Extrahepatic delivery

Abstract

New and improved methods for delivering siRNA molecules in vivo are provided. One aspect of the present invention relates to a compound comprising an antisense strand complementary to a target gene, a sense strand complementary to the antisense strand, and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand, optionally via a linker or carrier. Another aspect of the present invention relates to a method of gene silencing comprising administering to a cell or a subject in need thereof a therapeutically effective amount of a compound to which the lipophilic monomer is conjugated.

Description

[Background technology]

[0001] The efficient delivery of iRNA agents to cells in vivo requires specific targeting and substantial protection from extracellular environment, especially serum protein.RNAi-based therapy has shown promising clinical data for the treatment of liver-related diseases.However, the delivery of siRNA to tissues other than the liver remains an obstacle, limiting the use of siRNA-based therapy.

[0002] One of the factors limiting the experimental and therapeutic application of iRNA agents in vivo is the ability to efficiently deliver intact siRNA. Particular difficulties relate to non-viral gene transfer to the retina in vivo. One of the challenges is overcoming the inner limiting membrane, which prevents retinal transfection. Furthermore, negatively charged sugars in the vitreous have been shown to interact with cationic DNA-transfection reagent complexes, promoting their aggregation and hindering diffusion and cellular uptake.

[0003] The delivery of oligonucleotides into the central nervous system (CNS) presents particular challenges due to the blood-brain barrier (BBB), which free oligonucleotides cannot cross. One means of delivering oligonucleotides into the CNS is by intrathecal delivery. However, oligonucleotides also need to be efficiently taken up by target cells in the CNS to achieve the desired therapeutic effect. Previous studies have typically used delivery agents such as liposomes, cationic lipids, and nanoparticle-forming complexes to facilitate the intracellular uptake of oligonucleotides into neuronal-derived cells.

[0004] Thus, there remains a need for new and improved methods for delivering siRNA molecules in vivo without the use of tissue delivery reagents to achieve and enhance the therapeutic potential of iRNA agents. Summary of the Invention [Means for solving the problem]

[0005] One aspect of the invention provides compounds (e.g., oligonucleotides, which may be either single-stranded or double-stranded) that include one or more lipophilic monomers containing one or more lipophilic moieties conjugated, optionally via a linker or carrier, to one or more positions on at least one strand of the oligonucleotide. For example, some embodiments of the invention provide compounds (e.g., double-stranded iRNA agents) that include an antisense strand complementary to a target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties conjugated, optionally via a linker or carrier, to one or more positions on at least one strand.

[0006] In some embodiments, the octanol-water partition coefficient, log K ow The lipophilic moiety has a log K of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow may have:

[0007] In some embodiments, the hydrophobicity of the compound, as measured by the unbound fraction in a plasma protein binding assay of the compound, is greater than 0.2. In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the compound, as measured by the fraction of unbound siRNA in a binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for improved in vivo delivery of siRNA.

[0008] In some embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic, such as a steroid (e.g., a sterol) or a straight- or branched-chain aliphatic hydrocarbon. Exemplary lipophilic moieties are 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 group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0009] Suitable lipophilic moieties include saturated or unsaturated C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 Also included are those containing a saturated or unsaturated C6-C8 functional group (e.g., alkyl or alkenyl), and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. This functional group is useful for attaching a lipophilic moiety to an iRNA agent. In some embodiments, the lipophilic moiety is a saturated or unsaturated C6-C8 functional group. 18 Hydrocarbon chains (e.g., straight chain C6-C 18 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 In some embodiments, the lipophilic moiety comprises two or more carbon-carbon double bonds.

[0010] In some embodiments, the lipophilic moiety is a C6-C carboxylic acid having a free terminal carboxylic acid functional group (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid). 30It is a part.

[0011] In some embodiments, the lipophilic moiety is a C-C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.) or C6 to C 30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0012] A lipophilic monomer can include a lipophilic moiety conjugated to any part of an iRNA agent, such as a nucleobase, sugar moiety, or internucleoside linkage. If the lipophilic moiety is conjugated to an iRNA agent via a direct bond to the nucleobase, ribosugar, or internucleoside linkage of the iRNA agent, the lipophilic monomer includes the nucleobase, ribosugar, or internucleoside linkage and the lipophilic moiety. Alternatively, the lipophilic monomer can include a lipophilic moiety conjugated to a non-ribose-substituted unit, such as a linker or carrier. If the lipophilic moiety is conjugated to an iRNA agent via a non-ribose-substituted unit, such as a linker or carrier, the lipophilic monomer includes the non-ribose-substituted unit, such as a linker or carrier, and the lipophilic moiety.

[0013] In certain embodiments, the lipophilic monomer does not contain a nucleobase.

[0014] In certain embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via one or more linkers (tethers).

[0015] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the compound via a linker that includes 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 (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0016] In some embodiments, at least one of the linkers (tethers) is a redox-cleavable linker (a reductively cleavable linker; e.g., a disulfide group), an acidic 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).

[0017] In other embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0018] In certain embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via a non-ribose-substituting unit, i.e., a carrier that substitutes one or more nucleotides. The carrier can 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 decalin. In one embodiment, the acyclic group is a moiety based on a serinol backbone, a glycerol backbone, or a diethanolamine backbone.

[0019] In some embodiments, the carrier replaces one or more nucleotides in the double-stranded iRNA agent. In some embodiments, the carrier replaces one or more nucleotides at an internal position of the double-stranded iRNA agent. In other embodiments, the carrier replaces a nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine; for example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0020] In some embodiments, the lipophilic monomer has the following formula: [ka] and During the ceremony, J1 and J2 each independently represent O, S, or NR N , optionally substituted alkyl, OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R P )2)O, or OP(N(R P )2) and; [ka] is a cyclic or acyclic group; R N is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroaryl, or an amino protecting group; RP each, independently for each occurrence, is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, or optionally substituted heteroaryl; L 10 is a substituted or unsubstituted, saturated or unsaturated C3-C8 hydrocarbon (e.g., a C3-C8 alkyl, alkenyl, or alkynyl, or a C3-C8 hydrocarbon containing two or more double bonds); substituted groups include those already described herein for "substituted" hydrocarbon, alkyl, alkenyl, or alkynyl; L 11 is a substituted or unsubstituted, saturated or unsaturated C6-C26 hydrocarbon (e.g., a C6-C26 alkyl, alkenyl, or alkynyl, or a C3-C8 hydrocarbon containing two or more double bonds); substituted groups include those already described herein for "substituted" hydrocarbon, alkyl, alkenyl, or alkynyl; Q is absent when the nucleobase is not on the carrier, or is at least 10% in vivo, L 11 From L 10 For example, Q is a cleavable group that cleaves L from the lipophilic monomer by about 10-70%, about 15-50%, about 20-40%, or about 20-30%. 11 Exemplary cleavable groups include -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R 5 )=N-, -N=C(R 5 )-, -C(R 5 )=NO-, -ON=C(R 5 )-, -C(O)N(R 5 )-, -N(R 5 )C(O)-, -C(S)N(R 5 )-, -N(R 5 )C(S)-, -N(R 5 )C(O)N(R 5 )-, -N(R 5)C(O)C(R 3 )(R 4 )OC(O)-, -C(O)OC(R 3 )(R 4 )C(O)N(R 5 )-, -OC(O)O-, -OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O-, -OC(O)(CR 3 R 4 )C(O)-, [ka] or a combination thereof, R 11 is a C2-C8 alkyl or alkenyl. For each occurrence, R 3 , R 4 , and R 5 are each independently H or C1-C4 alkyl.

[0021] In one embodiment, the cleavability of Q is determined by the stability of the ligand in cerebrospinal fluid (CSF), the stability of the ligand in plasma, the stability of the ligand in brain homogenates or tissue homogenates (liver, eye, etc.), or the stability of the ligand in vitreous humor.

[0022] Cyclic and acyclic groups include those already described herein.

[0023] In one embodiment, the acyclic group is a serinol, glycerol, or diethanolamine backbone.

[0024] In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, hydroxyprolinyl, cyclopentyl, cyclohexyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0025] In one embodiment, the cyclic group is ribose or a ribose analogue. Examples of ribose analogues include arabinose, 4'-thioribose, 2'-O-methylribose, GNA, UNA, and LNA analogues.

[0026] In some embodiments, the lipophilic monomer conjugated to one or more positions on the chain of the compound is [ka] [ka] [ka] It has the following structure.

[0027] In the above structures of the lipophilic monomers, the monomers may also contain one or more asymmetric centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures, and all such isomers of the monomers are expressly included.

[0028] In the above structure of the lipophilic monomer, the alkylene chain may contain one or more unsaturated bonds.

[0029] The integer m is 0 to 8. The integer n is 1 to 21. R2' can be any functional group that is an acceptable 2'-modification for the ribose sugar, such as a 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl) modification, a 2'-O-allyl modification, a 2'-C-allyl modification, a 2'-fluoro modification, a 2'-ON-methylacetamido (2'-O-NMA) modification, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, a 2'-O-aminopropyl (2'-O-AP) modification, or a 2'-ara-F modification. For example, R2' can be H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase. W is an alkyl group such as C1 to C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl). R, R', and R" are each independently H or an alkyl group such as C1 to C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl).

[0030] In some embodiments, the lipophilic monomer conjugated to one or more positions on the chain of the compound is [ka] (R2' is 2'-F, 2'-OMe, 2'-NMA, 2'-deoxy, or 2'-OH); [ka] [ka] In these structures, B is a modified or unmodified nucleobase.

[0031] Particular embodiments of lipophilic monomers include: [ka] [ka] [ka] In these structures, B is a modified or unmodified nucleobase; R and R' are each independently H, methyl, ethyl, isopropyl, or t-butyl.

[0032] In some embodiments, the lipophilic monomer is [ka] The compound contains a lipophilic moiety conjugated to a strand of the compound (a single strand of a single-stranded oligonucleotide; or the sense and / or antisense strand of a double-stranded oligonucleotide) via a carrier of the formula (I). In these embodiments, R is a lipophilic moiety as defined herein. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0033] In some embodiments, the lipophilic monomer is [ka] The compound contains a lipophilic moiety conjugated to an internal position of a strand of the compound (a single strand of a single-stranded oligonucleotide; or the sense and / or antisense strand of a double-stranded oligonucleotide) via a carrier of the formula (I). In these embodiments, R is a lipophilic moiety as defined herein. n is an integer from 1 to 21. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0034] Further examples of lipophilic monomers can be found in the Examples.

[0035] In some embodiments, the sense and antisense strands of the compound are each 15 to 30 nucleotides in length.

[0036] In one embodiment, the sense and antisense strands of the compound are each 19 to 25 nucleotides in length.

[0037] In one embodiment, the sense and antisense strands of the compound are each 21-23 nucleotides in length.

[0038] In some embodiments, the compound comprises a single-stranded overhang at at least one of its ends, e.g., a 3' and / or 5' overhang of 1 to 10 nucleotides in length, e.g., an overhang of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length. In some embodiments, the compound may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. In one embodiment, the compound comprises a 3' overhang at the 3' end of the antisense strand and, optionally, a blunt end at the 5' end of the antisense strand. In one embodiment, the compound has a 5' overhang at the 5' end of the sense strand and, optionally, a blunt end at the 5' end of the antisense strand. In one embodiment, the compound has two blunt ends on either end of the iRNA duplex.

[0039] In one embodiment, the sense strand of the compound is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0040] In some embodiments, the sense strand further comprises at least one phosphorothioate bond at its 3'-end. In some embodiments, the sense strand further comprises at least two phosphorothioate bonds at its 3'-end. In some embodiments, one or more lipophilic monomers are located at the 3'-end of the sense strand. In one embodiment, one of the phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide from the 3'-end of the sense strand.

[0041] In some embodiments, the sense strand further comprises at least one phosphorothioate bond at the 5'-end. In some embodiments, the sense strand further comprises at least two phosphorothioate bonds at the 5'-end. In some embodiments, one or more lipophilic monomers are located at the 5'-end of the sense strand. In one embodiment, one of the phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide from the 5'-end of the sense strand.

[0042] In some embodiments, the antisense strand further comprises at least one phosphorothioate bond at its 3'-end.In some embodiments, the antisense strand further comprises at least two phosphorothioate bonds at its 3'-end.In some embodiments, one or more lipophilic monomers are located at the 3'-end of the antisense strand.In one embodiment, one of the phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide from the 3'-end of the antisense strand.

[0043] In some embodiments, the compound further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0044] In some embodiments, the 5' end of the antisense strand of the compound does not contain a 5'-vinylphosphonate (VP).

[0045] In some embodiments, the compound further comprises at least one terminal chiral phosphorus atom.

[0046] Site-specific chiral modifications of internucleotide linkages can occur at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the strand. This is referred to herein as a "terminal" chiral modification. Terminal modifications can occur at the 3'- or 5'-end position in the terminal region, for example, at the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. Chiral modifications can occur in the sense strand, the antisense strand, or both the sense and antisense strands. Each chirally pure phosphorus atom can be in either the Rp or Sp configuration, and combinations thereof. Further details regarding chiral modifications and chirally modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled "Chirally-Modified Double-Stranded RNA Agents," the entire contents of which are incorporated herein by reference.

[0047] In some embodiments, the compound further comprises a terminal chiral modification present at the first internucleotide linkage at the 3'-end of the antisense strand, the terminal chiral modification having the linked phosphorus atom in the Sp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, the terminal chiral modification having the linked phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, the terminal chiral modification having the linked phosphorus atom in either the Rp or Sp configuration.

[0048] In one embodiment, the compound further comprises a terminal chiral modification present at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0049] In one embodiment, the compound further comprises a terminal chiral modification present at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0050] In one embodiment, the compound further comprises a terminal chiral modification present at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification present at the third internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0051] In one embodiment, the compound further comprises terminal chiral modifications present at the first and second internucleotide linkages at the 3'-end of the antisense strand, having the linked phosphorus atom in the Sp configuration; terminal chiral modifications present at the first and second internucleotide linkages at the 5'-end of the antisense strand, having the linked phosphorus atom in the Rp configuration; and terminal chiral modifications present at the first internucleotide linkage at the 5'-end of the sense strand, having the linked phosphorus atom in either the Rp or Sp configuration.

[0052] In some embodiments, the compound has at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end).

[0053] In some embodiments, the antisense strand comprises two blocks of 1, 2, or 3 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.

[0054] In some embodiments, the compound further comprises a targeting ligand that targets a receptor that mediates delivery to a specific central nervous system tissue. In one embodiment, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

[0055] In some embodiments, the compound further comprises a targeting ligand that targets a receptor that mediates delivery to an ocular tissue. In one embodiment, the targeting ligand is selected from the group consisting of trans-retinol, an RGD peptide, an LDL receptor ligand, and a carbohydrate-based ligand. In one embodiment, the targeting ligand is an RGD peptide such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

[0056] In some embodiments, the compound further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is a GalNAc conjugate.

[0057] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the antisense and sense strands of a compound are modified. For example, if 50% of a compound is modified, then 50% of all nucleotides present in the compound contain a modification described herein.

[0058] In some embodiments, the antisense and sense strands of the compound comprise at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or substantially 100% 2'-O-methyl modified nucleotides.

[0059] In one embodiment, the compound is an oligonucleotide, e.g., a double-stranded dsRNA agent, wherein at least 50% of the nucleotides of the double-stranded dsRNA agent are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-deoxy, or 2'-fluoro.

[0060] In one embodiment, the oligonucleotide is antisense, and at least 50% of the nucleotides of the antisense are independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0061] In some embodiments, the sense and antisense strands of the compound contain fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modified nucleotides. In some embodiments, the compound has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications on the sense strand. In some embodiments, the compound has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications on the antisense strand.

[0062] In some embodiments, the compounds have one or more 2'-F modifications at any position in the sense or antisense strand.

[0063] In some embodiments, the compounds have less than 20%, less than 15%, less than 10%, less than 5%, or are substantially free of non-natural nucleotides. Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (2'-modified L-nucleosides, such as 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic, and open-chain alkyls.

[0064] In some embodiments, the compounds have more than 80%, more than 85%, more than 90%, more than 95%, or substantially 100% naturally occurring nucleotides. For purposes of these embodiments, naturally occurring nucleotides can include those with 2'-OH, 2'-deoxy, and 2'-OMe.

[0065] In some embodiments, the antisense strand contains at least one unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA) modification, e.g., in the seed region of the antisense strand. In one embodiment, the seed region is located at positions 2-8 (or 5-7) of the 5' end of the antisense strand.

[0066] In one embodiment, the compound comprises a sense strand and an antisense strand each having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the compound has less than 20%, less than 15%, less than 10%, less than 5%, or is substantially free of non-natural nucleotides.

[0067] In one embodiment, the compound comprises a sense strand and an antisense strand each having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the compound has more than 80%, more than 85%, more than 95%, or substantially 100% natural nucleotides, e.g., those having 2'-OH, 2'-deoxy, or 2'-OMe.

[0068] One aspect of the present invention provides a compound comprising: a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxy modifications on the sense strand and / or the antisense strand; wherein the compound has a double-stranded (duplex) region of 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand does not comprise glycol nucleic acid (GNA).

[0069] It is understood that the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, in other words, the compound is capable of inhibiting the expression of the target gene.

[0070] In one embodiment, the compound comprises at least three 2'-deoxy modifications: at positions 2 and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at position 11 of the sense strand, counting from the 5' end of the sense strand.

[0071] In one embodiment, the compound comprises at least five 2'-deoxy modifications: at positions 2, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0072] In one embodiment, the compound comprises at least seven 2'-deoxy modifications: at positions 2, 5, 7, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0073] In one embodiment, the antisense strand comprises at least five 2'-deoxy modifications, counting from the 5' end of the antisense strand, at positions 2, 5, 7, 12, and 14. The antisense strand has a length of 18 to 25 nucleotides, or a length of 18 to 23 nucleotides.

[0074] In one embodiment, the compound may contain one or more non-natural nucleotides. For example, the compound may contain less than 20%, e.g., less than 15%, less than 10%, or less than 5% of natural nucleotides, or the compound does not contain any non-natural nucleotides. For example, the compound contains all natural nucleotides. Some exemplary non-natural nucleotides include, but are not limited to, acyclic nucleotides, locked nucleic acids (LNAs), HNAs, CeNAs, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0075] In one embodiment, the compound comprises sense and antisense strands, each strand independently having a length of 15-35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense and / or antisense strand; wherein the compound has a duplex region of 19-25 base pairs; wherein the compound comprises a ligand; wherein the sense strand does not comprise glycol nucleic acid (GNA); wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or wherein the compound comprises all natural nucleotides.

