of a target nucleic acid (e.g., a target mRNA). 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, provided herein are bis-sciRNA molecules designed to target one or more target nucleic acids or two or more distinct target RNA sequences within one or more target nucleic acids, and exhibit delivery and surprising efficacy in a subject's CNS tissue upon contact. Two target RNA sequences within a single target RNA are considered "distinct" if the target RNA sequences do not overlap with each other.

[0430] Thus, the multi-targeting molecules disclosed herein are distinct from molecules in which one effector molecule is directed to two different targets, e.g., from double-stranded effector molecules in which each strand is directed to a different target, or from effector molecules in which at least a portion of the sequence comprises sequences complementary to or capable of hybridizing to two different target sequences.

[0431] For example, a circular or substantially circular sense strand may contain two sense nucleotide sequences forming a bis-sciRNA. A circular or substantially circular sense strand may contain two symmetrical nucleotide sequences or two asymmetrical nucleotide sequences. If symmetrical, each sense nucleotide sequence of a circular or substantially circular sense strand (e.g., each sense nucleotide sequence may have a length of about 19 to about 23 nucleotides, e.g., 20 to 21 nucleotides) may be annealed to two identical antisense nucleotide sequences (e.g., each may have a length of 21 nucleotides) targeting the same nucleotide sequence of an mRNA transcript. If asymmetrical, each sense nucleotide sequence of a circular or substantially circular sense strand (e.g., each sense nucleotide sequence may have a length of about 19 to about 23 nucleotides, e.g., 20 to 21 nucleotides) may be annealed to two different antisense nucleotide sequences (e.g., each may have a length of 23 nucleotides) targeting the nucleotide sequences of two different mRNA transcripts.

[0432] Exemplary circular or substantially circular sense strands (or bis-sense strands) and circular sciRNAs (or bis-sciRNAs) are shown in Schemes 1A-1C. Schemes 1A and 1B respectively show circular or substantially circular sense strands containing two symmetric (Scheme 1A) or asymmetric (Scheme 1B) sense nucleotide sequences (with a total bis-sense strand length of 42-45 nucleotides). The two sense nucleotide sequences are connected by a bis-linkage (e.g., a nucleotide-based or non-nucleotide-based linker (tether)). Scheme 1C shows a circular or substantially circular sense strand containing a long, dicer-cleavable sense nucleotide sequence (e.g., 30-45 nucleotides) annealed to a short antisense nucleotide sequence (e.g., 19-23 nucleotides).

[0433] Cyclic or substantially cyclic structures of the sense strand or bis-sense strand may be formed by click chemistry using the same reaction mechanism as shown in Scheme 1 above. In the case of the bis-sense strand, cyclic or substantially cyclic structures of the bis-sense strand may be formed by click chemistry reaction of the 5' end of one sense nucleotide sequence with the 3' end of the other sense nucleotide sequence. The cyclization linking moiety Z contains a combination of one or more of a phosphate linkage, an alkyl linkage, a triazole linkage, an amide linkage, and a pyrrolidinyl cyclic group, with or without a ligand (L) carried by the cyclic group. [ka]

[0434] Additional exemplary circular or substantially circular sense strands (or bis-sense strands) and circular sciRNAs (or bis-sciRNAs) are shown in Schemes 2A-2C, 3A-3C, 4A-4C, 5A-5C, 6A-6C, 7A-7C, and 8A-8C, illustrating various cyclization reactions and cyclization linkages. The sense and antisense strand nucleotide sequences in these schemes mirror those in Schemes 1A-1C. The cyclization reactions in these schemes differ from those in Schemes 1A-1C. For example, in Schemes 2A-2C, cyclization is by amide formation. The cyclization linkage Z contains a combination of one or more of a phosphate linkage, an alkyl linkage, an amide linkage, and a pyrrolidinyl cyclic group, with or without a ligand (L) carried by the cyclic group. In Schemes 3A-3C, cyclization is by disulfide formation. The cyclization linking moiety Z contains one or more combinations of phosphate linkages, alkyl linkages, disulfide linkages, amide linkages, and pyrrolidinyl cyclic groups, with or without a ligand (L) carried by the cyclic group. In Schemes 4A-4C, cyclization is by click chemistry. The cyclization linking moiety Z contains one or more combinations of phosphate linkages, alkyl linkages, triazole linkages, amide linkages, and PEG linkages. In Schemes 5A-5C and 6A-6C, cyclization is by oxime formation. The cyclization linking moiety Z contains one or more combinations of phosphate linkages, alkyl linkages, oxime linkages (aldoxime or ketoxime), amide linkages, and pyrrolidinyl cyclic groups, with or without a ligand (L) carried by the cyclic group. In Schemes 7A-7C and 8A-8C, cyclization is by hydrazone formation. The cyclized linking moiety Z contains a combination of one or more of phosphate linkages, alkyl linkages, hydrazo linkages, amide linkages, and pyrrolidinyl cyclic groups, with or without a ligand (L) carried by the cyclic group. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0435] Additional exemplary bis-sciRNA designs include those shown in Example 13, in which a circular or substantially circular sense strand contains two asymmetric sense nucleotide sequences (each 20-21 nucleotides in length). The two sense nucleotide sequences are connected by a bis-linkage (e.g., 3 nucleotides in length). Each sense nucleotide sequence is annealed to a longer, different antisense nucleotide sequence (each 23 nucleotides in length). The cyclization linkage moiety Z contains one or more combinations of phosphate, alkyl, and triazole linkages.

[0436] The examples shown in Schemes 1A-1C through 8A-8C and in Example 13 are for illustrative purposes only. The cyclization reactions and cyclization linkages shown for cyclization of a sense nucleotide sequence in the sense strand (or bis-sense strand) are also applicable to cyclization of an antisense nucleotide sequence in the antisense strand (or bis-antisense strand).

[0437] Linker / Tether The linker / tether is contained in the bis-sense strand or bis-antisense strand as part of the bis-linker, connecting two sense nucleotide sequences (forming the bis-sense strand) or antisense nucleotide sequences (forming the bis-antisense strand) of a multi-target molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA)).