[0076] In one embodiment, at least one of the sense strand and the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more 2'-deoxy modifications in the central region of the sense strand or antisense strand. Thus, in one embodiment, the compound comprises sense and antisense strands, each independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; wherein the compound has a duplex region of 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand and / or the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more 2'-deoxy modifications in the central region of the sense strand and / or the antisense strand.

[0077] In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications in a central region of the sense strand, e.g., the sense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications within positions 7, 8, 9, 10, 11, 12, and 13, counting from the 5' end of the sense strand.

[0078] In one embodiment, the antisense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications in the central region of the antisense strand, e.g., the antisense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications within positions 10, 11, 12, 13, 14, 15, and 16, counting from the 5' end of the antisense strand.

[0079] In one embodiment, the compound comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17 to 30 nucleotides and comprises at least one 2'-deoxy modification in the central region of the sense strand; and wherein the antisense strand independently has a length of 17 to 30 nucleotides and comprises at least two 2'-deoxy modifications in the central region of the antisense strand.

[0080] In one embodiment, the compound comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17 to 30 nucleotides and comprises at least two 2'-deoxy modifications in a central region of the sense strand; and wherein the antisense strand independently has a length of 17 to 30 nucleotides and comprises at least one 2'-deoxy modification in a central region of the antisense strand.

[0081] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; wherein the compound has a duplex region of 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense strand.

[0082] In one embodiment, the compound comprises sense and antisense strands, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense and / or antisense strand; wherein the compound has a duplex region of 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the antisense strand.

[0083] In one embodiment, the compound comprises sense and antisense strands, each strand independently having a length of 15-35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense and / or antisense strand; wherein the compound has a duplex region of 19-25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or wherein the compound comprises all natural nucleotides; and wherein the sense and / or antisense strands comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense and / or antisense strands.

[0084] In one embodiment, the compound comprises sense and antisense strands, each strand independently having a length of 15-35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense and / or antisense strand; wherein the compound has a duplex region of 19-25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense strand.

[0085] In one embodiment, the compound comprises sense and antisense strands, each strand independently having a length of 15-35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides, counting from the 5' end of the antisense strand; at least three, four, five, or six 2'-deoxynucleotides on the sense and / or antisense strand; wherein the compound has a duplex region of 19-25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the antisense strand.

[0086] In one embodiment, when the compound contains less than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA. For example, in any one of the embodiments of the present invention, when the compound contains less than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA.

[0087] In one embodiment, if the antisense comprises two deoxynucleotides, the nucleotides being at positions 2 and 14, counting from the 5' end of the antisense strand, the compound comprises eight or fewer (e.g., 8, 7, 6, 5, 4, 3, 2, 1, or 0) non-2'OMe nucleotides. For example, in any one of the embodiments of the present invention, if the antisense comprises two deoxynucleotides, the nucleotides being at positions 2 and 14, counting from the 5' end of the antisense strand, the compound comprises 0, 1, 2, 3, 4, 5, 6, 7, or 8 non-2'-OMe nucleotides.

[0088] In another aspect, the present invention further provides a method for delivering a compound of the present invention to a specific target in a subject by subcutaneous or intravenous administration. The present invention further provides a compound of the present invention for use in a method for delivering said agent to a specific target in a subject by subcutaneous or intravenous administration.

[0089] Another aspect of the present invention relates to a method of reducing expression of a target gene in a cell, comprising contacting the cell with a compound comprising: an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties, conjugated to one or more positions on at least one strand, optionally via a linker or carrier.

[0090] All of the above embodiments relating to lipophilic monomers, lipophilic moieties and their conjugation to compounds in the first aspect of the invention relating to compounds are suitable for this aspect of the invention relating to a method of reducing expression of a target gene in a cell.

[0091] In one embodiment, the cell is an extrahepatic cell.

[0092] In one embodiment, the cell is not a hepatocyte.

[0093] Another aspect of the present invention relates to a method of reducing expression of a target gene in a subject, comprising the step of administering to a subject a compound comprising contacting said cells with a compound comprising: an antisense strand complementary to the target gene; a sense strand complementary to said antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties, conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0094] All of the above embodiments relating to lipophilic monomers, lipophilic moieties and their conjugation to compounds in the first aspect of the invention relating to compounds are suitable for this aspect of the invention relating to a method of reducing expression of a target gene in a subject.

[0095] In some embodiments, the compound is administered extrahepatically.

[0096] In one embodiment, the compound is administered intrathecally or intracerebroventricularly. By administering the compound intrathecally or intracerebroventricularly, the method may reduce expression of the target gene in brain or spinal tissue, such as the cerebral cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0097] In some embodiments, exemplary target genes are APP, ATXN2, C9orf72, TARDBP, MAPT (tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and TTR.To reduce the expression of these target genes in subjects, compound can be administered directly to eye, for example, intravitreally.By administering compound intravitreally, this method can reduce the expression of target genes in ocular tissue.

[0098] Another aspect of the present invention relates to a method of treating a subject suffering from a central nervous system disease (or central nervous system disorder), comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, thereby treating the subject. The double-stranded RNAi agent comprises an antisense strand complementary to a target gene, a sense strand complementary to the antisense strand, and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0099] All of the above embodiments relating to the lipophilic monomer, one or more lipophilic moieties, and their conjugation to the compound in the first aspect of the invention relating to the compound are suitable for this aspect of the invention relating to the method of treating a subject suffering from a central nervous system disease. Exemplary central nervous system diseases that can be treated by the methods of the invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar ataxia (spinocerebellar disorders), prion diseases, and Lafora's disease. [Brief explanation of the drawings]

[0100] [Figure 1] 1 is a scheme showing the general structure of ceramides. [Figure 2] 1 is a graph showing the stability of siRNA conjugates in rat CSF after incubating the siRNA duplexes with rat CSF for 24 hours. [Figure 3] 1 is a graph showing the stability of siRNA conjugates in the vitreous humor of rabbits and cynomolgus monkeys (NHPs) over a 24-hour period, where the amount of remaining ligand-conjugated duplex is plotted. [Figure 4] 1 is a graph showing the stability of siRNA conjugates in the vitreous humor of rabbits and cynomolgus monkeys (NHPs) over a 24-hour period, where the amount of remaining ligand-conjugated duplex is plotted. [Figure 5A]1 is a graph showing the stability of siRNA conjugates in rat brain homogenate over a 4-hour period, with the amount of remaining ligand-conjugated duplex plotted. [Figure 5B] 1 is a graph showing the stability of siRNA conjugates in rat brain homogenate over a 4-hour period. The stability of PS binding is plotted. [Figure 6]

[0023] Figure 1 shows the stability of siRNA conjugates with esterase-cleavable conjugates in the vitreous humor of rabbits and cynomolgus monkeys (NHPs) over a 24-hour period. The percentage of ligand-conjugate duplexes hydrolyzed is plotted. [Figure 7]

[0023] Figure 1 is a graph showing the stability of siRNA conjugates with esterase-cleavable conjugates in rat plasma, CSF, and brain homogenate over 24 hours. The percentage of ligand-conjugate duplex hydrolyzed is plotted. [Figure 8] 1 is a graph showing human serum albumin binding of siRNA conjugates at various concentrations of HSA, where the percentage of bound siRNA is plotted against the concentration of human serum albumin. [Figure 9] 1 is a graph showing human serum albumin binding of siRNA conjugates with exposed carboxylic acids at various concentrations of HSA, where the percentage of bound siRNA is plotted against the concentration of human serum albumin. [Figure 10] 1 is a graph showing inhibition of intraocular TTR expression by qPCR in mouse eyes following intravitreal administration of a single 7.5 μg dose of siRNA duplex compared to a PBS control. [Figure 11] 1 is a graph showing inhibition of intraocular TTR expression by qPCR in rat eyes following intravitreal administration of a single 1 μg dose of siRNA duplex compared to a PBS control. [Figure 12] 1 is a graph showing inhibition of intraocular TTR expression by qPCR in mouse eyes following intravitreal administration of a single 7.5 μg dose of siRNA duplex compared to a PBS control. [Figure 13]1 is a graph showing inhibition of intraocular TTR expression by qPCR in rat eyes following intravitreal administration of a single 1 μg dose of siRNA duplex compared to a PBS control. [Figure 14] 1 is a graph showing inhibition of intraocular TTR expression by qPCR in mouse eyes following intravitreal administration of a single 7.5 μg dose of siRNA duplex compared to a PBS control. [Figure 15] 1 is a graph showing inhibition of intraocular TTR expression by qPCR in rat eyes following intravitreal administration of a single 1 μg dose of siRNA duplex compared to a PBS control. [Figure 16] 1 is a graph showing inhibition of TTR gene expression in primary mouse hepatocytes 24 hours after transfection of the cells with siRNA duplexes modified with Q367, compared to control duplex AD-900954 at three different concentrations. Each of the nucleotides was modified across the sense strand with Q367. [Figure 17] 1 is a graph showing inhibition of SOD1 gene expression in primary mouse hepatocytes 24 hours after transfection of the cells with siRNA duplexes modified with Q367 compared to control duplex AD-900954 at three different concentrations. Each of the nucleotides was modified across the sense strand with Q367. [Figure 18A] 10 is a graph showing inhibition of SOD1 expression by qPCR in the spinal cord of rats after a single IT dose of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 18B] 10 is a graph showing inhibition of SOD1 expression by qPCR in rat cerebellum following a single IT dose of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 18C] 10 is a graph showing inhibition of SOD1 expression by qPCR in the frontal cortex of rats after a single IT administration of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 18D]10 is a graph showing inhibition of SOD1 expression by qPCR in rat hearts after a single IT dose of 0.9 mg siRNA duplex / rat compared to artificial CSF-treated controls after 14 days. [Figure 19A] 10 is a graph showing inhibition of SOD1 expression by qPCR in the spinal cord of rats after a single IT dose of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 19B] 10 is a graph showing inhibition of SOD1 expression by qPCR in rat brainstem following a single IT dose of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 19C] 10 is a graph showing inhibition of SOD1 expression by qPCR in rat cerebellum following a single IT dose of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 19D] 10 is a graph showing inhibition of SOD1 expression by qPCR in the frontal cortex of rats after a single IT administration of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 19E] 10 is a graph showing inhibition of SOD1 expression by qPCR in rat hearts after a single IT dose of 0.9 mg siRNA duplex / rat compared to artificial CSF-treated controls after 14 days. [Figure 20] 1 is a graph showing inhibition of SOD1 expression by qPCR in rat brain (cerebellum and frontal lobe) and spinal cord (thoracic cord) after a single IT administration of 0.9 mg siRNA duplex / rat compared to an artificial CSF-treated control group after 14 days. [Figure 21A] 1 is a graph showing inhibition of SOD1 expression by qPCR in mouse brain (right hemisphere) following a single ICV administration of 50 μg of siRNA duplex / mouse compared to an artificial CSF-administered control group after 14 days. [Figure 21B]Graph showing inhibition of SOD1 expression by qPCR in mouse brain (right hemisphere) and heart after a single ICV administration of 110 μg siRNA duplex / mouse compared to an artificial CSF-treated control group after 7 days. DETAILED DESCRIPTION OF THE INVENTION

[0101] In particular, the inventors have found that conjugating a lipophilic monomer containing a lipophilic moiety to one or more positions on at least one strand of the compound provides unexpectedly good results for in vivo intraocular (e.g., intravitreal) and intrathecal or intracerebroventricular delivery of double-stranded iRNA, resulting in efficient entry into central nervous system and ocular tissues and efficient uptake by cells of the central nervous system and visual system.

[0102] One aspect of the present invention provides a compound comprising: an antisense strand complementary to a target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand, optionally via a linker or carrier.

[0103] The term "lipophilic moiety" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One method for characterizing the lipophilicity of a lipophilic moiety is the octanol-water partition coefficient, log K ow where K owis the ratio of a chemical's concentration in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the chemical's components calculated using first-principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its log K ow is greater than 0. Typically, a lipophilic moiety has a log K of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow For example, the log K of 6-aminohexanol ow For example, the log K of cholesteryl N-(hexan-6-ol) carbamate is predicted to be about 0.7. ow is predicted to be 10.7.

[0104] The lipophilicity of a molecule can be altered with respect to the functional groups it possesses. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value may be increased or decreased.

[0105] Alternatively, the hydrophobicity of a compound (e.g., a double-stranded iRNA agent) conjugated to one or more lipophilic monomers containing one or more lipophilic moieties can be measured by its protein binding properties.For example, the unbound fraction of a compound in a plasma protein binding assay can be determined to correlate with the relative hydrophobicity of the double-stranded iRNA agent, which can be positively correlated with the silencing activity of the double-stranded iRNA agent.

[0106] In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the double-stranded iRNA agent, as measured by the fraction of unbound siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for improved in vivo delivery of siRNA.

[0107] Therefore, conjugating a lipophilic monomer containing a lipophilic moiety to an internal position of the compound provides optimal hydrophobicity for improved in vivo delivery of siRNA.

[0108] In certain embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic, compounds such as steroids (e.g., sterols) or straight- or branched-chain aliphatic hydrocarbons. The lipophilic moiety generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, e.g., oxygen or nitrogen atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C6 30 Hydrocarbons (e.g., C6-C 18 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes and C 40 tetraterpenes), and other polyalicyclic hydrocarbons. For example, the lipophilic moiety may be C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 In some embodiments, the lipophilic moiety may contain a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., straight chain C6-C 18 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C16 alkyl or alkenyl).

[0109] A lipophilic monomer containing a lipophilic moiety can be attached to an iRNA agent by any method known in the art, such as through a functional group already present in the lipophilic monomer or introduced into the iRNA agent, such as a hydroxy group (e.g., -CO-CH-OH). Functional groups already present in the lipophilic monomer or introduced into the iRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0110] Conjugation of an iRNA agent and a lipophilic monomer can occur, for example, by formation of an ether or carboxyl or carbamoyl ester bond between a hydroxy and alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched-chain; and saturated or unsaturated). The alkyl group R can be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0111] In some embodiments, the lipophilic monomer containing the lipophilic moiety is conjugated to the compound via a linker that includes 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 (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0112] In another embodiment, the lipophilic moiety is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0113] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" refers broadly to monocyclic and polycyclic aromatic hydrocarbons. Aromatic groups include, but are not limited to, C6-C8 aromatic groups containing 1-3 aromatic rings, which may be optionally substituted. 14 "Aralkyl" or "arylalkyl" groups comprising an aryl group covalently bonded to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; 6, 10, or 14 pi-electrons shared in a cyclic arrangement; and, in addition to carbon atoms, 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0114] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group has 1 to about 4, preferably 1 to about 3, and more preferably 1 or 2 non-hydrogen substituents. Suitable substituents include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0115] In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulin, or α-1-glycoprotein.

[0116] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Pat. Nos. 3,904,682 and 4,009,197, the entire contents of which are incorporated herein by reference. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure: [ka] is.

[0117] In certain embodiments, the ligand is ibuprofen or an ibuprofen structural derivative. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, the entire contents of which are incorporated herein by reference. The structure of ibuprofen is: [ka] is.

[0118] Further exemplary aralkyl groups are set forth in US Pat. No. 7,626,014, the entire contents of which are incorporated herein by reference.

[0119] In another embodiment, suitable lipophilic moieties include 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, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0120] In some embodiments, the lipophilic moiety is a C-C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.) or C6 to C 30Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0121] In certain embodiments, lipophilic monomers containing two or more lipophilic moieties can be incorporated into a double-stranded iRNA agent, particularly when the lipophilic moieties have low lipophilicity or hydrophobicity. In one embodiment, lipophilic monomers containing two or more lipophilic moieties are incorporated into the same strand of a double-stranded iRNA agent. In one embodiment, each strand of a double-stranded iRNA agent incorporates a lipophilic monomer moiety containing one or more lipophilic moieties. In one embodiment, lipophilic monomers containing two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage) of a double-stranded iRNA agent. This can be achieved, for example, by using a carrier capable of linking two or more lipophilic moieties, and / or a branched linker, and / or a lipophilic monomer containing one or more linkers.

[0122] When the lipophilic moiety is conjugated to an iRNA agent via a direct bond to the nucleobase, ribosugar, or internucleoside linkage of the iRNA agent, the lipophilic monomer comprises the nucleobase, ribosugar, or internucleoside linkage and the lipophilic moiety. Alternatively, the lipophilic monomer can comprise the lipophilic moiety conjugated to a non-ribose-substituted unit, such as a linker or carrier. When the lipophilic moiety is conjugated to a double-stranded iRNA agent via a non-ribose-substituted unit, such as a linker or carrier, the lipophilic monomer comprises the non-ribose-substituted unit, such as a linker or carrier, and the lipophilic moiety.

[0123] In certain embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to an iRNA agent via one or more linkers (tethers).

[0124] In one embodiment, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0125] Linker / Tether The linker / tether is linked to the lipophilic moiety at a "tethering attachment point (TAP)." The linker / tether can 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 amido (NHC(O)-) group in the linker / tether that can serve as a point of attachment for a lipophilic moiety. Non-limiting examples of linkers / tethers (underlined) include: [ka] wherein 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 a 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 be optionally substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or may optionally contain one or more additional heteroatoms, e.g., N, O, or S. Preferred linking ligands include, for example, [ka] 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, for example, with C(O)CF.

[0126] 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: [ka] where n can be as described elsewhere. The tether can be optionally substituted, for example, with hydroxy, alkoxy, perhaloalkyl, and / or can optionally contain one or more additional heteroatoms, for example, N, O, or S. The double bond can be cis or trans or E or Z.

[0127] 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, such as an NHS ester, or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) include: [ka] where n is 1 to 6 and R"" is C1 to C6 alkyl; or [ka] where n is 1 to 6 and R'''' is C1 to C6 alkyl; [ka] where n is 1 to 11 and R"" is C1 to C6 alkyl; or [ka] where n can be as described elsewhere and R"" is a C1-C6 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.

[0128] 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]

[0129] In other embodiments, other protected amino groups can be at the terminal positions of the linker / tether, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety can be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0130] 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 comprise - or -(CH=CH)-.

[0131] Cleavable Linker / Tether In some embodiments, at least one of the linkers / tethers can be a redox-cleavable linker, an acidic-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0132] In one embodiment, at least one of the linkers / tethers can be a reductively cleavable linker (eg, a disulfide group).

[0133] 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).

[0134] In one embodiment, at least one of the linkers / tethers can be an esterase-cleavable linker (eg, an ester group).

[0135] In one embodiment, at least one of the linkers / tethers can be a phosphatase-cleavable linker (eg, a phosphate group).