[0438] The linker / tether may be included as part of the circularization linkage of the circular or substantially circular sense strand (or circular or substantially circular bis-sense strand) of the sciRNA (or bis-sciRNA).

[0439] Linkers / tethers can be used to connect the ligands, for example via a carrier, to multi-targeting molecules (e.g., effector molecules such as bis siRNA or scriRNA (or bis-sciRNA)).

[0440] The terms "linker," "bond," "linking group," and "tether" may be used interchangeably.

[0441] The linker in the sense strand (or bis-sense strand) or antisense strand (or bis-antisense strand) may be a nucleotide-based linker or a non-nucleotide-based linker. The linker may be a stable linker that is stable in biological fluids (e.g., plasma or artificial cerebrospinal fluid). Alternatively, the linker may be a cleavable linker (e.g., a biocleavable linker).

[0442] The linker / tether may be attached to the ligand at a "tethering attachment point (TAP)." The linker / tether may be any C1-C 100 Carbon-containing moieties (e.g., C1-C 75 , C1-C 50 , C1-C 20 , C1-C 10 , C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10) and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amide group (NHC(O)-) on the linker / tether, which may serve as an attachment point for the ligand. Non-limiting examples of linkers / tethers (underlined) include TAP-(CH2). n NH-;TAP-C(O)(CH2) n NH-;TAP-NR''''(CH2) n NH-,TAP-C(O)-(CH2) n -C(O)-;TAP-C(O)-(CH2) n -C(O)O-;TAP-C(O)-O-;TAP-C(O)-(CH2) n -NH-C(O)-;TAP-C(O)-(CH2) n -;TAP-C(O)-NH-;TAP-C(O)-;TAP-(CH2) n -C(O)-;TAP-(CH2) n -C(O)O-;TAP-(CH2) n -; or TAP-(CH2) n Examples include -NH-C(O)-, where n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R"" is C1-C6 alkyl. Preferably, N is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or may contain one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands include, for example, TAP-(CH2) n NH(LIGAND);TAP-C(O)(CH2) n NH(LIGAND);TAP-NR''''(CH2) n NH(LIGAND);TAP-(CH2) n ONH(LIGAND);TAP-C(O)(CH2) nONH(LIGAND);TAP-NR''''(CH2) n ONH(LIGAND);TAP-(CH2) n NHNH2(LIGAND),TAP-C(O)(CH2) n NHNH2(LIGAND);TAP-NR''''(CH2) n NHNH2(LIGAND);TAP-C(O)-(CH2) n -C(O)(LIGAND);TAP-C(O)-(CH2) n -C(O)O(LIGAND);TAP-C(O)-O(LIGAND);TAP-C(O)-(CH2) n -NH-C(O)(LIGAND);TAP-C(O)-(CH2) n (LIGAND);TAP-C(O)-NH(LIGAND);TAP-C(O)(LIGAND);TAP-(CH2) n -C(O)(LIGAND);TAP-(CH2) n -C(O)O(LIGAND);TAP-(CH2) n (LIGAND); or TAP-(CH2) n In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can be acylated with, for example, C(O)CF.

[0443] In some embodiments, the linker / tether may terminate in a mercapto group (i.e., SH) or an olefin (e.g., CH=CH). For example, the tether may be TAP-(CH). n -SH, TAP-C(O)(CH2) n SH, TAP-(CH2) n -(CH=CH2), or TAP-C(O)(CH2) n(CH=CH2), where n can be as otherwise described. The tether can be optionally substituted, for example, with hydroxy, alkoxy, perhaloalkyl, and / or optionally inserted with one or more additional heteroatoms, for example, N, O, or S. The double bond can be cis or trans or E or Z.

[0444] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at the terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, an aldehyde, an alkyl halide, a mesylate, a tosylate, a nosylate, or a brosylate, or an activated carboxylic acid ester, for example, an NHS ester, or a pentafluorophenyl ester. A preferred linker / tether (underlined) is TAP-(CH2). n CHO;TAP-C(O)(CH2) n CHO; or TAP-NR''''(CH2) n CHO, where n is 1 to 6 and R"" is C1-C6 alkyl; or TAP-(CH2) n C(O)ONHS;TAP-C(O)(CH2) n C(O)ONHS; or TAP-NR''''(CH2) n C(O)ONHS, where n is 1 to 6 and R"" is C1-C6 alkyl; TAP-(CH2) n C(O)OC6F5;TAP-C(O)(CH2) n C(O)OC6F5; or TAP-NR''''(CH2) n C(O)OCF, where n is 1 to 11 and R"" is C-C alkyl; or -(CH) n CH2LG;TAP-C(O)(CH2) n CH2LG; or TAP-NR''''(CH2) nExamples include CHLG, where n can be as otherwise described and R"" is C-C alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be performed by coupling a nucleophilic group, e.g., a thiol or amino group, of the ligand with an electrophilic group on the tether.

[0445] In other embodiments, it may be desirable for the monomer to contain a phthalimide group (K) at the terminal position of the linker / tether. [ka]

[0446] In other embodiments, other protected amino groups may be at the terminal positions of the linker / tether, such as an alloc group, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety may be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0447] Any of the linkers / tethers described herein may contain one or more additional linking groups, such as -O-(CH) n -, -(CH2) n -SS-, -(CH2) n It may further contain - or -(CH=CH)-.

[0448] Cleavable linker / tether In some embodiments, at least one of the linkers / tethers may be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0449] In one embodiment, at least one of the linkers / tethers may be a reductively cleavable linker (eg, a disulfide group).

[0450] In one embodiment, at least one of the linkers / tethers can be an acid-cleavable linker (eg, a hydrazone group, an ester group, an acetal group, or a ketal group).

[0451] In one embodiment, at least one of the linkers / tethers may be an esterase-cleavable linker (eg, an ester group).

[0452] In one embodiment, at least one of the linkers / tethers may be a phosphatase-cleavable linker (eg, a phosphate group).

[0453] In one embodiment, at least one of the linkers / tethers may be a peptidase-cleavable linker (eg, a peptide bond).

[0454] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are found to be more widespread or at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction; esterases; endosomes, or agents that can create an acidic environment, such as agents that cause a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0455] Cleavable linking groups, such as disulfide bonds, can be pH sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some tethers have a linking group that cleaves at a preferred pH, thereby releasing the iRNA agent from the ligand (e.g., a targeting or cell-permeable ligand such as cholesterol) into the cell or into a desired compartment of the cell.

[0456] The chemical bond (e.g., linking group) that connects the ligand to the iRNA agent may include a disulfide bond. When the iRNA agent / ligand complex is taken up into the cell by endocytosis, the acidic environment of the endosome cleaves the disulfide bond, releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand may also be a targeting ligand or a second therapeutic agent that can complement the therapeutic effect of the iRNA agent.

[0457] The tether may include a linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether may depend on the cell targeted by the iRNA agent. For example, an iRNA agent targeting mRNA in liver cells may be conjugated to a tether containing an ester group. Liver cells are rich in esterases, and in liver cells, the tether is cleaved more efficiently than in cell types that are not rich in esterases. When the tether is cleaved, the iRNA agent is released from the ligand attached to the distal end of the tether, thereby potentially enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0458] A tether containing a peptide bond can be conjugated to an iRNA agent that targets a cell type rich in peptidases, such as a hepatocyte or a synoviocyte. For example, an iRNA agent that targets a synoviocyte for the treatment of an inflammatory disease (e.g., rheumatoid arthritis) can be conjugated to a tether containing a peptide bond.

[0459] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degradation agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues, such as tissues to which the iRNA agent will be exposed when administered to a subject. Thus, the relative susceptibility to cleavage may be determined between first and second conditions, where the first condition is selected to exhibit cleavage in target cells and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0460] The cleavable linker may be cleavable in various tissues and cellular structures, for example, liver homogenate, liver tritosomes, liver lysosomes, liver cytosol, brain homogenate, brain tritosomes, brain lysosomes, or brain cytosol.

[0461] Redox-cleavable linking groups One type of cleavable linking group is a redox-cleavable linking group that is cleaved by reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group," or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can turn to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least about 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0462] Phosphate-cleavable linker Phosphate-based linkages are cleaved by agents that degrade or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate-based linkages are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0463] Acid-cleavable linking groups An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0464] Ester-based bonding groups Ester-based linking groups are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0465] Peptide-based cleavage groups Peptide-based linking groups are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to create oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to create peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to create peptides and proteins, and do not include all amide functional groups. Peptide cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)—, wherein R 1 and R 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0466] Biocleavable Linkers / Tethers Linker can also comprise biocleavable linker, which is nucleotide linker and non-nucleotide linker, or a combination thereof, that connects two parts of a molecule.For example, biocleavable linker can connect one or both strands of two individual siRNA molecules to produce bis(siRNA).In some embodiments, linker can also be represented by simple electrostatic interaction or stacking interaction between two individual siRNAs.

[0467] Non-nucleotidic linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides and derivatives thereof, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0468] In some embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0469] In one embodiment, the biocleavable carbohydrate linker may have 1 to 10 sugar units, which have at least one anomeric bond capable of connecting two siRNA units. When more than one sugar is present, the units can be linked via 1 to 3, 1 to 4, or 1 to 6 sugar bonds or via an alkyl chain.

[0470] Exemplary biocleavable linkers include, but are not limited to, the following endosomally cleavable linkers as well as phosphoramidites: [ka] [ka] [ka] [ka] In the formula, n=1 to 12 and m=1 to 12.

[0471] Further discussion regarding biocleavable linkers can be found in WO2018136620, the contents of which are incorporated herein by reference in their entirety.

[0472] Carrier In certain embodiments, the circularization linkage portion of the circular or substantially circular sense (or antisense) strand contains one or more carriers, which carry one or more ligands and serve to conjugate the ligands to the sciRNA (or bis-sciRNA).

[0473] In certain embodiments, one or more ligands may be conjugated to the sciRNA (or bis-sciRNA) via a carrier rather than as part of a cyclization binding moiety.

[0474] In certain embodiments, one or more ligands may be conjugated to an effector molecule (eg, a bis siRNA compound) via a carrier.

[0475] In some embodiments, the carrier may replace one or more nucleotides.

[0476] The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalinyl. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0477] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the effector molecule (eg, bis siRNA) or sciRNA (or bis-sciRNA) agent.

[0478] Ribonucleotide subunits with such substitutions of the ribose sugar of the subunit are referred to herein as ribose-substituted modified subunits (RRMS). The carrier may be a cyclic or acyclic moiety and may include two "backbone attachment points" (e.g., hydroxyl groups) and a ligand. The ligand may be directly attached to the carrier, as described above, or indirectly attached to the carrier by an intervening linker / tether. [ka]

[0479] The ligand-conjugated monomer subunit may be the 5'- or 3'-terminal subunit of an effector molecule (e.g., dsRNA) or sciRNA molecule. That is, one of the two "W" groups may be a hydroxyl group, and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit may occupy an internal position, and both "W" groups may be one or more unmodified or modified ribonucleotides. Two or more ligand-conjugated monomer subunits may be present in a sciRNA (or bis-sciRNA) agent.