[0136] In one embodiment, at least one of the linkers / tethers can be a peptidase-cleavable linker (eg, a peptide bond).

[0137] Cleavable linkers are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or present at higher levels or activity within cells than in serum or blood. Examples of such degrading agents include intracellular oxidases or reductases or reducing agents that can degrade redox-cleavable linkers by reduction, such as mercaptans, which are selective for specific substrates or have no substrate specificity; esterases; endosomes or agents that can create an acidic environment, such as a pH of 5 or less; enzymes that can hydrolyze or degrade acidic cleavable linkers by functioning as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0138] Cleavable linking groups, e.g., disulfide bonds, are sensitive to pH. The pH of human serum is 7.4, while 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 about 5.0. Some tethers have linking groups that are cleaved at a preferred pH, thereby releasing the iRNA agent from the ligand (e.g., a targeting or cell-permeable ligand, e.g., cholesterol) into the cell or into a desired compartment of the cell.

[0139] The chemical bond (e.g., linking group) linking the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is taken up into a cell by endocytosis, the acidic environment of the endosome causes the disulfide bond to be cleaved, thereby 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 can be a secondary therapeutic agent that can complement the therapeutic effect of the targeting ligand or iRNA agent.

[0140] The tether may contain a linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether may vary depending 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 therefore, the tether is cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether releases the iRNA agent 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.

[0141] Tethers that include peptide bonds can be conjugated to iRNA agents that target cell types rich in peptidases, such as hepatocytes and synovial cells. For example, iRNA agents targeted to synovial cells for the treatment of inflammatory diseases (e.g., rheumatoid arthritis) can be conjugated to tethers that include peptide bonds.

[0142] In general, 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 in contact with other non-target tissues, such as tissues to which the iRNA agent is exposed when administered to a subject. Thus, the relative ease of cleavage between a first condition and a second condition can be determined, with the first condition being selected to demonstrate cleavage within target cells and the second condition being selected to demonstrate cleavage in other tissues or body fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-fold, or 100-fold 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).

[0143] Redox-cleavable linking groups One class of cleavable linkers is redox-cleavable linkers, which are cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (—SS—). To determine whether a candidate cleavable linker is a suitable “reductively cleavable linker,” or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be verified. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic cleavage rates that may be observed in cells, e.g., 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 2-fold, 4-fold, 10-fold, or 100-fold faster inside cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a 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.

[0144] Phosphate-based cleavable linkers Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate groups. An example of an agent that cleaves phosphate groups within a cell is an intracellular enzyme, such as a phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, 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.

[0145] acidic cleavable linking group An acidic cleavable tether is a tether that is cleaved under acidic conditions. In a preferred embodiment, the acidic cleavable tether 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 that can function as a general acid, such as an enzymatic agent. In cells, certain organelles with low pH, such as endosomes and lysosomes, can provide a cleavage environment for the acidic cleavable tether. Examples of acidic cleavable tethers include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acidic cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when 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.

[0146] Ester-based linking group Ester-based linking groups are cleaved by intracellular 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.

[0147] Peptide-based cleavable linkers Peptide-based linking groups are cleaved by intracellular enzymes, such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linking 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, resulting in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. Peptide cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)—, where 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.

[0148] Biocleavable linkers / tethers Linkers can also include biocleavable linkers, which are nucleotide and non-nucleotide linkers or combinations thereof, that link two parts of a molecule, for example, one or both strands of two individual siRNA molecules to generate bis(siRNA). In some embodiments, simple electrostatic or stacking interactions between two individual siRNAs can represent a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0149] In some embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0150] In one embodiment, the biocleavable carbohydrate linker can have 1-10 sugar units with at least one aromatic linkage capable of linking two siRNA units. When more than one sugar is present, the units can be linked via 1-3, 1-4, or 1-6 sugar linkages or via alkyl chains.

[0151] Exemplary biocleavable linkers include: [ka] [ka] [ka] [ka] Examples include:

[0152] Further description of biocleavable linkers can be found in PCT Application No. PCT / US18 / 14213, entitled "Endosomal Cleavable Linkers," filed January 18, 2018, the entire contents of which are incorporated herein by reference.

[0153] Carrier In certain embodiments, the lipophilic monomer comprises a non-ribose-substituting unit, i.e., a lipophilic moiety conjugated to an iRNA agent via a carrier that substitutes one or more nucleotides.

[0154] 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 decalin. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0155] A carrier can replace one or more nucleotides of a double-stranded iRNA agent.

[0156] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the double-stranded iRNA agent.

[0157] In other embodiments, the carrier replaces a nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine. For example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0158] Ribonucleotide subunits in which the ribose sugar of the subunit has been replaced in this manner are referred to as ribose-replacement modified subunits (RRMS). The carrier may be a cyclic or acyclic moiety and comprises two "backbone attachment points" (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). The lipophilic moiety may be directly attached to the carrier or indirectly attached to the carrier by an intervening linker / tether, as described above. [ka]

[0159] The ligand-conjugated monomer subunit can be the 5' or 3' terminal subunit of an iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit can occupy an internal position, and both "W" groups can be one or more unmodified or modified ribonucleotides. Two or more ligand-conjugated monomer subunits can be present in an iRNA agent.

[0160] Monomers based on sugar substitutions, e.g., ligand-conjugated monomers (cyclic) Monomers based on cyclic sugar substitutions, e.g., ligand-conjugate monomers based on the substitutions, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) shown 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 (two positions may be selected from two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 The preferred tether attachment point is R 7 ; When X is CH2, R 5 or R 6 Carriers are described below as substances that can be incorporated into the chain. Thus, the structure may have one (for terminal positions) or two (for internal positions) attachment points, e.g., R 1 or R 2 ;R 3 or R 4 ; or R 9 or R 10 (Y is CR 9 R 10It is understood that the term "R" also encompasses cases where the R group is linked to a phosphate or modified phosphate, e.g., a sulfur-containing backbone. For example, one of the R groups listed above can be -CH-, where one bond is linked to the carrier and one is linked to a backbone atom, e.g., the linking oxygen or central phosphorus atom. [ka] 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 does not exist; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 each of which is independently H, OR a , or (CH2) n OR b where R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of the a and / or (CH2) n OR b and; R 5 , R 6 , R 11 , and R 12 each independently comprising a ligand, H, one to three R 13 optionally substituted C1-C6 alkyl, or C(O)NHR 7 or R 5 and R 11 Together, R 14 and optionally substituted C3-C8 cycloalkyl; R 7can be a ligand, e.g., R 7 is R d or R 7 is a ligand, e.g., NR, that is indirectly linked to a carrier, e.g., via a tether moiety. c R d C1~C substituted with 20 Alkyl; or NHC(O)R d C1~C substituted with 20 may be 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 cyano, optionally substituted C1-C6 alkyl, or C2-C6 alkenyl; R 16 is C1~C 10 is alkyl; R 17 is a liquid or solid phase carrier reagent; L is -C(O)(CH2) q C(O)- or -C(O)(CH2) q S- and; R a is a protecting group, such as CAr; (e.g., dimethoxytrityl group) or Si(X 5’ )(X 5” )(X 5”’ ), where (X 5’ ),(X 5” ), and (X 5”’ ) is as described above. R b is P(O)(O - )H, P(OR 15 )N(R 16 )2 or LR 17 and; R c is H or C1-C6 alkyl; R d is H or a ligand; Each Ar is independently selected from C1-C4 alkoxy, C6-C 10 is aryl; n is 1 to 4; q is 0 to 4.

[0161] 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 CH; 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 indane ring system, for example, X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 taken together to form a C5 cycloalkyl (H, z=1).

[0162] 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 CR9 R 10 and Z does not exist (D). [ka] OFG 1 is preferably a carbon atom in the five-membered ring (-CH2OFG in D) 1 ) is attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, which is linked to one of the OFG 2 is preferably a carbon atom in the five-membered ring (-OFG in D) 2 ) for pyrroline-based carriers, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; or -CH2OFG 1 may be attached to C-3, OFG 2 may be attached to C-4. In certain embodiments, CH2OFG 1 and OFG 2 may be geminally substituted at one of the above carbons. For 3-hydroxyproline-based carriers, -CHOFG 1 may be attached to C-2, OFG 2 may be attached to C-4. Thus, pyrroline-based and 4-hydroxyproline-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, CHOFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2(The centers having the formula (I) and (II) both have the R configuration; or both 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 tether attachment point is preferably nitrogen. Preferred examples of carrier D include the following: [ka] Examples include:

[0163] In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., 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 a carbon atom in the six-membered ring [—(CH) in E] n OFG 1 OFG is bonded to a primary carbon, for example, an exocyclic alkylene group, for example, a methylene group (n=1) or an ethylene group (n=2), which is linked to one of the following: 2 is preferably a carbon atom in the six-membered ring (-OFG in E) 2 ) is directly bonded to one of the -(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, e.g., at C-2, C-3, or C-4. Alternatively, -(CH) n OFG 1 and OFG 2 may be 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, OFG 2 may be attached to C-3; -(CH2) n OFG 1may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3. Thus, a piperidine-based monomer may contain a bond (e.g., a carbon-carbon bond) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, -(CH2) n OFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having both have the R configuration; or both 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 tether attachment point is preferably nitrogen.

[0164] 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 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 a carbon atom in a six-membered ring (-CH2OFG in F or G) 1 ) is attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, which is linked to one of the OFG 2 is preferably a carbon atom in a six-membered ring (-OFG in F or G) 2 ) for both F and G. 1 may be attached to C-2, OFG 2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG 1 and OFG 2 may be geminally substituted on one of the carbons. Thus, piperazine- and morpholine-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, CHOFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the R and S configurations may both have the R and S configurations, or one center may have the R and S configurations, or vice versa). R''' may be, for example, a C1-C6 alkyl, preferably CH3. The tether attachment points are preferably nitrogen in both F and G.

[0165] In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH; Y is CR 9 R 10 and Z is CR 11 R 12and R 5 and R 11 together form a C6 cycloalkyl (H, z=2) or indane ring system, e.g., X is CH2; 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 —(CH) at C-2, C-3, C-4, or C-5 [H in —(CH) n OFG 1 ], for example, an exocyclic methylene group (n=1) or an ethylene group (n=2). OFG 2 is preferably at C-2, C-3, C-4, or C-5 (H in -OFG 2 ) is directly bonded to one of the -(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, for example, at C-2, C-3, C-4, or C-5. Alternatively, -(CH) n OFG 1 and OFG 2 may be 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, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1may be attached to C-4, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-5; or -(CH2) n OFG 1 may be attached to C-5, OFG 2 may be attached to C-4. Thus, decalin or indane-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, -(CH2) n OFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the formula (I) and (II) both have the R configuration; or both 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 preferred embodiments, the substituents at C-1 and C-6 are trans relative to each other. The tether attachment point is preferably at C-6 or C-7.

[0166] Other carriers may include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2can be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having both have the R configuration; or both 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 tether attachment point is preferably nitrogen.

[0167] Details regarding more representative cyclic sugar-based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, the entire contents of which are incorporated herein by reference.

[0168] Monomers based on sugar substitution (acyclic) Acyclic sugar-substitution-based monomers, e.g., sugar-substitution-based ligand-conjugate monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are represented by formula LCM-3 or LCM-4: [ka] may have:

[0169] In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. When y and z are different in formula LCM-3, the tertiary carbon can have either the R or S configuration. In a preferred embodiment, in formula LCM-3 (e.g., based on serinol), x is 0, and y and z are each 1; in formula LCM-3, y and z are each 1. Each of formulas LCM-3 or LCM-4 below can be optionally substituted, for example, with hydroxy, alkoxy, or perhaloalkyl.

[0170] Details regarding more representative acyclic sugar-based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, the entire contents of which are incorporated herein by reference.

[0171] In some embodiments, the compounds comprise one or more lipophilic monomers containing a lipophilic moiety conjugated to the 5'-end of the sense strand or the 5'-end of the antisense strand.

[0172] In certain embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the 5' end of the chain via a carrier and / or linker. In one embodiment, the lipophilic monomer has the formula: [ka] The 5' end of the chain contains a lipophilic moiety conjugated to the chain via a carrier of the formula: where R is a ligand such as a lipophilic moiety.

[0173] In some embodiments, the compounds comprise one or more lipophilic monomers containing one or more lipophilic moieties conjugated to the 3'-end of the sense strand or the 3'-end of the antisense strand.

[0174] In certain embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the 3' end of the chain via a carrier and / or linker. In one embodiment, the lipophilic monomer has the formula: [ka] The hydroxyl group contains a lipophilic moiety conjugated to the 3' end of the chain via a carrier of the formula: where R is a ligand such as a lipophilic moiety.

[0175] In certain embodiments, the lipophilic monomer contains a lipophilic moiety conjugated via a carrier and / or linker to an internal position of the chain. In one embodiment, the lipophilic monomer has the formula: [ka] The lipophilic moiety is conjugated to an internal position of the chain via a carrier of formula: where R is a ligand such as a lipophilic moiety.

[0176] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to both ends of the sense strand.

[0177] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to both ends of the antisense strand.

[0178] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to an internal position of the sense strand or antisense strand. In some embodiments, the one or more lipophilic moieties are conjugated to the ribose, the nucleobase, and / or at an internucleotide linkage. In some embodiments, the one or more lipophilic moieties are conjugated to the ribose at the 2', 3', 4', and / or 5' position of the ribose. In some embodiments, the one or more lipophilic moieties are conjugated to a natural (such as A, T, G, C, or U) or modified nucleobase as defined herein. In some embodiments, the one or more lipophilic moieties are conjugated to a phosphate or modified phosphate group as defined herein.

[0179] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to the 5'- or 3'-end of the sense strand and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to the 5'- or 3'-end of the antisense strand.

[0180] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the end of the chain via one or more linkers (tethers) and / or carriers.

[0181] In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to the end of the chain via one or more linkers (tethers).

[0182] In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to the 5' end of the sense strand or antisense strand via a cyclic carrier, optionally via one or more intervening linkers (tethers).

[0183] In some embodiments, at least one lipophilic monomer is located at one or more terminal positions of the sense strand or the antisense strand. In one embodiment, at least one lipophilic monomer is located at the 3'-end or 5'-end of the sense strand. In one embodiment, at least one lipophilic monomer is located at the 3'-end or 5'-end of the antisense strand.

[0184] In some embodiments, lipophilic monomers containing lipophilic moieties are conjugated to one or more internal positions on at least one strand, which refers to nucleotides at any position on the strand, excluding the 3'- and 5'-terminal positions of the strand (e.g., excluding two positions: position 1 counting from the 3'-end and position 1 counting from the 5'-end).

[0185] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, including all but the two most terminal positions on each end of the chain (e.g., four positions: excluding positions 1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end). In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, including all but the three most terminal positions on each end of the chain (e.g., six positions: excluding positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).

[0186] In one embodiment, at least one lipophilic monomer is located at one or more positions at at least one end of the duplex region, including all positions within the duplex region but not including the overhang region, or on a carrier replacing the terminal nucleotide at the 3' end of the sense strand.

[0187] In one embodiment, at least one lipophilic monomer is located on the sense strand within the first 5, 4, 3, 2, or first base pairs at the 5' end of the antisense strand of the duplex region.

[0188] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one strand excluding the cleavage site region of the sense strand, e.g., no lipophilic monomer is located at positions 9-12 from the 5' end of the sense strand, e.g., no lipophilic monomer is located at positions 9-11 from the 5' end of the sense strand, or internal positions excluding positions 11-13 from the 3' end of the sense strand.

[0189] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one strand, excluding the cleavage site region of the antisense strand, e.g., internal positions excluding positions 12-14 from the 5' end of the antisense strand.

[0190] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one strand, excluding positions 11-13 on the sense strand, counting from the 3' end, and positions 12-14 on the antisense strand, counting from the 5' end.

[0191] In one embodiment, the one or more lipophilic monomers are located at one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0192] In one embodiment, the one or more lipophilic monomers are located at one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0193] definition Unless otherwise specified, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Some such techniques and procedures are described, for example, in "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications," Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2002. nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout this disclosure are incorporated herein by reference in their entirety.

[0194] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be regulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target protein (including, but not limited to, pre-mRNA and mRNA or portions thereof), and also cDNA and miRNA obtained from such RNA. For example, a target nucleic acid may be a cellular gene (or mRNA transcribed from that gene) whose expression is associated with a particular disorder or pathology. In some embodiments, a target nucleic acid may be a nucleic acid molecule derived from an infectious agent.

[0195] As used herein, the term "iRNA" refers to an agent that mediates targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or iRNA agents. Thus, these terms may be used interchangeably herein. As used herein, the term iRNA includes microRNAs and pre-microRNAs. Furthermore, as used herein, a "compound" or "compounds" of the invention also refer to an iRNA agent and may be used interchangeably with an iRNA agent.

[0196] An iRNA agent should include a region sufficiently homologous to a target gene and be of sufficient length, in nucleotides, so that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. (For ease of explanation, the terms nucleotide or ribonucleotide may be used herein in reference to one or more monomeric subunits of an iRNA agent. It will be understood herein that the use of the terms "ribonucleotide" or "nucleotide" herein, in the case of a modified RNA or nucleotide surrogate, can also refer to the modified nucleotide or surrogate replacement moiety at one or more positions.) Thus, an iRNA agent is or includes a region that is at least partially, and in some embodiments, completely, complementary to a target RNA. While perfect complementarity between an iRNA agent and a target is not required, the match must be sufficient to enable the iRNA agent, or its cleavage product, to direct sequence-specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. The degree of complementarity, or homology, with the target strand is most important in the antisense strand. While perfect complementarity is often desirable, particularly in the antisense strand, some embodiments may include one or more, or for example, 6, 5, 4, 3, 2, or fewer, mismatches (with respect to the target RNA), particularly in the antisense strand. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded nature of the molecule.

[0197] iRNA agents include molecules that are long enough to trigger an interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)); and molecules that are short enough not to trigger an interferon response (which can also be cleaved by Dicer and / or enter RISC), e.g., molecules of a size that allows entry into RISC, e.g., molecules similar to Dicer cleavage products. Molecules that are short enough not to trigger an interferon response are referred to herein as siRNA agents or short iRNA agents. As used herein, "siRNA agent or short iRNA agent" refers to an iRNA agent, e.g., a double-stranded RNA agent or a single-stranded agent, that is short enough not to induce a deleterious interferon response in human cells, e.g., which has a duplex region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or its cleavage product, can downregulate a target gene, for example, by inducing RNAi against the target RNA, where the target can include an endogenous or pathogen target RNA.