[0480] Sugar-substituted monomers, e.g., ligand-conjugated monomers (cyclic) Cyclic sugar-substituted monomers, e.g., sugar-substituted ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) provided below (in which the preferred backbone attachment points are R 1 Or R 2 ;R 3 Or R 4 ; or Y is CR 9 R 10 If R 9 and R 10 (The two positions may be selected from two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 ) is selected to obtain the desired tethering point. 7 ; when X is CH2, R 5 or R 6 The carriers are generally described below and can be incorporated into the chain. Thus, the structure can be attached at one (for terminal positions) or two (for internal positions) of the attachment points, e.g., R 1 Or R 2 ;R 3 Or R4 ; or R 9 Or R 10 (Y is CR 9 R 10 It is understood that the R group (where R is a phosphate or modified phosphate, e.g., a sulfur-containing backbone) also includes the case where R is attached to a phosphate or modified phosphate, e.g., a sulfur-containing backbone. For example, one of the R groups above may be -CH-, in which case one bond is attached to the carrier and one is attached to a backbone atom, e.g., the oxygen bond or the central phosphorus atom. [ka] (LCM-2) During the ceremony, X is N(CO)R 7 , N.R. 7 , or CH2; Y is NR 8 ,O,S,CR 9 R 10 and; Z is CR 11 R 12 is or is not present; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 each independently represents H, OR a , or (CH2) n OR b However, R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of the following are true: OR a and / or (CH2) n OR b and; R 5 , R 6 , R 11 , and R 12 each independently represents a ligand, H, 1 to 3 R 13 C1-C6 alkyl optionally substituted with, or C(O)NHR 7 or R5 and R 11 Together, R 14 is a C3-C8 cycloalkyl optionally substituted with R 7 may be a ligand, e.g., R 7 is R d or R 7 For example, NR c R d C1-C substituted with 20 alkyl; or NHC(O)R d C1-C substituted with 20 It may be a ligand that is indirectly tethered to a carrier through a tethering moiety, such as an alkyl; R 8 is H or C1-C6 alkyl; R 13 is hydroxy, C1-C4 alkoxy, or halo; R 14 is NR c R 7 and; R 15 is C1-C6 alkyl or C2-C6 alkenyl optionally substituted with cyano; R 16 is C1-C 10 is alkyl; R 17 is a liquid or solid phase supported reagent; L is -C(O)(CH2) q C(O)- or -C(O)(CH2) q S- and; R a is a protecting group, such as CAr3 (e.g., a dimethoxytrityl group) or Si(X 5’ )(X 5” )(X 5’’’ ) in which (X 5’ ), (X 5” ), and (X 5’’’ ) are as otherwise stated. R b is P(O)(O - )H, P(OR 15 )N(R16 )2, or LR 17 and; R c is H or C1-C6 alkyl; R d is H or a ligand; Each Ar independently represents a C-C alkyl group optionally substituted with C-C alkoxy. 10 is aryl; n is 1 to 4, and q is 0 to 4.

[0481] Exemplary carriers include, for example, those in which X is N(CO)R 7 Or NR 7 and Y is CR 9 R 10 and Z is absent; or X is N(CO)R 7 Or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 or X is N(CO)R 7 Or NR 7 and Y is NR 8 and Z is CR 11 R 12 or X is N(CO)R 7 Or NR 7 and Y is O and Z is CR 11 R 12 or X is CH2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2), or an indane ring system, e.g., X is CH2 and Y is CR 9 R 10 , Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1).

[0482] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z does not exist (D). [ka] OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, attached to one of the carbons in the five-membered ring (-CHOFG in D). 1 ). OFG 2 is preferably directly bonded to one of the carbon atoms in the five-membered ring (-OFG of D 2 For pyrroline-based carriers, -CH2OFG 1 is attached to C-2 and OFG 2 may be attached to C-3; or -CH2OFG 1 is attached to C-3 and OFG 2 may be attached to C-4. In certain embodiments, CH2OFG 1 and OFG 2 may be geminally substituted at one of the carbons mentioned above. For 3-hydroxyproline-based carriers, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached to C-4. Thus, pyrroline-based monomers and 4-hydroxyproline-based monomers may contain a bond (e.g., a carbon-carbon bond), in which case bond rotation is restricted to that particular bond, e.g., restricted by restrictions resulting from the presence of a ring. Thus, CHOFG 1 and OFG 2may be cis or trans to each other in any of the above pairings. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 Both centers carrying D can have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, or vice versa). The tethering attachment point is preferably nitrogen. Preferred examples of carrier D include: [ka]

[0483] In certain embodiments, the carrier may be based on a piperidine ring system (E), for example, where X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 is. [ka] OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or an ethylene group (n=2), attached to one of the carbons of the six-membered ring [E's -(CH2) n OFG 1 ]. OFG 2 is preferably directly bonded to one of the carbon atoms in the six-membered ring (-OFG of E) 2 ). -(CH2) n OFG 1 and OFG 2may be geminally positioned on the ring, i.e., both groups may be attached to the same carbon, such as C-2, C-3, or C-4. Alternatively, -(CH) n OFG 1 and OFG 2 may be arranged vicinal on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, and OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, and OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, and OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, and OFG 2 may be bonded to C-3. Thus, the piperidine-based monomer may contain a bond (e.g., a carbon-carbon bond), in which case bond rotation is restricted to that particular bond, e.g., restricted by the restrictions imposed by the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 may be cis or trans relative to each other in any of the above pairings. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2(The centers bearing the can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, or vice versa). The tethering attachment point is preferably nitrogen.

[0484] In certain embodiments, the carrier may be based on a piperazine ring system (F), e.g., X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or may be based on a morpholine ring system (G), for example, X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 is. [ka] OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, attached to one of the carbons in the six-membered ring (-CHOFG of F or G). 1 ). OFG 2 is preferably directly bonded to one of the carbons in the six-membered ring (-OFG of F or G) 2 ) for both F and G, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached to C-3, or vice versa. 1 and OFG 2 may be geminally substituted on one of the aforementioned carbons. Thus, piperidine-based and morpholine-based monomers may contain a bond (e.g., a carbon-carbon bond), in which case bond rotation is restricted to that particular bond, e.g., restricted by restrictions resulting from the presence of a ring. Thus, CH2OFG 1 and OFG 2may be cis or trans relative to each other in any of the above pairings. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers bearing F and G can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other the S configuration, or vice versa). The tethering attachment points are preferably nitrogen in both F and G.

[0485] In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 may be taken together to form a C6 cycloalkyl (H, z=2), or may be based on an indane ring system, e.g., X is CH2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1). [ka] OFG 1 is preferably attached to a primary carbon, such as an exocyclic methylene group (n=1) or an ethylene group (n=2), attached to one of C-2, C-3, C-4, or C-5, for example [H of -(CH2) n OFG 1 ]. OFG 2is preferably directly bonded to any one of C-2, C-3, C-4 or C-5 (-OFG of H 2 ).-(CH2) n OFG 1 and OFG 2 may be geminally positioned on the ring, i.e., both groups may be attached to the same carbon, such as, for example, C-2, C-3, C-4, or C-5. Alternatively, -(CH) n OFG 1 and OFG 2 may be arranged vicinal on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, and OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, and OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, and OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, and OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-4, and OFG 2 may be attached to C-5; or -(CH2) n OFG 1 may be attached to C-5, and OFG 2 may be bonded to C-4. Thus, the decalin-based or indane-based monomer may contain a bond (e.g., a carbon-carbon bond), in which case bond rotation is restricted to that particular bond, e.g., restricted by restrictions resulting from the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2may be cis or trans relative to each other in any of the above pairings. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 Both centers carrying the aryl groups can have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, or vice versa). In a preferred embodiment, the substituents at C-1 and C-6 are trans relative to each other. The tethering attachment point is preferably at C-6 or C-7.

[0486] Other carriers include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2 may be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. The monomers may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers bearing the can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, or vice versa). The tethering attachment point is preferably nitrogen.

[0487] Details regarding more representative cyclic sugar-substituted carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0488] Sugar-substituted monomers (acyclic) Acyclic sugar-substituted monomers, such as sugar-substituted ligand-conjugated monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers may have the following formula: LCM-3 or LCM-4: [ka]

[0489] In some embodiments, each of x, y, and z, independently of one another, can be 0, 1, 2, or 3. When y and z are different in formula LCM-3, the tertiary carbon can have either an R or S configuration. In preferred embodiments, x is zero, y and z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z are each 1 in formula LCM-3. Each of formulas LCM-3 or LCM-4 below can be optionally substituted, for example, with hydroxy, alkoxy, or perhaloalkyl.

[0490] Details regarding more representative acyclic sugar-substituted carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0491] In some embodiments, a multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) comprises one or more ligands conjugated to the 5' end of the sense nucleotide sequence or the 5' end of the antisense nucleotide sequence.

[0492] In certain embodiments, the ligand is conjugated to the 5' end of the nucleotide sequence via a carrier and / or a linker. In one embodiment, the ligand is conjugated to the 5' end of the nucleotide sequence via a carrier of the following formula: [ka] R is a ligand.

[0493] In some embodiments, a multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) comprises one or more ligands conjugated to the 3' end of the sense nucleotide sequence or the 3' end of the antisense nucleotide sequence.

[0494] In certain embodiments, the ligand is conjugated to the 3' end of the nucleotide sequence via a carrier and / or linker. In one embodiment, the ligand is conjugated to the 3' end of the nucleotide sequence of the strand via a carrier of the following formula: [ka] R is a ligand.

[0495] In certain embodiments, at least one of the ligands is conjugated to a chain having a cyclic or substantially cyclic structure. In certain embodiments, at least one of the ligands is conjugated to a chain that does not have a cyclic or substantially cyclic structure. In one embodiment, at least one of the ligands is conjugated to a chain having a cyclic or substantially cyclic structure and at least one of the ligands is conjugated to a chain that does not have a cyclic or substantially cyclic structure.

[0496] In some embodiments, a multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) comprises one or more ligands conjugated to both ends of a sense nucleotide sequence. In some embodiments, a multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) comprises one or more ligands conjugated to both ends of an antisense nucleotide sequence.

[0497] In some embodiments, a multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) comprises one or more ligands conjugated to the 5' or 3' end of a sense nucleotide sequence, or one or more ligands conjugated to the 5' or 3' end of an antisense nucleotide sequence.

[0498] In some embodiments, the ligand is conjugated to the chain via one or more linkers (tethers) and / or carriers, hi one embodiment, the ligand is conjugated to the chain via one or more linkers (tethers).

[0499] In one embodiment, the ligand is conjugated to the 5' or 3' end of the sense or antisense nucleotide sequence by a cyclic carrier, optionally via one or more intervening linkers (tethers).

[0500] In some embodiments, the ligand is conjugated to one or more internal positions in at least one nucleotide sequence. An internal position of a nucleotide sequence refers to a nucleotide at any position in the nucleotide sequence, excluding the terminal positions from the 3' and 5' ends of the nucleotide sequence (e.g., excluding the two positions 1 from the 3' end and 1 from the 5' end).

[0501] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence. The internal positions include all positions except the last two positions from each end of the nucleotide sequence (e.g., excluding four positions: positions 1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end). In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one nucleotide sequence. The internal positions include all positions except the last three positions from each end of the nucleotide sequence (e.g., excluding six positions: positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).

[0502] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence, excluding the cleavage site region of the sense nucleotide sequence. For example, the ligand is not conjugated to positions 9-12, counting from the 5' end of the sense nucleotide sequence. For example, the ligand is not conjugated to positions 9-11, counting from the 5' end of the sense nucleotide sequence. Alternatively, the internal positions exclude positions 11-13, counting from the 3' end of the sense nucleotide sequence.

[0503] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence excluding the cleavage site region of the antisense nucleotide sequence, e.g., internal positions excluding positions 12-14 from the 5' end of the antisense nucleotide sequence.

[0504] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence excluding positions 11-13 from the 3' end of the sense nucleotide sequence and positions 12-14 from the 5' end of the antisense nucleotide sequence.

[0505] In one embodiment, the one or more ligands are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 of the sense nucleotide sequence, and positions 6-10 and 15-18 of the antisense nucleotide sequence, counting from the 5' end of each nucleotide sequence.

[0506] In one embodiment, the one or more ligands are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15 and 17 of the sense nucleotide sequence, and positions 15 and 17 of the antisense sequence, counting from the 5' end of each nucleotide sequence.

[0507] In some embodiments, the ligand is conjugated to a nucleobase, sugar moiety, or internucleotide linkage of a multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent).