[0198] As used herein, a "single-stranded iRNA agent" is an iRNA agent that is composed of a single molecule. It may include a duplex region formed by intrastrand pairing; for example, it may be or include a hairpin or panhandle structure. A single-stranded iRNA agent may be antisense to a target molecule. A single-stranded iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of a target mRNA. A single-stranded iRNA agent is at least 14, and in other embodiments, at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.

[0199] A loop refers to a region of an iRNA strand that, when base-pairing with another strand or another portion of the same strand, does not pair with the opposite nucleotide in the duplex.

[0200] Hairpin iRNA agents have a duplex region of at least or equal to 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be 200, 100, or 50 or less in length. In certain embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. The hairpin has a single-stranded overhang or terminal unpaired region at the 3' end in some embodiments, and in certain embodiments, on the antisense side of the hairpin. In some embodiments, the overhang is 2-3 nucleotides in length.

[0201] As used herein, a "double-stranded (ds) iRNA agent" is an iRNA agent that includes two or more, and in some cases two, strands that can form a region of duplex structure by hybridization between the strands.

[0202] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0203] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in mRNA levels in a cell of a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between these, of the mRNA levels found in a cell in the absence of the miRNA or RNA interference molecule. In a preferred embodiment, the mRNA levels are reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between 5% and 100%.

[0204] As used herein, the term "modulate gene expression" means up-regulating or down-regulating the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, such that the expression, level, or activity is higher or lower than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," although the use of the word "modulate" is not limited to this definition.

[0205] As used herein, gene expression modulation occurs when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The percentage and / or fold difference may be calculated relative to a control or non-control, for example, as follows:

number

[0206] As used herein, the terms "inhibit," "down-regulate," or "reduce," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced by at least 10% compared to a corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably, 100% (i.e., no gene expression).

[0207] As used herein, the terms "increase" or "up-regulate," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased by at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more, relative to a corresponding unmodulated control.

[0208] As used herein, the term "increased" or "increasing" generally refers to an increase by a statistically significant amount; for the avoidance of doubt, "increased" means an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference sample, or an increase of up to 100%, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a reference level, or any increase from 2-fold to 10-fold or more.

[0209] As used herein, the term "reduced" or "reducing" generally refers to a statistically significant reduction. However, for the avoidance of doubt, "reduced" refers to a reduction of at least 10% compared to the reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of 100% or less (i.e., zero level compared to the reference sample), or any reduction between 10% and 100% compared to the reference level.

[0210] A double-stranded iRNA comprises two oligonucleotide strands sufficiently complementary to hybridize to form a duplex structure. Typically, the duplex structure is 15-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded iRNAs, 25-30 base pairs in length, are preferred. In some embodiments, shorter double-stranded iRNAs, 10-15 base pairs in length, are preferred. In another embodiment, the double-stranded iRNA is at least 21 nucleotides in length.

[0211] In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity that is complementary to at least a portion of a target sequence, and the duplex region is 14-30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 nucleotides in length.

[0212] As used herein, "compound" refers to an oligomeric compound, which may be an oligonucleotide, antisense, or iRNA agent, such as an siRNA.

[0213] As used herein, the phrase "antisense strand" refers to an oligomeric compound that is substantially or 100% complementary to the intended target sequence. The phrase "antisense strand" includes the antisense region of both oligomeric compounds that are formed from two separate strands, as well as unimolecular oligomeric compounds that can form hairpin or dumbbell structures. The terms "antisense strand" and "guide strand" are used interchangeably.

[0214] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence that is wholly or partially identical to a target sequence, such as a sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably.

[0215] "Specifically hybridizable" and "complementary" mean that a nucleic acid can form hydrogen bonds with another nucleic acid sequence, either through Watson-Crick or other non-traditional types. In the context of the nucleic acid molecules of the present invention, the binding free energy between a nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, such as RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, Am. Chem. Soc. 109: 3783-3785). Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to a situation in which some (but not all) nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected to be non-complementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0216] In some embodiments, the double-stranded region of the compound is at least equal to or equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0217] In some embodiments, the antisense strand of the compound is at least or equal to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0218] In some embodiments, the sense strand of the compound is at least equal to or equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0219] In one embodiment, the sense and antisense strands of the compound are each 15 to 30 nucleotides in length.

[0220] In one embodiment, the sense and antisense strands of the compound are each 19 to 25 nucleotides in length.

[0221] In one embodiment, the sense and antisense strands of the compound are each 21-23 nucleotides in length.

[0222] In some embodiments, one strand has at least one stretch of 1 to 5 single-stranded nucleotides within the double-stranded region. A "single-stranded nucleotide stretch within a double-stranded region" means that there is at least one nucleotide base pair on both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. When both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides can be highly opposite (e.g., a mismatched stretch), or they can be positioned such that the second strand does not have a single-stranded nucleotide opposite the single-stranded iRNA of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides from either end, for example, 8, 7, 6, 5, 4, 3, or 2 nucleotides from either the 5' or 3' end of the region of complementarity between the two strands.

[0223] In one embodiment, the compound comprises a single-stranded overhang on at least one of its termini, hi one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0224] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

[0225] In some embodiments, each strand of the double-stranded iRNA has a ZXY structure, such as that described in PCT Publication No. WO2004080406, the entire contents of which are incorporated herein by reference.

[0226] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked to each other. The two strands may be linked to each other at both ends or only one end. Linked at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands may be, but are not limited to, (N) n (where N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker may participate in base-pairing interactions with other nucleotides in the linker. The two strands can also be linked to each other by a non-nucleoside linker, such as the linkers described herein. It will be understood by those skilled in the art that any of the oligonucleotide chemical modifications or alterations described herein can be used in the oligonucleotide linker.

[0227] Hairpin and dumbbell-shaped oligomeric compounds have duplex regions of at least or equal to 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be up to 200, 100, or 50 nucleotide pairs in length. In some embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0228] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3', and in some embodiments, at the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4, more usually 2 to 3, nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0229] In certain embodiments, two oligomer strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0230] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound hybridizes to its target sequence but to a minimal number of other sequences. Stringent conditions are sequence-dependent and vary in various 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 tested.

[0231] It is understood in the art that incorporating nucleotide affinity modifications can allow a greater number of 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 an oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443).

[0232] siRNA design In one embodiment, the iRNA agent is a 19-nt long double-ended bluntmer, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0233] In one embodiment, the iRNA agent is a 20 nt long blunt-ended duplex, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0234] In one embodiment, the iRNA agent is a 21 nt long blunt-ended duplex, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0235] In one embodiment, the iRNA agent comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the iRNA is blunt while the other end comprises a 2-nt overhang. Preferably, the 2-nt overhang is at the 3' end of the antisense strand. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0236] In one embodiment, the iRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and wherein the 5' end of the antisense strand comprises 10 to 30 consecutive ribonucleotides that are not paired with the sense strand. The double-stranded nucleic acid comprises at least 19 ribonucleotides of the sense strand, forming a 10-30 nucleotide single-stranded 5' overhang; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, at least one of the motifs being located at or near the cleavage site; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

[0237] In one embodiment, the iRNA agent comprises a sense strand and an antisense strand, wherein the iRNA agent comprises a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand including at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1-4 nucleotides longer than the first strand at its 3' end, the duplex region is at least 25 nucleotides long, and the second strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the second strand such that the iRNA agent reduces target gene expression when introduced into a mammalian cell, and wherein Dicer cleavage of the iRNA preferentially results in siRNA including the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the iRNA agent further includes a ligand (eg, GalNAc3).

[0238] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs is at the cleavage site of the sense strand. For example, the sense strand can contain at least one motif of three 2'-F modifications in three consecutive nucleotides within positions 7-15 from the 5' end.

[0239] In one embodiment, the antisense strand of an iRNA agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs is at or near the cleavage site of the antisense strand. For example, the antisense strand can contain at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides within positions 9-15 from the 5' end.

[0240] For iRNA agents having a duplex region 17-23 nt in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the iRNA from the 5' end.

[0241] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand includes at least two motifs of three identical modifications in three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site within the strand, and at least one of the motifs is in another portion of the strand separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand can also include at least one motif of three identical modifications in three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site within the strand. The modifications in the motifs at or near the cleavage site in the sense strand are different from the modifications in the motifs at or near the cleavage site in the antisense strand.

[0242] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, where the sense strand includes at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs is at or near the site of cleavage in the strand. In one embodiment, the antisense strand also includes at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the site of cleavage.

[0243] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, where the sense strand includes at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand includes at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0244] In one embodiment, the iRNA agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., relative to the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

[0245] In one embodiment, the iRNA agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group A:U, G:U, I:C, and a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing including a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0246] In one embodiment, the nucleotide at position 1 from the 5' end of the antisense strand into the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand into the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand into the duplex region is an AU base pair.

[0247] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the dsRNA agent is modified. For example, if 50% of the dsRNA agent is modified, then 50% of all nucleotides present in the dsRNA agent contain a modification described herein.

[0248] In one embodiment, the sense strand and the antisense strand are each independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0249] In one embodiment, the sense and antisense strands of the dsRNA agent each contain at least two different modifications.

[0250] In one embodiment, the dsRNA agent does not include a 2'-F modification.

[0251] In one embodiment, the sense strand and / or antisense strand of a dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0252] In one embodiment, the sense and antisense strands of the dsRNA agent each have 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0253] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0254] In one embodiment, the antisense strand of a dsRNA agent is 100% complementary to a target RNA for hybridizing to the target RNA and inhibiting its expression by RNA interference. In another embodiment, the antisense strand of a dsRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0255] In one aspect, the invention relates to a dsRNA agent, as defined herein, capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand comprises at least one thermolabile nucleotide, at least one of which is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand).

[0256] For example, if the sense strand is 21 nucleotides long, the thermally destabilizing nucleotide may be located between positions 14 and 17 at the 5' end of the sense strand. The antisense strand includes at least two modified nucleic acids that are less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids that are less than sterically demanding 2'-OMe are spaced 11 nucleotides apart. For example, the two modified nucleic acids are located at positions 2 and 14 at the 5' end of the antisense strand.

[0257] In one embodiment, the dsRNA agent is (a) a sense strand comprising: (i) 18–23 nucleotides long; (ii) a sense strand having three consecutive 2'-F modifications at positions 7 to 15; and (b) an antisense strand: (i) 18–23 nucleotides long; (ii) at least a 2'-F modification anywhere on the strand; and (iii) comprises an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); The dsRNA agent has either one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the duplex.

[0258] In one embodiment, the dsRNA agent is (a) a sense strand comprising: (i) 18–23 nucleotides long; (ii) the sense strand, having fewer than four 2'-F modifications; (b) an antisense strand: (i) 18–23 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) comprises an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); The dsRNA agent has either one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the duplex.

[0259] In one embodiment, the dsRNA agent is (a) a sense strand comprising: (i) 19–35 nucleotides long; (ii) the sense strand, having fewer than four 2'-F modifications; (b) an antisense strand: (i) 19–35 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) comprises an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); wherein the duplex region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the duplex.

[0260] In one embodiment, the dsRNA agent has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; and the dsRNA agent has less than 20%, less than 15%, and less than 10% non-naturally occurring nucleotides.

[0261] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, and others.

[0262] In one embodiment, the dsRNA agent has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; and the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% naturally occurring nucleotides, e.g., 2'-OH, 2'-deoxy, and 2'-OMe are naturally occurring nucleotides.

[0263] In one embodiment, the dsRNA agent has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; and the dsRNA agent has 100% naturally occurring nucleotides, e.g., 2'-OH, 2'-deoxy, and 2'-OMe are naturally occurring nucleotides.

[0264] In one embodiment, the dsRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, where the sense strand sequence is represented by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) During the ceremony: i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b represents an oligonucleotide sequence containing, independently, 1, 2, 3, 4, 5, or 6 modified nucleotides; each n p and n q independently represent overhanging nucleotides; N b and Y do not have the same modification; XXX, YYY and ZZZ each independently represent a motif of three identical modifications in three consecutive nucleotides; The dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions in at least one strand; The antisense strand of the dsRNA contains two blocks of 1, 2, or 3 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.

[0265] Various publications describe multimeric siRNAs, all of which may be used with the iRNAs of the present invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of which are incorporated herein by reference.

[0266] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agents of the invention are modified with 2'-OMe.

[0267] In some embodiments, each of the sense and antisense strands of an iRNA agent is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0268] In some embodiments, the sense and antisense strands of an iRNA agent each contain at least two different modifications.

[0269] In some embodiments, the compounds of the invention do not contain any 2'-F.

[0270] In some embodiments, the compounds of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, the compounds of the invention comprise 9 or 10 2'-F modifications.

[0271] The iRNA agent of the present invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present at any nucleotide in the sense strand, antisense strand, or both strands at any position. For example, the internucleotide bond modification may be present at every nucleotide in the sense strand or antisense strand; each internucleotide bond modification may be present in an alternating pattern in the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification in the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of the internucleotide bond modification in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modification in the antisense strand.

[0272] In one embodiment, the iRNA agent includes a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification can also be formed to link the overhang nucleotide to the terminal base-pairing nucleotide in the duplex region. For example, at least two, three, four, or all of the overhanging nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, an additional phosphorothioate or methylphosphonate internucleotide bond can be present linking the overhang nucleotide to the next base-pairing nucleotide. For example, at least two phosphothioate internucleotide bonds can be present between the terminal three nucleotides, two of which are overhanging nucleotides and the third nucleotide is the next base-pairing nucleotide to the overhanging nucleotide. Preferably, these terminal three nucleotides can be at the 3' end of the antisense strand.

[0273] In some embodiments, the sense strand and / or antisense strand of an iRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0274] In some embodiments, the antisense strand of an iRNA agent is 100% complementary to the target RNA in order to hybridize to the target RNA and inhibit its expression by RNA interference, hi other embodiments, the antisense strand of an iRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0275] Nucleic acid modification In some embodiments, the compound comprises at least one nucleic acid modification as described herein.For example, at least one modification is selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combination thereof.Without being limited, such modification can be present anywhere in the compound.For example, the modification can be present in one of the RNA molecules.

[0276] Nucleic acid modifications (nucleobases) The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common types of such heterocyclic bases are purines and pyrimidines. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0277] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art can be used in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is substituted with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases, including conjugate moieties, e.g., ligands, as described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl, or amide bonds.

[0278] The oligomeric compounds described herein may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6-(Isopentenyl)adenine, 6-(Alkyl)adenine, 6-(Methyl)adenine, 7-(Deaza)adenine, 8-(Alkenyl)adenine, 8-(Alkyl)adenine, 8-(Alkynyl)adenine, 8-(Amino)adenine, 8-(Halo)adenine, 8-(Hydroxyl)adenine, 8-(Thioalkyl)adenine, 8-(Thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 , N 6 -(Dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(Acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil , 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil Uracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl allyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stivenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2 -substituted purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used.

[0279] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an iRNA duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, and imidizopyridinyl. In some embodiments, the aryl groups include phenyl, 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stibenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0280] No. 3,687,808; those disclosed in International Application No. PCT / US09 / 038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30,613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, all of which are incorporated herein by reference.

[0281] In certain embodiments, the modified nucleobase is a nucleobase that is structurally very similar to the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, the nucleobase mimic comprises a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase mimic. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.

[0282] Nucleic acid modification (sugar) The compounds of the invention provided herein can contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers, including nucleosides or nucleotides, having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent group or the bridging of two non-geminal ring atoms to form a locked nucleic acid or a bicyclic nucleic acid. In certain embodiments, an oligomeric compound contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers that are LNAs.

[0283] In some embodiments of the locked nucleic acid, the 2' position of the furanosyl is -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -C(=NR1)-, -C(=NR1)- O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) x - and -N(R1)-; During the ceremony: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, substituted heteroaryl, a C5-C7 cycloaliphatic radical, a substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0284] In some embodiments, each of the linkers in the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In other embodiments, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or a C1-C12 alkyl.

[0285] Certain LNAs have been prepared and disclosed in the patent and scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al. al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Patent Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0286] Also provided herein are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bond to form a bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage may be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, and the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; if there is an ethylene group at this position, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy(4'-CH2-O-2')LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0287] A similarly described isomer of methyleneoxy(4'-CH2-O-2')LNA is α-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have superior stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras, which have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0288] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0289] Analogs of methyleneoxy(4'-CH2-O-2')LNA, phosphorothioate-methyleneoxy(4'-CH2-O-2')LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been reported.

[0290] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars, including methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars having a 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituent; and 4'-thio modified sugars. Sugars can also be substituted with sugar mimetic groups, among others. Methods for preparing modified sugars are well known to those of skill in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; and 5,576,427. Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO 2005 / 121371.

[0291] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) nCH2CH2OR, n=1-50; "locked" nucleic acids (LNA) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2) n AMINE (n=1-10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0292] "Deoxy" modifications include hydrogen (i.e., deoxyribose sugars particularly associated with single-stranded overhangs); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Included are CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl, and alkynyl, which may be optionally substituted, for example, with an amino function.

[0293] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0294] Modifications at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of the ribose in the same configuration as the 2'-OH in arabinose.

[0295] A sugar may contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar group may also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, an oligomeric compound may contain, for example, one or more monomers containing arabinose as the sugar. The monomer may have an α-linkage at the 1'-position of the sugar, e.g., an α-nucleoside. The monomer may also have the opposite configuration at the 4'-position, e.g., C5' and H4' or the substituents replacing them are interchanged. When C5' and H4' or the substituents replacing them are interchanged, the sugar is said to be modified at the 4'-position.

[0296] The compounds of the present invention disclosed herein may also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having other chemical groups at C1' instead of a nucleobase. See, for example, U.S. Pat. No. 5,998,203, the entire contents of which are incorporated herein. These abasic sugars may also contain modifications to one or more of the constituent sugar atoms. The compounds of the present invention may also contain one or more sugars in the L-isomer, e.g., L-nucleoside. Modifications to the sugar group may also include replacing the 4'-O with sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0297] Sugar modifications can also include "acyclic nucleotides," which refers to any nucleotide having an acyclic ribose sugar in which, for example, a C-C bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0298] In some embodiments, the sugar modification is selected from the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl, 2'-O-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0299] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be located at the 3' position of the sugar for that particular nucleotide, e.g., the nucleotide linked through its 2' position. The modification at the 3' position can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the 3'-OH in a xylose sugar.

[0300] The hydrogen attached to C4' and / or C1' can be replaced with a straight or branched chain, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein the backbone of the alkyl, alkenyl, and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, wherein R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 21 R 31 ,CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is independently at each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 , or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is independently at each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , or CO2R 11 , or NR 11 R 11 ' and ;R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is substituted.