[0508] Ligand In certain embodiments, multi-targeting molecules (e.g., effector molecules such as bis siRNA or sciRNA agents (or bis-scriRNAs)) are further modified by the covalent attachment of one or more conjugate groups. Generally, conjugate groups modify one or more performance properties of the attached effector molecule (e.g., bis siRNA) or sciRNA agent (or bis-scriRNA), including, but not limited to, pharmacology, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in chemistry and are attached directly to parent compounds, such as oligomeric compounds, or via optional linking moieties or groups. A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0509] In some embodiments, multi-targeting molecules (e.g., effector molecules such as bis siRNA or sciRNA (or bis-sciRNA) agents) further comprise targeting ligands that target receptors that mediate delivery to specific CNS tissues. These targeting ligands can be conjugated in combination with lipophilic moieties that allow for specific intrathecal and systemic delivery.

[0510] Examples of targeting ligands for receptor-mediated delivery to CNS tissues are peptide ligands such as, for example, Angiopep-2, lipoprotein receptor-related protein (LRP) ligands, bEnd.3 cell-binding ligands; transferrin receptor (TfR) ligands (which can utilize the iron transport system in the brain and transport cargo into the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transport proteins, and LDL receptor ligands.

[0511] In some embodiments, the multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) further comprises a targeting ligand that targets a receptor that mediates delivery to a specific ocular tissue. These targeting ligands can be conjugated in combination with a lipophilic moiety, allowing for specific intravitreal and systemic delivery. Examples of targeting ligands that target receptor-mediated delivery to ocular tissues include all-trans-retinol (targets retinoic acid receptors); RGD peptides (target retinal pigment epithelial cells), such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or Cyclo(-Arg-Gly-Asp-D-Phe-Cys); LDL receptor ligands; and carbohydrate-based ligands (target endothelial cells of the posterior eye).

[0512] Preferred conjugate groups suitable for the present invention include, for example, cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains such as dodecanediol 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, e.g., 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), adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or lipid moieties such as octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0513] As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or compartment, e.g., a cellular, tissue, or organ compartment. For example, CNS targeting ligands include lipophilic ligands herein, such as C16-modified.

[0514] Generally, a wide variety of entities, e.g., ligands, can be attached to the oligomeric compounds described herein. Ligands can include naturally occurring molecules, or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, thiazolinone ... Examples of antibodies include thrombin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralens, mitomycin), and the like. Synthon 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, zeranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., alpha helical peptides, amphipathic peptides, RGD peptides, cell penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 These include activators of MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-penetrating agents (e.g., helical cell-penetrating agents).

[0515] Peptide and peptidomimetic ligands include those containing natural or modified peptides, such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides containing one or more amides, i.e., peptides with peptide bonds substituted with one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands are about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0516] Exemplary amphipathic peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforins, CPFs, bombinin-like peptides (BLPs), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainins, brevinins-2, dermaseptins, melittin, pleurocidins, H2A peptides, Xenopus peptides, esculentinis-1, and caerins.

[0517] As used herein, the term "endosomolytic ligand" refers to a molecule that has endosomolytic properties. An endosomolytic ligand promotes lysis and / or transport of a composition of the invention or its components from a cellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other vesicles, into the cytoplasm of a cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, masked or unmasked linear or branched polymers with cationic or anionic charges, masked or unmasked dendrimers with cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0518] Exemplary endosomolytic / fusogenic peptides include, but are not limited to, AALEALAEALAEALEALAEAAAAGGC (GALA); AALEALAEALAEALAEALAEALAAAAGGC (EALA); ALEALEALEALAEA; GLFEAIEGFIENGWEGMIWDYG (INF-7); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3); GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, where n is norleucine); LFEALLELLESLWELLLEA (JTS-1); GLFKALLKLLKSLWKLLLKA (ppTG1); GLFRALLRLLRSLWRLLLRA (ppTG20); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA); GLFFEAIAEFIEGGWEGLIEGC (HA); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin); H5WYG; and CHK6HC.

[0519] Without wishing to be bound by theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids typically have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0520] Synthetic polymers with endosomolytic activity suitable for the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410, 2009 / 0023890, 2008 / 0287630, 2008 / 0287628, 2008 / 0281044, 2008 / 0281041, 2008 / 0269450, 2007 / 0105804, 20070036865, and 2004 / 0198687, the contents of which are incorporated herein by reference in their entireties.

[0521] Exemplary cell-penetrating peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin); GRKKRRQRRRPPQC (Tat fragment 48-60); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence-based peptide); LLIILRRRIRKQAHAHSK (PVEC); GWTLNSAGYLLKINLKALAALAKKIL (transportan); KLALKLALKALKAALKLA (amphipathic model peptide); RRRRRRRRR (Arg9); KFFKFFKFFK (bacterial cell wall-penetrating peptide); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE S(LL-37); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39); ILPWKWPWWPWRR-NH2 (indolicidin); AAVALLPAVLLALLAP (RFGF); AALLPVLLAAP (RFGF analog); and RKCRIVVIRVCR (bactenecin).

[0522] Exemplary cationic groups include, but are not limited to, protonated amino groups derived from, for example, O-amines (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, for example, O(CH2) n Amines (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH2CH2NH)n Included are CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0523] As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a cell compartment, tissue compartment, or organ compartment.Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.For example, CNS targeting ligands include lipophilic ligands herein, such as C16-modified.

[0524] Carbohydrate targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, for example, GalNAc2 and GalNAc3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, glycosylated polyamino acid, and lectin.The term multivalent indicates the presence of multiple monosaccharide units.These monosaccharide subunits can be linked to each other via glycosidic bonds or can be linked to scaffold molecules.

[0525] Numerous folates and folate analogs suitable as ligands 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 incorporated herein by reference in their entireties.

[0526] As used herein, the terms "PK-modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of a composition. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and transthyretin-binding ligands (e.g., tetraiidothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligomeric compounds containing multiple phosphorothioate intersugar linkages are also known to bind to serum proteins. Thus, short oligomeric compounds, e.g., oligonucleotides containing approximately 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) in the present invention. PK-modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in the PK-modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-modulating ligands in the present invention. Binding to serum components (eg, serum proteins) can be predicted from albumin binding assays, such as the assay described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0527] When there are two or more ligands, the ligands all have the same performance, or all have different properties, or some ligands have the same properties, and other ligands have different properties.For example, the ligand can have targeting properties, have endosomolytic activity, or have PK regulation properties.In a preferred embodiment, all ligands have different properties.

[0528] A ligand or tethered ligand may be present on a monomer when that monomer is incorporated into an effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) agent component. In some embodiments, a ligand may be incorporated via coupling to a "precursor" monomer after that "precursor" monomer has been incorporated into an effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) agent component. For example, a monomer with an amino-terminal tether (i.e., no associated ligand), e.g., monomer-linker-NH, may be incorporated into an effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) agent component. Subsequent operations, i.e., after the precursor monomer is incorporated into an effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) component, a ligand bearing an electrophilic group, e.g., a ligand bearing a pentafluorophenyl ester or aldehyde group, can then be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the precursor monomer.

[0529] In another example, one can incorporate monomers bearing chemical groups suitable for participating in click chemistry reactions, such as azide- or alkyne-terminated tethers / linkers. In a subsequent operation, i.e., after incorporating the precursor monomers into a chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer by coupling the alkyne and azide together.

[0530] In some embodiments, the ligand may be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of an effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) agent. Conjugation to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is attached to a conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. When a ligand is conjugated to a nucleobase, preferred positions are those that do not interfere with hybridization, i.e., do not interfere with the hydrogen bonding interactions required for base pairing.

[0531] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Illustrative carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as an abasic residue. The internucleoside linkage can also bear a conjugate moiety. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. For internucleoside linkages containing amines or amides (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0532] There are many methods for preparing oligonucleotide conjugates.Generally, oligonucleotides are linked to conjugate moieties by contacting the reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotides with the reactive group of conjugate moieties.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0533] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or a thiol.Methods for conjugating nucleic acids and related oligomeric compounds, with or without linking groups, are described in, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0534] Representative U.S. patents that teach the preparation of nucleic acid conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5, 608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,149,782;5,214,136;5,245, 022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,371; Nos. 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559,279, the contents of which are incorporated herein by reference in their entireties.

[0535] In some embodiments, the multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) further comprises one or more targeting ligands that target liver tissue. In some embodiments, at least one of the targeting ligands is a carbohydrate-based ligand. In some embodiments, the carbohydrate-based ligand is an ASGPR ligand. In one embodiment, at least one of the targeting ligands is a GalNAc-based conjugate.

[0536] In certain embodiments, the multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) further comprises a ligand having the structure shown below: [ka] During the ceremony, L G is, independently for each occurrence, a ligand, e.g., a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, polysaccharide; and Z', Z", Z''', and Z'''' are independently for each occurrence O or S.

[0537] In certain embodiments, the multi-targeting molecule (e.g., an effector molecule such as a bis siRNA or sciRNA (or bis-sciRNA) agent) comprises a ligand of formula (II), (III), (IV), or (V): [ka] During the ceremony, q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B , and q 5C represents, independently for each occurrence, 0 to 20, and the repeat units may be the same or different; Q and Q' are, independently for each occurrence, absent, -(P 7 -Q 7 -R 7 ) p -T 7 -, or -T 7 -Q 7 -T 7’ -BT 8’ -Q 8 -T 8 and; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , P 7 , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C , T 7 , T 7’ , T 8 , and T 8’ is, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; B is -CH2-N(B L )-CH2-; B L -T B -Q B -T B’ -R x and; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 7 , Q 8 , and Q Bis, independently for each occurrence, absent, alkylene, substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R'), C≡C, or C(O); T B and T B’ is, independently for each occurrence, absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH2, CH2NH, or CH2O; R x may contain lipophilic substances (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxyethanol. noxadin), vitamins (e.g., folic acid, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomolytic components, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid), or cationic lipids; R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 is independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C is, independently for each occurrence, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide; R' and R" are each independently H, C1-C6 alkyl, OH, SH, or N(R N )2; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; R a is H or an amino acid side chain; Z', Z", Z''', and Z'''' are, independently for each occurrence, O or S; p, independently for each occurrence, is 0 to 20.

[0538] As discussed above, because a ligand can be conjugated to an effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) agent via a linker or carrier, and because the linker or carrier can contain a branched linker, the effector molecule (e.g., bis siRNA) or sciRNA (or bis-sciRNA) agent can then contain multiple ligands via the sa...

Claims

**Claim 1** A nucleic acid composition for modulating one or more target RNAs, comprising one or more distinct target RNA sequences in a subject's central nervous system (CNS), wherein the nucleic acid composition comprises a first double-stranded RNA (dsRNA) molecule, and a single-stranded nucleic acid agent or a second dsRNA molecule, wherein the first dsRNA molecule, and the single-stranded nucleic acid agent or the second dsRNA molecule are connected together by a linker and do not overlap with each other, wherein the first dsRNA comprises at least one conjugated lipophilic moiety, wherein the single-stranded nucleic acid agent or the second dsRNA molecule comprises at least one conjugated lipophilic moiety, wherein the first dsRNA molecule of the nucleic acid composition, and the single-stranded nucleic acid agent or the second dsRNA molecule can each modulate the activity or expression of one or more target RNAs by at least 15% in the subject's CNS tissue as compared to a suitable control. A nucleic acid composition. **Claim 2** The nucleic acid composition according to claim 1, comprising the first dsRNA molecule and the second dsRNA molecule connected together by the linker. **Claim 3** At least one lipophilic moiety is a saturated or unsaturated C ４ -C ３０ hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne, the nucleic acid composition according to claim 1. **Claim 4** Each lipophilic moiety is a saturated or unsaturated C １６ or C ２２ hydrocarbon chain-containing nucleic acid composition according to claim 1. **Claim 5** The nucleic acid composition according to claim 1, wherein the sense strand of the first dsRNA molecule is covalently linked to the sense strand of the second dsRNA molecule. **Claim 6** The nucleic acid composition according to claim 1, wherein one or more of the first and second dsRNA molecules, if present, contain a lipophilic moiety conjugated independently at position 6 of the sense strand of each dsRNA molecule, counted from the 5' end of the sense strand of each dsRNA molecule. **Claim 7** The nucleic acid composition according to claim 1, wherein one or more of the first and second dsRNA molecules, if present, contain one or more lipophilic moieties conjugated independently at one or more of the following non-terminal positions: positions 5, 6, 7, 15, and 17 of the sense strand, and positions 15 and 17 of the antisense strand, counting the 5' end of each strand as position 1. **Claim 8** The nucleic acid composition according to claim 1, wherein one or more of the first and second dsRNA molecules, if present, contain a sense strand of a length of 19 - 30 nucleotides and an antisense strand of a length of 19 - 30 nucleotides. **Claim 9** The nucleic acid composition according to claim 1, wherein each of the first dsRNA molecule and the second dsRNA molecule, when present, has a sense strand having a length of 21 to 25 nucleotides and an antisense strand having a length of 21 to 25 nucleotides.