[0301] In some embodiments, C4' and C5' together preferably form an optionally substituted heterocycle containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, where M is independently for each occurrence an alkali metal or transition metal having a total charge of +1; and Y is O, S, or NR', where R' is hydrogen or an optionally substituted aliphatic. Preferably, this modification is at the 5' end of the iRNA.

[0302] In certain embodiments, compounds of the present invention comprise at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, compounds of the present invention comprise a gap motif. In certain embodiments, compounds of the present invention comprise at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, compounds of the present invention comprise at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0303] In certain embodiments, the compounds of the present invention have the formula: [ka] wherein Bx is a heterocyclic base moiety. and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer.

[0304] In certain embodiments, the monomer comprises a sugar mimetic. In certain such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while the nucleobase is maintained for hybridization with a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some examples, the mimetics are used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those skilled in the art.

[0305] Nucleic acid modification (sugar bond) Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, e.g., oligonucleotides. Such linking groups are also referred to as intersugar linkages. Two major classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphodiester bonds, modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In certain embodiments, linkages with chiral atoms can be prepared as separate enantiomers or as racemic mixtures. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0306] The phosphate group in the linking group can be modified by replacing one of the oxygen atoms with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced with any of the following: S, Se, BR3 (R is hydrogen, alkyl, or aryl), C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, or aryl), or (R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or atomic groups makes the phosphorus atom chiral; in other words, the phosphorus atom in such a modified phosphate group is a stereocenter. The stereogenic phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

[0307] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in phosphorodithioates is chiral, which prevents the formation of oligonucleotide diastereomers. Therefore, without wishing to be bound by theory, modifications to both non-bridging oxygens may be desirable because they eliminate asymmetric centers, such as phosphorodithioate formation, which cannot produce diastereomeric mixtures. Thus, the non-bridging oxygens can independently be any one of O, S, Se, B, C, H, N, or OR (where R is alkyl or aryl).

[0308] Phosphate linkers can also be modified by substitution of the bridging oxygen (i.e., the oxygen linking the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). This substitution can occur at either or both of the linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution with nitrogen is preferred.

[0309] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced, or the phosphate group is replaced by a non-phosphorus group, are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0310] In certain embodiments, the phosphate group can be replaced by a non-phosphorus-containing connector, such as a dephosphoryl linker. Dephosphoryl linkers are herein referred to as non-phosphodiester linkers. Without wishing to be bound by theory, it is believed that because the charged phosphodiester group is the reactive center of nucleic acid degradation, its replacement with a neutral structural mimic should confer improved nuclease stability. Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the charged phosphate group is replaced by a neutral moiety.

[0311] Examples of moieties that can replace the phosphate group include, but are not limited to, amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methyl Examples include carbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-O-P(O)-O-S-S-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5' and nonionic linkages containing mixed N, O, S and CH2 moieties. For example, Carbohydrate See Modifications in Antisense Research; YS Sanghvi and PDCook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0312] Those skilled in the art will appreciate that, in certain instances, substitution of a non-bridging oxygen can result in enhanced cleavage of the intersugar bond by the adjacent 2'-OH, and therefore, in many cases, modification of the non-bridging oxygen may require modification of the 2'-OH, e.g., a modification that does not involve cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0313] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates containing an enantiomeric excess of the Sp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorothioates containing an enantiomeric excess of the Rp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0314] In some embodiments, the compounds of the invention contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to and including all) modified or non-phosphodiester linkage. In some embodiments, the compounds of the invention contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and up to and including all) phosphorothioate linkage.

[0315] The compounds of the present invention can also be constructed in which the phosphate linker and sugar are replaced by nuclease-resistant nucleoside or nucleotide surrogates. Without wishing to be bound by theory, it is believed that the absence of a repeatedly charged backbone weakens binding to proteins that recognize polyanions (e.g., nucleases). Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce modifications in which the bases are linked by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and backbone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0316] The compounds of the invention described herein can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined with respect to absolute stereochemistry as (R) or (S), such as in the case of sugar anomers, or as (D) or (L), such as in the case of amino acids. The compounds of the invention provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0317] Nucleic acid modification (end modification) In some embodiments, the compound further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0318] In some embodiments, the 5' end of the antisense strand of the compound does not contain a 5'-vinylphosphonate (VP).

[0319] The termini of the iRNA agents of the invention can be modified. Such modifications can be at one or both termini. For example, the 3' and / or 5' termini of the iRNA can be conjugated to a labeling moiety, e.g., a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entity, such as a protecting group (e.g., sulfur, silicon, boron, or ester-based). The functional molecular entity can be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker can be linked to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker can be linked to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

[0320] When a linker / phosphate-functional molecular entity-linker / phosphate array is placed between the two strands of a double-stranded oligomeric compound, the array can take the place of a hairpin loop in a hairpin-shaped oligomeric compound.

[0321] Terminal modifications useful for modulating activity include modification of the 5' end of an iRNA with phosphate or a phosphate analog. In certain embodiments, the 5' end of an iRNA is phosphorylated or contains a phosphoryl analog. Exemplary 5'-phosphate modifications include modifications compatible with RISC-mediated gene silencing. Modifications at the 5' end may also be useful for stimulating or inhibiting a subject's immune system. In some embodiments, the 5' end of an oligomeric compound is modified [ka] wherein W, X, and Y are each independently O, OR (wherein R is hydrogen, alkyl, or aryl), S, Se, BR3 (wherein R is hydrogen, alkyl, or aryl), BH3 - , C (i.e., alkyl, aryl, etc.), H, NR (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl, or aryl); A and Z are each, independently at each occurrence, absent, O, S, CH, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can include internally and / or terminally one or more of O, S, SS, and NR (R is hydrogen, alkyl, aryl); and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen linked to the 5' carbon of the sugar. When n is 0, W and Y, together with the P to which they are attached, can form an optionally substituted 5-8 membered heterocycle, where W and Y are each independently O, S, NR', or alkylene. Preferably, the heterocycle is substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on the C5' of the 5' terminal nucleotide are replaced with a halogen, eg, F.

[0322] Exemplary 5'-modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); 5'-alpha-thiotriphosphate; 5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkyl phosphonates (R(OH)(O)PO-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)PO-5', R = alkyl ether, e.g., methoxymethyl (CHOMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is alkyl, optionally substituted at least once, e.g., ((HO)(X)PO[-(CH) a -OP(X)(OH)-O] b -5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl-terminated phosphates and phosphate mimetics: HO[-(CH2) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b -5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O]b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5', where a and b are each independently 1 to 10. Other embodiments include BH3, BH3 - and / or containing substitution of oxygen and / or sulfur by Se.

[0323] Terminal modifications may also be useful for monitoring distribution; in such cases, preferred groups to be added include fluorophores, such as fluorescein, or Alexa dyes, such as Alexa 488. Terminal modifications may also be useful for promoting uptake; useful modifications for this purpose include targeting ligands. Terminal modifications may also be useful for crosslinking the oligonucleotide to another moiety; useful modifications for this purpose include mitomycin C, psoralens, and their derivatives.

[0324] Thermal destabilization modification Compounds of the invention, such as iRNA or dsRNA agents, can be optimized for RNA interference by introducing a thermostabilizing modification into the sense strand opposite the antisense strand's seed region (i.e., positions 2-8 at the 5' end of the antisense strand) to enhance the dissociation or melting properties of the iRNA duplex (reducing the free energy of duplex binding). This modification can enhance the dissociation or melting properties of the duplex at the antisense strand's seed region.

[0325] Thermally destabilizing modifications can include abasic modifications; mismatches with opposing nucleotides in the opposing strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycerol nucleic acids (GNAs).

[0326] Exemplary abasic modifications are as follows: [ka]

[0327] Exemplary sugar modifications are as follows: [ka]

[0328] The term "UNA" refers to an unlocked acyclic nucleic acid in which all sugar bonds have been removed to form an unlocked "sugar" residue. In one example, a UNA includes a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' sugar bond (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) has been removed (see Mikhailov et al., Tetrahedron Letters, 26 (17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), the entire contents of each of which are incorporated herein by reference). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick base pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0329] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" as it consists of repeating glycerol units linked by phosphodiester bonds: [ka]

[0330] The thermolabile modification can be a mismatch (i.e., non-complementary base pair) between the thermolabile nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pairings known in the art are also suitable for the present invention. Mismatches can occur between nucleotides that are naturally occurring or modified nucleotides, i.e., mismatch base pairing can occur between nucleobases from each nucleotide regardless of the modification in the ribose sugar of the nucleotide. In certain embodiments, the compounds of the present invention, such as siRNA or iRNA agents, contain at least one nucleobase that is a 2'-deoxynucleobase in the mismatch pairing; for example, the 2'-deoxynucleobase is in the sense strand.

[0331] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, the entire contents of which are incorporated herein by reference.

[0332] Thermodestabilizing modifications can include universal bases that have reduced or eliminated the ability to form hydrogen bonds with opposing bases, and phosphate modifications.

[0333] Nucleobase modifications that reduce or completely eliminate the ability to form hydrogen bonds with bases in the opposing strand were evaluated for destabilizing the central region of the dsRNA agent duplex, as described in International Publication No. WO 2010 / 0011895, the entire contents of which are incorporated herein by reference. Exemplary nucleobase modifications are as follows: [ka]

[0334] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are: [ka]

[0335] In some embodiments, compounds of the invention may contain 2'-5' linkages (with 2'-H, 2'-OH, and 2'-OMe, and with P=O or P=S). For example, 2'-5' linkage modifications may be used to increase nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to avoid activation of the sense strand by RISC.

[0336] In another embodiment, the compounds of the invention may contain L-sugars (e.g., L-ribose, L-arabinose, including 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications may be used to increase nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0337] In one embodiment, an iRNA agent of the invention is conjugated to a ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0338] In some embodiments, at least one strand of an iRNA agent disclosed herein is 5' phosphorylated or includes a phosphoryl analog at the 5' terminus. 5'-phosphate modifications include modifications that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO -5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0339] Target gene Non-limiting examples of target genes for siRNA include genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, gene expression kinases, adaptor protein genes, genes encoding G-protein superfamily molecules, genes encoding transcription factors, genes that mediate angiogenesis, viral genes, genes required for viral replication, cellular genes that mediate viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes that mediate unwanted immune responses, genes that mediate pain processing, genes that mediate neurological disorders, alleles found in cells characterized by loss of heterozygosity, or one allele of a polymorphic gene.

[0340] Specific exemplary target genes for siRNA include, but are not limited to, PCSK-9, ApoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGF bet gene; Erb-B gene, Src gene; CRK gene; GRB2 gene; RAS gene; MEKK gene; JNK gene; RAF gene; Erk1 / 2 gene; PCNA (p21) gene; MYB gene; c-MYC gene; JUN gene; FOS gene; BCL-2 gene; cyclin D gene; VEGF gene; EGFR gene; cyclin gene; cyclin E gene; WNT-1 gene; β-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; topoisomerase I gene; and topoisomerase II gene. α gene; p73 gene; p21 (WAF1 / CIP1) gene, p27 (KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor genes; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, e.g., MLL-AF9, BCR / ABL fusion gene; TEL / AML1 fusion gene; EWS / FLI1 fusion gene; TLS / FUS1 fusion gene; PAX3 / FKHR fusion gene; AML1 / ETO fusion gene; α v-integrin gene; Flt-1 receptor gene; tubulin gene; human papillomavirus gene, gene required for human papillomavirus replication, human immunodeficiency virus gene, gene required for human immunodeficiency virus replication, hepatitis A virus gene, gene required for hepatitis A virus replication, hepatitis B virus gene, gene required for hepatitis B virus replication, hepatitis C virus gene, gene required for hepatitis C virus replication, hepatitis D virus gene, gene required for hepatitis D virus replication, hepatitis E virus gene, gene required for hepatitis E virus replication, hepatitis F virus gene, gene required for hepatitis F virus replication, hepatitis G virus gene, gene required for hepatitis G virus replication, hepatitis H virus gene, gene required for hepatitis H virus replication, respiratory syncytial virus geneGenes required for respiratory syncytial virus replication, Herpes simplex virus genes, Genes required for herpes simplex virus replication, Herpes cytomegalovirus genes, Genes required for herpes cytomegalovirus replication, Herpes Epstein-Barr virus genes, Genes required for herpes Epstein-Barr virus replication, Kaposi's sarcoma-associated herpesvirus genes, Genes required for Kaposi's sarcoma-associated herpesvirus replication, JC virus genes, Human genes required for JC virus replication, Myxovirus genes, Genes required for myxovirus gene replication, Rhinovirus genes, Genes required for rhinovirus replication, Coronavirus genes, Genes required for coronavirus replication, West Nile virus genes, Genes required for West Nile virus replication, St. Louis encephalitis genes, Genes required for St. Louis encephalitis replication, Tick-borne encephalitis virus genes, Genes required for tick-borne encephalitis virus replication, Murray Valley encephalitis virus genes, Genes required for Murray Valley encephalitis virus replication Dengue virus genes, genes required for dengue virus replication, simian virus 40 genes, genes required for simian virus 40 replication, human T-cell lymphotropic virus genes, genes required for human T-cell lymphotropic virus replication, Moloney murine leukemia virus genes, genes required for Moloney murine leukemia virus replication, encephalomyocarditis virus genes, genes required for encephalomyocarditis virus replication, measles virus genes, genes required for measles virus replication, varicella-zoster virus genes, genes required for varicella-zoster virus replication, adenovirus genes, genes required for adenovirus replication, yellow fever virus genes, genes required for yellow fever virus replication, poliovirus genes, genes required for poliovirus replication, poxvirus genes, genes required for poxvirus replication, plasmodium genes, genes required for plasmodium replication, Mycobacterium ulcerans ulcerans genes, genes required for Mycobacterium ulcerans replication, Mycobacterium tuberculosis genes,Genes required for Mycobacterium tuberculosis replication, Mycobacterium leprae genes, Genes required for Mycobacterium leprae replication, Staphylococcus aureus genes, Genes required for Staphylococcus aureus replication, Streptococcus pneumoniae genes, Genes required for Streptococcus pneumoniae replication, Streptococcus pyogenes genes, Genes required for Streptococcus pyogenes replication, Chlamydia pneumoniae genes, Genes required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, Mycoplasma pneumoniae) replication-required genes, integrin genes, selectin genes, complement system genes, chemokine genes, chemokine receptor genes, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene, proplatelet basic protein gene, MIP-1I gene, MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene, CMBKR3 gene, CM Examples include BKR5v, AIF-1 gene, I-309 gene, genes for components of ion channels, genes for neurotransmitter receptors, genes for neurotransmitter ligands, amyloid family genes, presenilin genes, HD genes, DRPL genes, SCA1 genes, SCA2 genes, MJD1 genes, CACNL1A4 genes, SCA7 genes, SCA8 genes, alleles found in loss of heterozygosity (LOH) cells, single alleles of polymorphic genes, and combinations thereof.

[0341] Loss of heterozygosity (LOH) can result in hemizygous sequences, such as genes, in the region of LOH. This can result in significant genetic differences between normal cells and diseased cells, such as cancer cells, providing useful differentiation between normal cells and diseased cells, such as cancer cells. This difference can occur because a gene or other sequence is heterozygous in diploid cells but hemizygous in cells with LOH. Regions of LOH often contain genes whose loss promotes unwanted proliferation, such as tumor suppressor genes, and other sequences, including other genes, in some cases genes essential for normal function, such as growth. The methods of the present invention rely, in part, on the specific regulation of one allele of an essential gene by the compositions of the present invention.

[0342] In certain embodiments, the present invention provides compounds of the present invention that modulate microRNAs.

[0343] Central Nervous System Targeting In some embodiments, the present invention provides compounds that target APP for early-onset familial Alzheimer's disease, ATXN2 for spinocerebellar ataxia 2 and ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia.

[0344] In some embodiments, the present invention provides compounds that target TARDBP for ALS, MAPT (tau) for frontotemporal dementia, and HTT for Huntington's disease.

[0345] In some embodiments, the present invention provides compounds that target SNCA for Parkinson's disease, FUS for ALS, ATXN3 for spinocerebellar ataxia 3, ATXN1 for SCA1, genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, PRNP for prion diseases, recessive central nervous system disorders: Lafora disease, DMPK (central nervous system and skeletal muscle) for DM1, and TTR (central nervous system, intraocular and systemic) for hATTR.

[0346] Spinocerebellar ataxia is a genetic brain dysfunction. Dominantly inherited spinocerebellar ataxias, such as SCA1-8, are severe diseases with no disease-modifying treatment. Exemplary targets include SCA2, SCA3, and SCA1.

[0347] Targeting ATXN2 for SCA2 Spinocerebellar ataxia 2 (SCA2), a progressive ataxia, is the second most common form of SCA. Another disease related to this target is amyotrophic lateral sclerosis (ALS). These diseases are debilitating and ultimately fatal, with no disease-modifying treatment available. The prevalence of SCA is 2-6 per 100,000; ATXN2 causes 15% of the SCA population worldwide and a much higher SCA population in some countries, particularly Cuba (40 per 100,000). Targeting ATXN2 through human molecular genetics may be promising; for example, a coding CAG repeat expansion in ATXN2 has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA and ALS. The mechanism of this targeting may be due to the autosomal dominant coding CAG expansion in ATXN2, which leads to the expression of harmful misfolded proteins and Purkinje cell and neuronal cell death. Efficacy was demonstrated by a 70% knockdown (KD) of ATXN2 mRNA; proof of concept was demonstrated in mATXN2 mice. Regarding safety, mATXN2 knockout (KO) mice were reported to be healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0348] Targeting ATXN3 for SCA3 Spinocerebellar ataxia 3 (SCA3), a progressive ataxia, is the most common type of SCA worldwide. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. It is the most common cause of SCA, with a prevalence of 2-6 per 100,000 people; ATXN3 causes 21% of SCA cases in the United States and is much more prevalent in Europe, particularly Portugal. Targeting ATXN3 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATXN3 has been found in tissues such as the spinal cord, brainstem, and cerebellum in familial and sporadic SCA. The mechanism of this targeting may be due to the autosomal dominant coding CAG expansion in ATXN3, which leads to the expression of a deleterious misfolded protein, resulting in Purkinje cell and neuronal death. Efficacy was demonstrated by a 70% knockdown of ATXN3 mRNA; proof-of-concept in ATXN3 knockdown mice was demonstrated. Regarding safety, mATXN3 KO mice were reported to be healthy. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0349] Targeting ATXN1 for SCA1 Spinocerebellar ataxia 1 (SCA1), a progressive ataxia, was the first SCA gene discovered in 1993. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. The prevalence of SCA is 2-6 per 100,000 people; ATXN1 causes 6% of the SCA population in the United States and worldwide, with much higher prevalence in some countries (25% in Japan), particularly Poland (64%) and Siberia (100%). Targeting ATXN1 through human molecular genetics may be promising; for example, a coding CAG repeat expansion in ATXN1 has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA. The mechanism of this targeting may be due to the autosomal dominant coding CAG expansion in ATXN1, which leads to the expression of harmful misfolded proteins and Purkinje cell and neuronal cell death. Efficacy was demonstrated by a 70% reduction in ATXN1 mRNA; proof-of-concept in mATXN1 mice was demonstrated. Regarding safety, mATXN1 KO mice were reported to be healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0350] Targeting ATXN7 for SCA7 Spinocerebellar ataxia 7 (SCA7) causes progressive ataxia and retinal degeneration. It is a debilitating, ultimately fatal retinal and cerebellar disorder with no disease-modifying treatment. The prevalence of SCA is 2-6 per 100,000 people; ATXN7 causes 5% of the global SCA population, with a much higher prevalence in some countries, particularly South Africa. Targeting ATXN7 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATXN7 has been found in tissues such as the spinal cord, brainstem, cerebellum, and retina in familial and sporadic SCA. The mechanism of this targeting may be through the autosomal dominant coding CAG expansion in ATXN1, which causes the expression of a deleterious misfolded protein, leading to cone-rod degeneration, Purkinje cell death, and neuronal death. Efficacy was demonstrated by a 70% reduction in ATXN1 mRNA levels following intrathecal (IT) and intravitreal (IVT) administration. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0351] Targeting ATXN8 for SCA8 Spinocerebellar ataxia 8 (SCA8), a progressive neurodegenerative disorder, is caused by a CTG repeat expansion in ATXN8. It is a debilitating, ultimately fatal disorder with no disease-modifying treatment. Prevalence: SCA is 2-6 per 100,000 individuals; ATXN8 causes 3% of the global SCA population, with a much higher incidence in some countries, particularly Finland. Targeting ATXN8 through human molecular genetics may be promising. For example, coding CTG repeat expansions in ATXN8 have been found in tissues such as the spinal cord, brainstem, and cerebellum in familial and sporadic SCA. The mechanism of this targeting may be through the autosomal dominant coding CTG expansion of ATXN8, which leads to the expression of a deleterious misfolded protein, causing Purkinje cell and neuronal death. Efficacy was demonstrated by a 70% reduction in ATXN8 mRNA expression. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CTG mRNA and peptide repeat proteins.

[0352] Targeting CACNA1A for SCA6 Spinocerebellar ataxia 6 (SCA6) is a progressive ataxia. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. The prevalence of SCA is 2-6 per 100,000 people; CACNA1A causes 15% of the SCA population worldwide. Targeting CACNA1A through human molecular genetics may be promising. For example, a coding CAG repeat expansion in CACNA1A has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in CACNA1A causes the expression of a deleterious misfolded protein and Purkinje cell and neuronal cell death. Efficacy was demonstrated by a 70% KD of the CACNA1A CAG expansion. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0353] Exemplary targets of inherited polyglutamine disorders include Huntington's disease (HD).

[0354] Targeting HTT for Huntington's disease Huntington's disease mutations cause HD, a progressive central nervous system degenerative disorder. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. The prevalence of HD is 5-10 per 100,000 people worldwide, with much higher rates in some countries, particularly Venezuela. Targeting HTT through human molecular genetics may be promising. For example, a coding CAG repeat expansion in HTT has been found in tissues such as the striatum or cerebral cortex in familial and sporadic HD. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in HTT leads to the expression of a deleterious misfolded protein and neuronal death. Efficacy was demonstrated by a 70% knockdown of the HTT CAG expansion alone; proof-of-concept in mice has been demonstrated. Regarding safety, knockdown of HTT in mice can be lethal; knockdown in humans has been demonstrated. Potential diagnostic criteria include family history, genetic testing, and early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0355] Targeting ATN1 for DRPLA Atrophin 1 mutations cause dentatorubral-pallidoluysian atrophy (DRPLA), a progressive spinocerebellar disease similar to HD. This disease is debilitating and ultimately fatal, with no disease-modifying treatment available. The prevalence of DRPLA is 2-7 per 1,000,000 people in Japan. Targeting ATN1 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATN1 has been found in tissues such as the spinal cord, brainstem, cerebellum, and cerebral cortex in familial and sporadic SCA. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in ATN1 leads to the expression of harmful misfolded proteins and neuronal death. Efficacy was demonstrated by a 70% knockout of ATN1. Regarding safety, ATN1 KO mice were reported to be healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0356] Targeting AR for spinal and bulbar muscular atrophy Androgen receptor mutations cause spinal and bulbar muscular atrophy (SBMA, Kennedy's disease), a progressive muscle-wasting disorder, and other diseases. This disease is debilitating and ultimately fatal, with no disease-modifying treatment. The prevalence of SBMA is 2 per 100,000 men; women have a milder phenotype. Targeting the AR through human molecular genetics may be promising; for example, a coding CAG repeat expansion in the AR has been discovered in tissues such as the spinal cord or brainstem in familial SBMA. The mechanism of this targeting may be that the X-linked coding CAG expansion in the AR causes deleterious gain of function and motor neuron death. Efficacy has been demonstrated by a 70% reduction in AR KD. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0357] Targeting FXN for Friedreich's ataxia A recessive loss-of-function GAA expansion in FXN causes Friedreich's ataxia (FA), a progressive degenerative ataxia. It is a debilitating, ultimately fatal disorder with no disease-modifying treatment. The prevalence of FA is 2 per 100,000 people worldwide. Targeting FXN through human molecular genetics may be promising. For example, an intronic GAA repeat expansion in FXN has been found in familial FA in tissues such as the spinal cord, cerebellum, and possibly the retina and heart. The mechanism of this targeting may be that the autosomal recessive noncoding FAA expansion in FXN reduces the expression of FXN, an important mitochondrial protein. Efficacy was demonstrated by a 70% knockdown of the FXN intronic GAS expansion. Regarding safety, knockdown of the intronic GAA expansion was safe and effective in mice. Potential diagnostics include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0358] Targeting FMR1 for FXTAS Fragile X-associated tremor / ataxia syndrome (FXTAS), a progressive disorder of adult ataxia and cognitive impairment, is caused by FMR1 overexpression. This disease is severe and has no disease-modifying treatment. The prevalence of FMR1 premutations is 1 in 500 males. Targeting FMR1 through human molecular genetics may be promising; for example, a coding CCG repeat expansion premutation in FMR1 has been found in FXTAS in tissues such as the spinal cord, cerebellum, and cerebral cortex. The mechanism of this targeting may be that the X-linked coding CCG expansion of FMR1 causes toxic mRNA. Efficacy was demonstrated by a 70% reduction in toxic mRNA. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0359] Targeting FMR1 upstream for fragile X syndrome Fragile X syndrome (FRAXA), a progressive disorder of mental retardation, may be treated by targeting the upstream mRNA of FMR1. This disease is debilitating and has no disease-modifying treatment. The prevalence of FRAXA is 1 in 4,000 men and 1 in 8,000 women. Targeting FMR1 through human molecular genetics may be promising; for example, a coding CCG repeat expansion in FMR1 has been discovered in FRAXA in tissues such as the central nervous system. The mechanism of this targeting may be that the X-linked coding CCG expansion in FMR1 causes loss of function; normal FMR1 functions to export specific mRNAs from the nucleus. Efficacy was demonstrated by a 70% reduction in the toxic mRNA. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0360] Dominantly inherited amyotrophic lateral sclerosis is a severe disease with no disease-modifying treatment. Exemplary targets include C9orf72, ATXN2 (which also causes SCA2), and MAPT.

[0361] Targeting C9orf72 for ALS C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These are fatal motor neuron disorders with no disease-modifying treatments. The prevalence of ALS is 2-5 per 100,000 people (10% are familial); C9orf72 causes 39% of familial ALS cases and 7% of sporadic ALS cases in the United States and Europe. Targeting of C9orf72 through human molecular genetics may be promising; for example, hexanucleotide expansions have been found in familial and sporadic ALS in tissues such as upper and lower motor neurons (for ALS) or the cerebral cortex (for FTD). The mechanism of this targeting may be that the autosomal dominant hexanucleotide expansion triggers repeat-associated non-AUG-dependent translation of toxic dipeptide repeat proteins and neuronal death. Efficacy was demonstrated by a 70% KD of C9orf72. Regarding safety, heterozygous LOF mutations of C9orf72 appear to be safe in humans and mice. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF hexanucleotide repeat mRNA and dipeptide repeat protein.

[0362] Targeting TARDBP for ALS TARDBP mutations cause ALS and frontotemporal dementia (FTD). These diseases are fatal motor neuron disorders with no disease-modifying treatment. The prevalence of ALS is 2-5 per 100,000 people (10% are familial); TARDBP causes 5% of familial ALS cases and 1.5% of sporadic ALS cases. Targeting TARDBP through human molecular genetics may be promising; for example, mutations have been found in familial and sporadic ALS in tissues such as upper and lower motor neurons (for ALS) or the cerebral cortex (for FTD). The mechanism of this targeting may be through autosomal dominant TRDBP mutations, which cause toxic TRDBP protein and neuronal death. Efficacy was demonstrated by a 70% KD of TARDBP mutant alleles. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF protein.

[0363] Targeting FUS for ALS FUS mutations cause ALS and FTD. These diseases are fatal motor neuron disorders with no disease-modifying treatment. The prevalence of ALS is 2-5 per 100,000 people (10% are familial); FUS causes 5% of familial ALS cases; FUS inclusions are often found in sporadic ALS. Targeting FUS through human molecular genetics may be promising; for example, mutations have been found in tissues such as upper and lower motor neurons in familial ALS. The mechanism of this targeting may be that autosomal dominant FUS mutations cause abnormal protein folding and neuronal death. Efficacy was demonstrated by 70% knockdown of FUS mutant alleles. Regarding safety, knockout mice exhibit a writhing response but survive and have an ADHD phenotype. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF protein.

[0364] Targeting SOD1 for ALS Dominant and recessive mutations in SOD1 cause ALS, a fatal disease of motor neurons with no disease-modifying treatment. The prevalence of ALS is 2-5 per 100,000 people (10% are familial); SOD1 causes 5-20% of familial ALS cases. Targeting SOD1 through human molecular genetics may be promising; for example, many SOD1 mutations have been associated with familial AD and AR ALS in tissues such as upper and lower motor neurons for ALS. Efficacy of this targeting may require mutation-specific knockdown. Potential diagnoses include family history; genetic testing; or early symptoms. Biomarkers may be mutation-specific.

[0365] Dominantly inherited frontotemporal dementia and progressive supranuclear palsy. Targets include MAPT, as MAPT may be important for AD or C9orf72.

[0366] Targeting the microtubule-associated protein tau for FTD-17 and PSP Familial frontotemporal dementia 17 (FTD-17), a familial form of FTD linked to chromosome 17, and familial progressive supranuclear palsy can be caused by MAPT mutations. MAPT mutations can also cause rare forms of progressive supranuclear palsy, corticobasal degeneration, tauopathy with respiratory failure, and dementia with seizures. These diseases are fatal neurodegenerative disorders with no disease-modifying treatments. The prevalence of FTD is 15-22 per 100,000 people; the prevalence of FTD-17 in the Netherlands is 1 per 1,000,000 people. Targeting MAPT through human molecular genetics may be promising; for example, MAPT GOF and splice site mutations have been found in tissues such as the frontal and temporal lobes in familial and sporadic FTD. The mechanism of this targeting may be due to the autosomal dominant GOF mutation in MAPT, which causes harmful tau peptides and neuronal cell death. Efficacy was demonstrated by a 70% KO of MAPT. Regarding safety, MAPT KO mice were reported to be healthy. Possible diagnoses include family history; genetic testing; and early symptoms. Biomarkers that can be used include, for example, CSF tau mRNA and protein.

[0367] Targeting sequestosome 1 for FTD and ALS Sporadic FTD / ALS is associated with dominant SQSTM1 mutations. This disease is a fatal neurodegenerative disorder with no disease-modifying treatment. It is an extremely rare disease. Targeting sequestosome 1 through genetic association with human molecules is rational in sporadic cases in tissues such as the frontal and temporal lobes or the cerebellum and spinal cord. Potential diagnostics include genetic testing; early symptoms.

[0368] Dominantly inherited Parkinson's disease is a severe disease with no disease-modifying treatment. Targets include SNCA.

[0369] Targeting SNCA for Parkinson's disease Alpha-synuclein mutations cause familial Parkinson's disease (PD) and dementia with Lewy bodies. These diseases are fatal neurodegenerative disorders with no disease-modifying treatment. PD prevalence is 4 million worldwide; one-third of PD cases are familial; and 1% of fPD cases are caused by SNCA. Targeting SNCA through human molecular genetics may be promising. For example, SNCA point mutations and duplications cause familial PD in tissues such as the medulla oblongata or the substantia nigra of the midbrain. The mechanism of this targeting may be through overexpression or abnormal expression of SNCA protein, which leads to toxic peptides and neuronal death. Efficacy was demonstrated by a 70% knockdown of SNCA. Regarding safety, SNCA KO mice are healthy. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF SNCA mRNA and protein.

[0370] Targeting LRRK2 for Parkinson's disease Leucine-rich repeat kinase 2 mutations cause familial Parkinson's disease (PD). This is a fatal neurodegenerative disorder with no disease-modifying treatment. PD prevalence is 4 million worldwide; one-third of PD cases are familial; and 3-7% of fPD cases are caused by LRRK2. Targeting LRRK2 through human molecular genetics may be promising; for example, LRRK2 point mutations cause familial PD in tissues such as the medulla oblongata or the substantia nigra of the midbrain. Potential diagnostics include family history, genetic testing, and early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0371] Targeting GARS for spinal muscular atrophy V Autosomal dominant glycyl-tRNA synthetase mutations cause spinal muscular atrophy V (SMAV) or distal hereditary motor neuropathy Va. These diseases are neurodegenerative disorders with no disease-modifying treatment. They are extremely rare. Targeting GAR through human molecular genetics may be successful; for example, GARS point mutations cause familial SMA in tissues such as the spinal cord. Potential diagnosis includes family history; genetic testing; and early symptoms.

[0372] Targeting seipin for spinal muscular atrophy Autosomal dominant seipin mutations cause spinal muscular atrophy (SMA) or distal hereditary motor neuropathy. These diseases are neurodegenerative disorders with no disease-modifying treatment. They are extremely rare. Targeting seipin through human molecular genetics may be successful; for example, seipin point mutations cause familial SMA in tissues such as the spinal cord. The mechanism of this targeting is likely GOF and toxic peptides. Efficacy was demonstrated by a 50% KD. Regarding safety, recessive LOF mutations cause progressive encephalopathy with or without lipodystrophy. Possible diagnoses include family history; genetic testing; or early symptoms.

[0373] Dominantly inherited Alzheimer's disease is a devastating disease with no disease-modifying treatment. Targets include APP because of its central mechanistic role in familial disease and its possible role in general AD.

[0374] Targeting APP for Alzheimer's disease Amyloid precursor protein mutations cause early-onset familial Alzheimer's disease (EOFAD); AD in Down syndrome; or AD. These diseases are fatal neurodegenerative disorders with no disease-modifying treatment. The prevalence of EOFAD-APP is 1% of AD; the prevalence of trisomy 21 is 1% of AD; the prevalence of AD is approximately 2.5 to 5 million people in the United States. Targeting APP through human molecular genetics may be promising; for example, APP duplications and point mutations cause EOFAD in tissues such as the cerebral cortex or hippocampus. The mechanism of this targeting may be through APP overexpression or expression of toxic metabolites, which cause progressive neuronal death. Efficacy was demonstrated by 70% KD of APP. Regarding safety, KD mice were reported to be healthy, albeit with some behavioral abnormalities; KD mice were reported to be healthy, albeit with some spatial memory deficits. Possible diagnostic criteria include family history; genetic testing; early symptoms; or MRI. Biomarkers that can be used include, for example, CSF APP mRNA and peptides.

[0375] Targeting PSEN1 for Alzheimer's disease Presenilin 1 mutations cause early-onset familial Alzheimer's disease (EOFAD), or AD. These diseases are fatal neurodegenerative disorders with no disease-modifying treatment. Targeting PSEN1 through human molecular genetics may be promising; for example, PSEN1 point mutations cause EOFAD in tissues such as the cerebral cortex or hippocampus. The mechanism of this targeting may be that autosomal dominant mutations in PSEN1 cause abnormal APP metabolism, resulting in toxic peptides that cause progressive neuronal death. Efficacy has been demonstrated by APP KD, which may eliminate the need for PSEN1-specific therapy. Possible diagnoses include family history, genetic testing, early symptoms, or MRI. Biomarkers that may be used include, for example, CSF PSEN1 and APP peptides.

[0376] Targeting PSEN2 for Alzheimer's disease Presenilin 2 mutations cause early-onset familial Alzheimer's disease (EOFAD), or AD. These diseases are fatal neurodegenerative disorders with no disease-modifying treatment. Targeting PSEN2 through human molecular genetics may be promising; for example, PSEN2 point mutations cause EOFAD in tissues such as the cerebral cortex or hippocampus. The mechanism of this targeting may be that autosomal dominant mutations in PSEN2 cause abnormal APP metabolism, resulting in toxic peptides that cause progressive neuronal death. Possible diagnoses include family history; genetic testing; early symptoms; or MRI. Biomarkers that can be used include, for example, CSF PSEN2 and APP peptides.

[0377] Targeting Apo E for Alzheimer's disease Apolipoprotein E4 has been linked to sporadic AD in elderly people. This disease is a fatal neurodegenerative disorder with no disease-modifying treatment. The prevalence of AD is 2.5 to 5 million in the United States. Targeting Apo E may be effective because genomic evidence supporting the association between ApoE4 and AD is strong in many populations. The target tissue may be the cerebral cortex. Despite the strong association in many populations, it is still unclear whether Apo E4 contributes to the development of AD. To date, data indicate that Apo E4 homozygosity indicates an increased risk of AD in elderly people, but is not sufficient to cause AD, even in elderly people. Regarding safety, Apo E KD in the central nervous system may be safe because human LOF mutations in Apo E are not associated with obvious neurological abnormalities, but systemic exposure may cause type III hyperlipoproteinemia. Possible diagnostic criteria include a clinical diagnosis of AD; exclusion of EOFAD mutations; and genetic testing for Apo E4 genotype. Biomarkers that may be used include, for example, CSF APP, tau mRNA and peptides.