10. The nucleic acid composition according to claim 1, wherein each dsRNA molecule of the nucleic acid composition contains at least one modified nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing glycol nucleic acid (GNA), and nucleotides containing vinyl phosphonate.

11. The nucleic acid composition according to claim 1, wherein each dsRNA molecule contains 2 to 8 phosphorothioate nucleotide internucleotide linkages or methylphosphonate nucleotide internucleotide linkages.

12. All or substantially all of the nucleotides of each dsRNA molecule are modified with a modification selected from the group consisting of 2'-O-methyl modification, 2'-fluoro modification, and 2'-C ６ -C １８ The nucleic acid composition according to claim 1, which contains a modification selected from the group consisting of hydrocarbon chain modifications.

13. The nucleic acid composition according to claim 1, wherein the nucleic acid composition contains two nucleic acid dsRNA molecules, the sense strand of each dsRNA molecule has a length of 21 nucleotides, the antisense strand of each dsRNA molecule has a length of 23 nucleotides, the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is a nucleic acid linker having a length of three nucleotides that connects the sense strands of each dsRNA molecule, and the lipophilic moiety is conjugated to the 6th position of the sense strand of each dsRNA molecule.

14. The nucleic acid composition according to claim 1, wherein the linker is a cleavable linker in vivo.

15. The nucleic acid composition according to claim 1, wherein the linker contains a moiety selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

16. The nucleic acid composition according to claim 1, wherein the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is a nucleic acid linker having a length of 1 to 15 nucleotides.

17. The nucleic acid composition according to claim 16, wherein the linker has a length of three nucleotides.

18. The nucleic acid composition according to claim 1, wherein the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is a nucleic acid linker containing one or more nucleotides selected from the group consisting of 2'-O-methyl nucleotide, 2'-fluoro nucleotide, deoxyribonucleotide (dNTP), and ribonucleotide.

19. The nucleic acid composition according to claim 1, wherein the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is a polynucleotide containing one or more modifications selected from the group consisting of 2'-O-methyl ribonucleotide modification, 2'-fluoro-ribonucleotide modification, 2'-5'-linked nucleotide having different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, hexanol nucleic acid (HNA) modification, abasic ribose modification, abasic deoxyribose modification, and abasic hydroxyprolinol modification.

20. The nucleic acid composition according to claim 1, wherein the linker connecting the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule is selected from the group consisting of: 【Chemical 1】 【Chemical 2】 (Y195), and 【Chemical Formula 3】 (Y254).

21. The nucleic acid composition according to claim 1, wherein the nucleic acid composition regulates the gene expression of at least two target nucleic acids by at least 75% each, compared to the case where the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule are not connected together.

22. The nucleic acid composition according to claim 1, wherein the first dsRNA molecule regulates the gene expression of a first target nucleic acid, and the single-stranded nucleic acid agent or the second dsRNA molecule regulates the gene expression of a second nucleic acid.

23. A method for modulating one or more target RNAs comprising one or more distinct target RNA sequences in a subject's central nervous system (CNS), said method comprising contacting a nucleic acid composition comprising a first dsRNA molecule and a single-stranded nucleic acid agent or a second dsRNA molecule with the CNS cells of said subject, wherein said first dsRNA molecule and said single-stranded nucleic acid agent or second dsRNA molecule are connected together by a linker and do not overlap with each other, and each of said first dsRNA molecule and said second dsRNA molecule, if present, comprises at least one conjugated lipophilic moiety, and said nucleic acid composition inhibits the activity or expression of said one or more target RNAs comprising one or more distinct target RNA sequences in said CNS cells of said subject by at least 15% each as compared to a suitable control, thereby modulating said one or more target RNAs comprising one or more distinct target RNA sequences in the central nervous system (CNS) of said subject. Claim 24 A method for treating or preventing a disease or disorder of the CNS of a subject, said method comprising administering an injection to said subject, said injection comprising a nucleic acid composition comprising at least a first dsRNA molecule and a single-stranded nucleic acid agent or a second dsRNA molecule, wherein said first dsRNA molecule and said single-stranded nucleic acid agent or second dsRNA molecule are connected together by a linker and do not overlap with each other, and each of said at least first dsRNA molecule and said second dsRNA molecule, if present, comprises at least one conjugated lipophilic moiety, and said nucleic acid composition inhibits the activity or expression of one or more target RNAs comprising one or more distinct target RNA sequences in the CNS tissue of said subject by at least 15% each as compared to a suitable control, thereby treating or preventing said disease or disorder of the CNS of said subject. Claim 25 The method according to claim 24, wherein said injection is an ICV injection or an intrathecal injection. Claim 26 A pharmaceutical composition for inhibiting the expression of one or more target genes associated with a CNS disease or disorder, wherein the pharmaceutical composition is formulated for administration to the CNS of a subject and comprises a nucleic acid composition containing at least a first dsRNA molecule and a single-stranded nucleic acid agent or a second dsRNA molecule, and a pharmaceutically acceptable carrier, wherein the first dsRNA molecule and the single-stranded nucleic acid agent or the second dsRNA molecule are connected together by a linker and do not overlap with each other, and each of the at least first dsRNA molecule and the second dsRNA molecule, if present, comprises at least one conjugated lipophilic moiety, and the nucleic acid composition is capable of inhibiting the activity or expression of one or more distinct target RNAs, each containing one or more distinct target RNA sequences, in the CNS tissue of the subject by at least 15% each, as compared to an appropriate control.

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