[0378] Central nervous system gene duplication disorders. Consistent knockout of half of these disorders may improve these disorders. Targets include MeCP2.

[0379] Targeting MeCP2 for X-linked mental retardation Methyl-CpG-binding protein 2 gene duplication causes X-linked mental retardation (XLMR). This disease is a fatal cognitive disorder with no disease-modifying treatment. 1-15% of X-linked MR cases are caused by MeCP2 duplication; 2-3% of the population has MR. Targeting MeCP2 through human molecular genetics may be successful; for example, MeCP2 duplication causes XLMR in tissues such as the cerebral cortex. The mechanism of this targeting may be that MeCP2 overexpression causes dysregulation of other genes and neurodegeneration. Efficacy is demonstrated by 50% knockdown of MeCP2; ASO knockdown in mouse models reverses the phenotype. Regarding safety, MeCP2 LOF mutations can cause Rett syndrome. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF MeCP2 mRNA and peptides.

[0380] Dominantly inherited cerebral amyloid angiopathy is a severe disease with no disease-modifying treatment. Targets include TTR.

[0381] Targeting TTR for hATTR CAA This targeting may result in low-risk introduction of siRNA into the central nervous system. Cerebral amyloid angiopathy (CAA) and meningeal amyloidosis are fatal diseases with no disease-modifying treatments. Targeting TTR based on human genetics and pharmacology may be advantageous. The target tissue may be the central nervous system vasculature or the central nervous system. The mechanism of this targeting may be through accumulation of mutant protein in the vascular adventitia, causing central nervous system bleeding. Efficacy was demonstrated by a 70% reduction in TTR expression. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that may be used include, for example, CSF mRNA and protein.

[0382] Targeting ITM2B for CAA Integral membrane protein 2B mutations cause cerebral amyloid angiopathy (CAA), British type, or familial British dementia (FBD). Specific mutations can also cause dominant retinal degeneration. This disease is fatal, with no disease-modifying treatment. It is a rare disease. Targeting ITM2B through human molecular genetics may be advantageous. Target tissues may be the central nervous system vasculature or the central nervous system. The mechanism of this targeting likely involves GOF mutations. Efficacy was demonstrated by a 70% KD of ITM2B mutant alleles. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and potentially protein.

[0383] Targeting CST3 for CAA Cystatin C mutations cause Icelandic-type familial cerebral amyloid angiopathy. This disease is fatal and has no disease-modifying treatment. It is rare except in Iceland and Denmark. Targeting CST3 through human genetics may be promising. The target tissue may be the central nervous system vasculature. The mechanism of this targeting may be that the mutant protein accumulates in the vascular adventitia, causing central nervous system bleeding. Efficacy is likely demonstrated by 70% knockout of the mutant allele. Regarding safety, CST3 KO mice may be at risk for arthritis. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and potentially protein.

[0384] Targeting SPAST for spastic paraplegia SPASTIN mutations cause spastic paraplegia (SP)4 with cognitive impairment. This disease is a lower motor neurodegenerative disorder with no disease-modifying treatment. The prevalence of SP is 5 per 100,000 population; SP4 accounts for 45% of dominant SP. Targeting of SPAST through human molecular genetics may be promising; for example, SPAST trinucleotide mutations cause familial SP in tissues such as the spinal cord or central nervous system. The mechanism of this targeting may be through nonsense and putative dominant-negative mutations, which cause abnormal microtubule metabolism and neurodegeneration. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF SPAST mRNA and potentially protein.

[0385] Targeting KIF5A for spastic paraplegia Kinesin family member 5A mutations cause spastic paraplegia (SP)10, which is accompanied by peripheral neuropathy and other disorders. This disease is a lower motor neurodegenerative disorder with no disease-modifying treatment. The prevalence of SP is 5 per 100,000 people; SP10 is 1 per 1,000,000 people. Targeting KIF5A through human molecular genetics may be advantageous. For example, a KIF5A amino-terminal missense mutation causes SP10; KIF5A is expressed in the central nervous system and encodes a microtubule motor protein. The target tissue may be the spinal cord. The mechanism of this targeting may be through an autosomal dominant missense mutation causing SP10, possibly affecting the binding of microtubules to the motor. Efficacy may be provided by the potential knockdown of the mutant allele. Regarding safety, KIF5A frameshift mutations cause neonatal intractable myoclonus, and splice site mutations are associated with familial ALS, possibly via a loss of function (LOF) mechanism. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and potentially protein.

[0386] Targeting ATL1 for spastic paraplegia Atlastin mutations cause spastic paraplegia 3A and sensory neuropathy 1D, hereditary sensory neuropathy (HSN). This disease is a lower motor neurodegenerative disorder with no disease-modifying treatment. The prevalence of SP is 5 per 100,000; SP3A is a rare dominant form. Targeting ATL1 through human molecular genetics may be successful; for example, ATL1 point mutations cause familial SP. The target tissue may be the spinal cord. The mechanism of this targeting may be autosomal dominant expression of a dominant-negative ATL1 protein, which causes SP3A; LOF mutations cause sensory neuropathy 1D. Efficacy was demonstrated by a 70% KD of specific ATL1 alleles. Regarding safety, ATL1 heterozygous LOF mutations cause HSN1D. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that may be used include, for example, CSF ATL1 mRNA and protein.

[0387] Targeting NIPA1 for spastic paraplegia LOF NIPA1 mutations cause spastic paraplegia6 with epilepsy and seizures. This disease is a lower motor neurodegenerative disorder with no disease-modifying treatment. The prevalence of SP is 5 per 100,000; SP6 is a rare dominant form. Targeting NIPA1 through human molecular genetics may be promising; for example, NIPA1 point mutations cause familial SP. The target tissue may be the spinal cord or central nervous system. The mechanism of this targeting may be autosomal dominant expression of a defective membrane protein, causing SP3A and possibly LOF. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and possible protein.

[0388] Dominantly inherited myotonic dystrophies are diseases of the central nervous system, skeletal muscle, and cardiac muscle that require central nervous system and systemic treatment. Targets include MPK for DM1.

[0389] Targeting DMPK for myotonic dystrophy 1 Central nervous system and systemic treatments were needed for effective treatments targeting myotonic dystrophy protein kinase. Myotonic dystrophy 1 (DM1) is a degenerative disease of the muscles and central nervous system. It is a fatal disease with no disease-modifying treatment. The prevalence of DM1 is 1 in 8,000 people worldwide. Targeting DMPK through human molecular genetics may be promising; for example, DMPK CTG repeat expansions cause familial DM1. Target tissues may be skeletal muscle, cardiac muscle, or the central nervous system. The mechanism of this targeting may be that autosomal dominant noncoding CTG repeats cause abnormal RNA processing and a dominant-negative effect; extreme expansions may result in early-onset disease. Efficacy was demonstrated by 70% DMPK suppression; ASO efficacy was demonstrated in mice. Safety in mice was demonstrated by knockout and ASO knockout. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, blood and CSF mRNA and protein.

[0390] Targeting ZNF9 for myotonic dystrophy 2 Mutations in zinc finger protein 9 cause myotonic dystrophy 2 (DM2), a degenerative disease of skeletal muscle. This is a severe disease with no disease-modifying treatment. The prevalence of DM2 is 1 in 8,000 people worldwide; it is the most common muscular dystrophy in adults. Targeting ZNF9 through human molecular genetics may be promising; for example, a ZNF9 CTTG repeat expansion in intron 1 causes familial DM2. The target tissue may be skeletal muscle or cardiac muscle. The mechanism of this targeting may be due to an autosomal dominant CTTG repeat expansion in intron 1 causing abnormal RNA metabolism and a dominant-negative effect. Efficacy was demonstrated in 70% of ZNF9 cases. Safe KD in mice has been demonstrated. Potential diagnoses include family history, genetic testing, or early symptoms. Biomarkers that may be used include, for example, blood mRNA and protein.

[0391] Dominantly inherited prion diseases are inherited, sporadic, and transmissible PRNP diseases. Targets include PRNP.

[0392] Targeting PRNP for myotonic prion disease Myotonic prion disease is a dominantly inherited prion disease that also includes PRNP-associated cerebral amyloid angiopathy, Gerstmann-Straussler disease (GSD), Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Huntington's disease-like 1 (HDL1), and susceptibility to kuru. These diseases are fatal neurodegenerative disorders with no disease-modifying treatments. The prevalence of this type of disease is 1 in 1,000,000. Targeting PRNP through human molecular genetics may be promising; for example, PRNP mutations cause familial and sporadic prion diseases. The target tissue may be the central nervous system. The mechanism of this targeting may be through autosomal dominant protein misfolding, which causes neurotoxicity. Efficacy is demonstrated by a 70% PRNP KD; PRNP polymorphisms appear to be protective against kuru. Regarding safety, PRNP KO mice were reported to be healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0393] Targeting glycogen synthase for myoclonic epilepsy in Lafora disease Mutations in the laforin (EPM2A) gene cause AR myoclonic epilepsy, a hereditary progressive seizure disorder. This disease is fatal, accompanied by seizures and cognitive decline, with no disease-modifying treatment. The prevalence of this disease is 4 per 1,000,000 people. Targeting glycogen synthase through human molecular genetics may be promising; for example, mutations cause AR familial myoclonic epilepsy, known as Lafora disease. The target tissue may be the central nervous system. The mechanism of this targeting may be through autosomal recessive dysfunction of laforin, which causes glycogen misfolding and seizure foci. Efficacy was demonstrated by a 70% knockdown of glycogen synthase GYS1. Regarding safety, GYS1 deficiency causes skeletal and cardiac muscle glycogen deficiency; surviving GYS1 mice have muscle abnormalities. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0394] In some embodiments, the present invention provides compounds that target genes for diseases including, but not limited to, age-related macular degeneration (AMD) (dry and wet), birdshot chorioretinitis, dominant retinitis pigmentosa 4, Fuchs' dystrophy, hATTR amyloidosis, hereditary and sporadic glaucoma, and Stargardt's disease.

[0395] In some embodiments, the present invention provides compounds that target VEGF for wet (or exudative) AMD.

[0396] In some embodiments, the present invention provides compounds that target C3 for dry (or non-exudative) AMD.

[0397] In some embodiments, the present invention provides compounds that target the CFB for dry (or non-exudative) AMD.

[0398] In some embodiments, the present invention provides compounds that target MYOC for glaucoma.

[0399] In some embodiments, the present invention provides compounds that target ROCK2 for glaucoma.

[0400] In some embodiments, the present invention provides compounds that target ADRB2 for glaucoma.

[0401] In some embodiments, the present invention provides compounds that target CA2 for glaucoma.

[0402] In some embodiments, the present invention provides compounds that target CRYGC for cataracts.

[0403] In some embodiments, the present invention provides compounds that target PPP3CB for dry eye syndrome.

[0404] Ligand In certain embodiments, the compounds of the present invention are further modified by the covalent attachment of one or more conjugate groups. Generally, the conjugate group modulates one or more properties of the conjugated compounds of the present invention, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in the chemical arts and are linked to parent compounds, such as oligomeric compounds, directly or via an optional linking moiety or linking group. A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0405] In some embodiments, the compounds further comprise targeting ligands that target receptors that mediate delivery to specific central nervous system tissues. These targeting ligands can be conjugated in combination with lipophilic moieties to enable specific intrathecal and systemic delivery.

[0406] Exemplary targeting ligands for receptor-mediated delivery to central nervous system tissues are peptide ligands such as 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 to the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transporter proteins, and LDL receptor ligands.

[0407] In some embodiments, the compound 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 to enable specific intraocular (e.g., intravitreal) and systemic delivery. Exemplary targeting ligands that target receptor-mediated delivery to ocular tissues include lipophilic ligands such as all-trans-retinol (targeting retinoic acid receptors); RGD peptides such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or Cyclo(-Arg-Gly-Asp-D-Phe-Cys) (targeting retinal pigment epithelial cells); LDL receptor ligands; and carbohydrate-based ligands (targeting endothelial cells in the posterior segment of the eye).

[0408] Preferred conjugate groups suitable for the present invention include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains, e.g., 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, such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0409] 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 polyamines Salts, thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralens, mitomycin), 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, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., α-helical peptides, amphipathic peptides, RGD peptides, cell-penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen , aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, 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, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor α (TNFα), interleukin-1β, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-penetrating agents (e.g., alpha-helical cell-penetrating agents).

[0410] Peptide and peptidomimetic ligands include natural or modified peptides, e.g., D- or L-peptides; α-, β-, or γ-peptides; N-methylpeptides; azapeptides; peptides with one or more amide bonds, i.e., peptides, one or more urea, thiourea, carbamate, or sulfonylurea bonds substituted; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0411] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0412] As used herein, the term "endosomolytic ligand" refers to a molecule that has endosomolytic properties. An endosomolytic ligand promotes lysis of a composition of the invention, or a component thereof, and / or transport of a composition of the invention, or a component thereof, from a cellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular endoplasmic reticulum, to the cytoplasm of a cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear or branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charge, dendrimers with masked or unmasked cationic or anionic charge, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0413] 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, n is norleucine); LFEALLELLESLWELLLEA (JTS-1); GLFKALLKLLKSLWKLLLKA (ppTG1); GLFRALLRLLRSLWRLLLRA (ppTG20); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA); GLFFEAIAEFIEGGWEGLIEGC (HA); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin); H5WYG; and CHK6HC.

[0414] 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-dioleoyl-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).

[0415] 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 entire contents of which are incorporated herein by reference.

[0416] 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); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVP RTES (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).

[0417] Exemplary cationic groups include, but are not limited to, O-AMINE (AMINE = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, e.g., O(CH) n AMINE, (e.g., AMINE = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CHCHNH)n Included are protonated amino groups derived from CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0418] As used herein, the term "targeting ligand" refers to any molecule that provides increased affinity for a selected target, e.g., a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0419] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g., 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 acids, and lectins. The term multivalent indicates the presence of two or more monosaccharide units. Such monosaccharide subunits can be linked to each other via glycosidic bonds or to a backbone molecule.

[0420] As ligands, several folates and folate analogs suitable for the present invention are described in U.S. Pat. Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the entire contents of which are incorporated herein by reference.

[0421] As used herein, the terms "PK-modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and transthyretin-binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligomeric compounds containing several phosphorothioate intersugar linkages are also known to bind to serum proteins. Therefore, short oligomeric compounds, e.g., oligonucleotides containing 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., PK-modulating ligands) for the present invention. PK-modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in a PK-modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-modulating ligands for the present invention. Binding to serum components (eg, serum proteins) can be predicted from albumin binding assays such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0422] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while other ligands have different properties. For example, the ligands may have targeting properties, endosomolytic activity, or PK modulating properties. In a preferred embodiment, the ligands all have different properties.

[0423] A ligand or linking ligand may be present on a monomer when the monomer is incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). In some embodiments, a ligand may be incorporated into a "precursor" monomer by coupling after the "precursor" monomer has been incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). For example, a monomer having an amino-terminated tether (i.e., no ligand attached), e.g., monomer-linker-NH, may be incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). In a subsequent operation, i.e., after incorporation of the precursor monomer into a component of a compound of the invention (e.g., a compound or linker of the invention), a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, may be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the tether of the precursor monomer.

[0424] In another example, monomers bearing chemical groups suitable for participating in click chemistry reactions can be incorporated into tethers / linkers, for example, azide- or alkyne-terminated tethers / linkers. Subsequent to this, i.e., after the precursor monomers are incorporated into the chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer by linking the alkyne and azide together.

[0425] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside bond of the compound of the present invention. 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 the purine nucleobase is bound to the 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 the pyrimidine nucleobase can be substituted with the conjugate moiety. When a ligand is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions required for base pairing.

[0426] Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Examples of 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 carry a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, and phosphoramidate), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of 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.

[0427] There are many methods for preparing the conjugate of oligonucleotide.Generally, oligonucleotide is linked to conjugate moiety by contacting the reactive group (for example, OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotide with the reactive group of conjugate moiety.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0428] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or thiol. Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in literature, such as, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, the entire contents of which are incorporated herein by reference.

[0429] The ligand is attached to the compound of the present invention via a linker or carrier monomer, e.g., a ligand carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, a "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond available and suitable for incorporation of a carrier monomer into the backbone of an oligonucleotide, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. A "tether attachment point" (TAP) refers to an atom of the carrier monomer, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), to which a selected moiety is attached. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the carrier monomer by an intervening tether. Thus, the carrier often contains a functional group, e.g., an amino group, or generally to allow a bond suitable for incorporation or linkage to a constituent atom of another chemical entity, e.g., a ligand.

[0430] Representative United States patents that teach the preparation of nucleic acid conjugates include, but are not limited to, U.S. Pat. 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 ... Specification No. 5,109,124; Specification No. 5,118,802; Specification No. 5,138,045; Specification No. 5,414,077; Specification No. 5,486,603; Specification No. 5,512,439; Specification No. 5,578,718; Specification No. 5,608,046 ; Specification No. 4,587,044; Specification No. 4,605,735; Specification No. 4,667,025; Specification No. 4,762,779 Specifications; Specification No. 4,789,737; Specification No. 4,824,941; Specification No. 4,835,263; Specification No. 4,876,335 Specification; Specification No. 4,904,582; Specification No. 4,958,013; Specification No. 5,082,830; Specification No. 5,112,96 Specification No. 3; Specification No. 5,214,136; Specification No. 5,082,830; Specification No. 5,112,963; Specification No. 5,149,7 Specification No. 82; Specification No. 5,214,136; Specification No. 5,245,022; Specification No. 5,254,469; Specification No. 5,258 ,506 specification; 5,262,536 specification; 5,272,250 specification; 5,292,873 specification; 5,31 Specification No. 7,098; Specification No. 5,371,241, Specification No. 5,391,723; Specification No. 5,416,203, Specification No. 5, Specification No. 451,463; Specification No. 5,510,475; Specification No. 5,512,667; Specification No. 5,514,785; Specification No. 5 ,565,552; 5,567,810; 5,574,142; 5,585,481; Specification No. 5,587,371; Specification No. 5,595,726; Specification No. 5,597,696; Specification No. 5,599,923;Nos. 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.

[0431] In some embodiments, the compound further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is a GalNAc conjugate.

[0432] Because a ligand can be conjugated to an iRNA agent via a linker or carrier, and because the linker or carrier can include a branched linker, an iRNA agent can include multiple ligands via the same or different backbone attachment points to the carrier or via a branched linker. For example, the branch point of a branched linker can be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom or group exhibiting such multivalency. In certain 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 optionally substituted alkyl. In other embodiments, the branch point is glycerol or a glycerol derivative.

[0433] Evaluation of candidate iRNAs Candidate iRNA agents, e.g., modified RNAs, can be evaluated for a selected property by exposing the agent or modified molecule and a control molecule to appropriate conditions and assessing the presence of the selected property. For example, resistance to a degradative agent can be assessed as follows: The candidate modified RNA (and a control molecule, usually in unmodified form) can be exposed to degradative conditions, e.g., an environment containing a degradative agent, e.g., a nuclease. For example, a biological sample, e.g., one resembling an environment that may be encountered during therapeutic use, e.g., blood or a cell fraction, e.g., a cell-free homogenate or disrupted cells, can be used. The candidate and control can then be evaluated for resistance to degradation by any of several techniques. For example, the candidate and control can be labeled prior to exposure, e.g., with a radioactive or enzymatic label, or a fluorescent label, e.g., Cy3 or Cy5. The control and modified RNA can be incubated with the degradative agent and, optionally, a control, e.g., an inactivated, e.g., heat-inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules is then determined. They can be determined by physical methods, such as polyacrylamide gel electrophoresis or sizing columns, to assess whether the molecule maintains its original length, or functionally assessed. Alternatively, Northern blot analysis can be used to assay the length of unlabeled modified molecules.

[0434] Functional assays can also be used to evaluate candidate agents. Functional assays can be applied first or after a previous non-functional assay (e.g., an assay for resistance to degradation) to determine whether a modification alters the ability of a molecule to silence gene expression. For example, cells, e.g., mammalian cells, e.g., mouse or human cells, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00 / 44914). For example, a modified dsiRNA homologous to GFP mRNA can be assayed for its ability to inhibit GFP expression by monitoring a decrease in cellular fluorescence compared to control cells, where the transfection did not include the candidate dsiRNA, e.g., a control to which no agent was added and / or a control to which unmodified RNA was added. The effectiveness of a candidate agent on gene expression can be evaluated by comparing the fluorescence of cells in the presence of modified and unmodified dssiRNA compounds.

[0435] In an alternative functional assay, candidate dssiRNA compounds homologous to endogenous mouse genes, such as maternally expressed genes, such as c-mos, can be injected into immature mouse oocytes to assess the agent's ability to inhibit gene expression in vivo (see, for example, International Publication No. 01 / 36646). The oocyte phenotype, such as the ability to maintain metaphase II arrest, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by dssiRNA compounds causes oocytes to exit metaphase arrest and initiate parthenogenesis (Colledge et al. Nature 370:65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of a modifying agent on target RNA levels can be verified by Northern blot to assay for a reduction in target mRNA levels compared to a negative control, or by Western blot to assay for a reduction in target protein levels. Controls may include cells to which no agent has been added and / or cells to which unmodified RNA has been added.

[0436] Physiological effects The siRNA compounds described herein can be designed so that determining therapeutic toxicity is facilitated by the complementarity of the siRNA to both human and non-human animal sequences. By these methods, the siRNA can consist of a nucleic acid sequence from a human and a sequence that is completely complementary to a nucleic acid sequence from at least one non-human animal, such as a non-human mammal, for example, a rodent, ruminant, or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, bonobo (Pan paniscus), chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatto), or cynomolgus monkey. The sequence of the siRNA compound can be complementary to a sequence within a homologous gene in a non-human mammal and a human, such as an oncogene or tumor suppressor gene. By determining the toxicity of the siRNA compound in a non-human mammal, the toxicity of the siRNA compound in humans can be estimated. For more stringent toxicity testing, the siRNA can be complementary to a human and two or more non-human animals, for example, two or three or more non-human animals.

[0437] The methods described herein can be used to associate any physiological effect of an siRNA compound on humans, for example, any undesirable effect, such as a toxic effect, or any positive or desired effect.

[0438] Increased cellular uptake of siRNA Described herein are various siRNA compositions and / or intracellular targeting of siRNA that include covalent conjugates that increase cellular uptake.

[0439] Further provided are methods of the present invention that include administering an siRNA compound and a drug that affects cellular uptake of siRNA. The drug can be administered before, after, or at the same time as the siRNA compound. The drug can be covalently or non-covalently bound to the siRNA compound. The drug can be, for example, lipopolysaccharide, a p38 MAP kinase activator, or an NF-κB activator. The drug can have a transient effect on cells. The drug can increase cellular uptake of the siRNA compound, for example, by disrupting the cellular cytoskeleton, e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The drug can also increase cellular uptake of the siRNA compound, for example, by activating an inflammatory response. Exemplary drugs that may have such an effect include tumor necrosis factor alpha (TNFα), interleukin-1β, CpG motifs, gamma interferon or more generally agents that activate toll-like receptors.

[0440] siRNA production siRNAs can be produced, e.g., in large quantities, by a variety of methods. Exemplary methods include organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0441] Organic Synthesis: siRNAs can be made by separately synthesizing a single-stranded RNA molecule, or each respective strand of a double-stranded RNA molecule, and then allowing the constituent strands to anneal.

[0442] Large bioreactors, such as OligoPilot II from Pharmacia Biotec AB (Uppsala, Sweden), can be used to produce large quantities of specific RNA strands for a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. Ribonucleotide amidites are used to generate the RNA strands. Standard cycles of monomer addition can be used to synthesize 21-23 nucleotide strands for siRNA. Typically, two complementary strands are produced separately and then annealed, for example, after release from the solid support and deprotection.

[0443] Organic synthesis can be used to produce distinct siRNA species.The complementarity of species to specific target gene can be precisely specified.For example, species can be complementary to the region containing polymorphism, for example, single nucleotide polymorphism.Furthermore, the position of polymorphism can be precisely defined.In some embodiments, polymorphism is located in the internal region, for example, at least 4, 5, 7 or 9 nucleotides from one or both ends.

[0444] dsiRNA cleavage. siRNAs can also be generated by cleaving larger siRNAs. Cleavage can be mediated in vitro or in vivo. For example, the following method can be used to generate iRNAs by in vitro cleavage.

[0445] In vitro transcription. dsiRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe™ RNAi Transcription Kit (New England Biolabs) provides a method for producing dsiRNA from a nucleic acid segment cloned into a p vector and flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for dsiRNA. The templates are transcribed in vitro by adding T7 RNA polymerase to produce dsiRNA. Similar methods using PCR and / or other RNA polymerases (e.g., T3 or SP6 polymerase) may also result in endotoxins that can contaminate recombinant enzyme preparations.

[0446] In vitro cleavage. dsiRNA can be cleaved into siRNA in vitro, for example, using Dicer or equivalent RNAse III-based activity. For example, dsiRNA can be incubated in an in vitro extract from Drosophila or with purified components, for example, purified RNAse or RISC complex (RNA-induced silencing complex). For example, see Ketting et al. Genes Dev 2001 Oct 15; 15(20): 2654-9 and Hammond Science 2001 Aug 10; 293(5532): 1146-50.

[0447] dsiRNA cleavage generally produces multiple siRNA species, each a specific 21-23 nt fragment of the original dsiRNA molecule. For example, there may be siRNAs that contain sequences complementary to overlapping and flanking regions of the original dsiRNA molecule.

[0448] Regardless of the synthesis method, siRNA preparation can be prepared in a suitable solution (for example, aqueous solution and / or organic solution) for formulation.For example, siRNA preparation can be precipitated and redissolved in pure double-distilled water, and then lyophilized.The dried siRNA can then be resuspended in a suitable solution for the intended formulation process.

[0449] Preparation of compounds conjugated to lipophilic moieties In some embodiments, the lipophilic monomer containing the lipophilic moiety is conjugated to the compound via a nucleobase, a sugar moiety, or an internucleoside linkage.

[0450] Conjugation to purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms.In some embodiments, the 2nd, 6th, 7th, or 8th position of purine nucleobase is bound to the conjugate moiety.Conjugation to pyrimidine nucleobase or its derivative can occur at any position.In some embodiments, the 2nd, 5th, and 6th positions of pyrimidine nucleobase can be substituted with the conjugate moiety.When lipophilic moiety is conjugated to nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bond interaction required for base pairing.In one embodiment, the lipophilic monomer containing lipophilic moiety can be conjugated to nucleobase via a linker that includes alkyl, alkenyl, or amide bond.

[0451] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety to which a lipophilic moiety can be attached include the 2', 3', and 5' carbon atoms. A lipophilic moiety can also be attached to the 1' position, such as at an abasic residue. In one embodiment, a lipophilic moiety can be conjugated to the sugar moiety via a 2'-O modification, with or without a linker.

[0452] Internucleoside linkages can also have lipophilic moieties. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the lipophilic moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the lipophilic moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0453] There are many methods for preparing the conjugate of oligonucleotide.Generally, oligonucleotide is linked to conjugate moiety by contacting the reactive group (for example, OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotide with the reactive group of conjugate moiety.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0454] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or thiol. Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in literature, such as, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, the entire contents of which are incorporated herein by reference.

[0455] In one embodiment, a first (complementary) RNA strand and a second (sense) RNA strand can be synthesized separately, one of the RNA strands can contain a pendant lipophilic moiety, and the first and second RNA strands can be mixed to form the dsRNA. The step of synthesizing the RNA strands preferably involves solid-phase synthesis, in which individual nucleotides are joined end-to-end by formation of internucleotide 3'-5' phosphodiester bonds in successive synthesis cycles.

[0456] In one embodiment, a lipophilic molecule having a phosphoramidite group is attached to the 3' or 5' end of either the first (complementary) or second (sense) RNA strand in the final synthesis cycle. In solid-phase synthesis of RNA, nucleotides are initially in the form of nucleoside phosphoramidites. In each synthesis cycle, additional nucleoside phosphoramidites are linked to the -OH group of previously incorporated nucleotides. If the lipophilic molecule has a phosphoramidite group, it can be attached to the free OH end of pre-synthesized RNA in solid-phase synthesis in the same manner as nucleoside phosphoramidites. Synthesis can be carried out in an automated and standardized manner using a conventional RNA synthesizer. The synthesis of a lipophilic molecule having a phosphoramidite group can include phosphitylation of a free hydroxyl to generate a phosphoramidite group.

[0457] Generally, oligonucleotides can be synthesized using protocols known in the art, for example, as described in Caruthers et al., Methods in Enzymology (1992) 211:3-19; International Publication No. WO 99 / 54459; Wincott et al., Nucl. Acids Res. (1995) 23:2677-2684; Wincott et al., Methods Mol. Bio., (1997) 74:59; Brennan et al., Biotechnol. Bioeng. (1998) 61:33-45; and U.S. Patent No. 6,001,311, the entire contents of each of which are incorporated herein by reference. Generally, oligonucleotide synthesis requires conventional nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidite at the 3'-end. In a non-limiting example, small-scale synthesis is performed on an Expedite 8909 RNA synthesizer sold by Applied Biosystems, Inc. (Weiterstadt, Germany) using ribonucleoside phosphoramidites sold by ChemGenes Corporation (Ashland, Mass.). Alternatively, synthesis can be carried out in a 96-well plate synthesizer such as the instrument manufactured by Protogene (Palo Alto, Calif.), or by methods such as those described in Usman et al., J. Am. Chem. Soc. (1987) 109:7845; Scaringe, et al., Nucl. Acids Res. (1990) 18:5433; Wincott, et al., Nucl. Acids Res. (1990) 23:2677-2684; and Wincott, et al., Methods Mol. Bio. (1997) 74:59, the entire contents of each of which are incorporated herein by reference.

[0458] The nucleic acid molecules of the invention can be synthesized separately and joined to each other post-synthetically, for example, by ligation (Moore et al., Science (1992) 256:9923; WO 93 / 23569; Shabarova et al., Nucl. Acids Res. (1991) 19:4247; Bellon et al., Nucleosides & Nucleotides (1997) 16:951; Bellon et al., Bioconjugate Chem. (1997) 8:204; or by hybridization after synthesis and / or deprotection. The nucleic acid molecules can be purified by gel electrophoresis using conventional methods, or by high pressure liquid chromatography (HPLC; see Wincott et al., supra, incorporated herein by reference in its entirety), and resuspended in water.

[0459] Pharmaceutical Composition In one aspect, the invention features a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an ssiRNA compound, or a DNA encoding an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound, or a precursor thereof, that includes a nucleotide sequence complementary, e.g., substantially and / or exactly complementary, to a target RNA). The target RNA can be a transcript of an endogenous human gene. In one embodiment, the siRNA compound (a) is 19-25 nucleotides in length, e.g., 21-23 nucleotides in length; (b) is complementary to an endogenous target RNA; and, optionally, (c) includes at least one 3' overhang 1-5 nt in length. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

[0460] In one example, the pharmaceutical composition comprises a siRNA compound mixed with a topical delivery agent. The topical delivery agent can be a plurality of microvesicles. The microvesicles can be liposomes. In some embodiments, the liposomes are cationic liposomes.

[0461] In another aspect, the pharmaceutical composition comprises an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an ssiRNA compound, or a DNA encoding an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound, or a precursor thereof), mixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. Fatty acids include arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~10 It may be an alkyl ester, a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof.

[0462] In another embodiment, the topical penetration enhancer is a bile salt, which can be cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether, or a pharmaceutically acceptable salt thereof.

[0463] In another embodiment, the penetration enhancer is a chelating agent, which can be EDTA, citric acid, salicylate, N-acyl derivatives of collagen, laureth-9, N-aminoacyl derivatives of β-diketones, or mixtures thereof.

[0464] In another embodiment, the penetration enhancer is a surfactant, such as an ionic or non-ionic surfactant, which can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, perfluorochemical emulsion, or a mixture thereof.

[0465] In another embodiment, the penetration enhancer may be selected from the group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alacanones, steroidal anti-inflammatory drugs, and mixtures thereof. In yet another embodiment, the penetration enhancer may be a glycol, pyrrole, azone, or terpene.

[0466] In one aspect, the invention features a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a DNA encoding a double-stranded siRNA compound, or an ssiRNA compound, or precursor thereof, that can be processed into an ssiRNA compound) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver the siRNA compound composition to cells or regions of the gastrointestinal tract, e.g., the small intestine, colon (e.g., to treat colon cancer), etc. The oral delivery form can be a tablet, capsule, or gel capsule. In one embodiment, the siRNA compound of the pharmaceutical composition modulates expression of a cell adhesion protein, modulates cell proliferation rate, or has biological activity against a eukaryotic pathogen or retrovirus. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablet, capsule, or gel capsule in a mammalian stomach. In some embodiments, the enteric material is a coating. The coating may be acetate phthalate, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate phthalate.

[0467] In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can also be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

[0468] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example, the excipient is polyethylene glycol. In another example, the excipient is Precirol.

[0469] In another embodiment, the oral dosage form of the pharmaceutical composition comprises a plasticizer, which can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0470] In one aspect, the invention features a pharmaceutical composition including a siRNA compound and a delivery vehicle. In one embodiment, the siRNA compound (a) is 19-25 nucleotides in length, e.g., 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3' overhang 1-5 nucleotides in length.

[0471] In one embodiment, the delivery vehicle can deliver an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound, or a DNA encoding a precursor thereof, which can be processed into an ssiRNA compound) to a cell via a local administration route. The delivery vehicle can be a microvesicle. In one example, the microvesicle is a liposome. In some embodiments, the liposome is a cationic liposome. In another example, the microvesicle is a micelle. In one aspect, the invention features a pharmaceutical composition comprising an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a DNA encoding a double-stranded siRNA compound, or an ssiRNA compound, or a precursor thereof, which can be processed into an ssiRNA compound) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile solution may include diluents such as water; saline solution; fixed oils, polyethylene glycol, glycerol, or propylene glycol.

[0472] In one aspect, the invention features a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound, or a precursor thereof, that can be processed into an ssiRNA compound) in an oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of a tablet, a capsule, and a gel capsule. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablet, capsule, or gel capsule in a mammalian stomach. In some embodiments, the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

[0473] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example, the excipient is polyethylene glycol. In another example, the excipient is Precirol.

[0474] In another embodiment, the oral dosage form of the pharmaceutical composition comprises a plasticizer, which can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0475] In one aspect, the invention features a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a DNA encoding a double-stranded siRNA compound, or an ssiRNA compound, or precursor thereof, that can be processed into an ssiRNA compound) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

[0476] In one aspect, the invention features a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a DNA encoding a double-stranded siRNA compound, or an ssiRNA compound, or precursor thereof, that can be processed into an ssiRNA compound) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

[0477] In one aspect, the invention features a pharmaceutical composition comprising an siRNA compound, e.g., a double-stranded siRNA compound, or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound, or an siRNA compound, e.g., a DNA encoding a double-stranded siRNA compound, or an ssiRNA compound, or precursor thereof, that can be processed into an ssiRNA compound), in a pulmonary or nasal dosage form. In one embodiment, the siRNA compound is incorporated into particles, e.g., macroparticles, e.g., microspheres. The particles can be produced by spray drying, freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination thereof. The microspheres can be formulated as a suspension, a powder, or an implantable solid.

[0478] Treatment Methods and Delivery Routes Another aspect of the invention relates to a method of reducing expression of a target gene in a cell, comprising contacting said cell with a compound of the invention, hi one embodiment, the cell is an extrahepatic cell.

[0479] Another aspect of the present invention relates to a method of reducing expression of a target gene in a subject, comprising administering to the subject a compound of the present invention.

[0480] Another aspect of the present invention relates to a method for treating a subject suffering from a central nervous system disease, comprising administering a therapeutically effective amount of a double-stranded RNAi agent of the present invention to the subject, thereby treating the subject. Exemplary central nervous system diseases that can be treated by the method of the present invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar ataxia, prion diseases, and Lafora's disease.

[0481] The compounds of the present invention can be delivered to a subject by various routes, depending on the type of gene being targeted and the type of disease being treated. In some embodiments, the compounds are administered extrahepatically, such as intraocularly (e.g., intravitreal), intrathecally, or intracerebroventricularly.

[0482] In one embodiment, the compound is administered intrathecally or intracerebroventricularly. By administering the compound intrathecally or intracerebroventricularly, the method can reduce expression of the target gene in brain or spinal tissue, such as the cerebral cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0483] In some embodiments, exemplary target genes are APP, ATXN2, C9orf72, TARDBP, MAPT (tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and TTR.To reduce the expression of these target genes in a subject, compound can be administered directly to the eye (for example, intravitreally).By administering compound intravitreally, this method can reduce the expression of target genes in ocular tissue.

[0484] For ease of explanation, the formulations, compositions, and methods in this section will be described primarily with respect to modified siRNA compounds. However, it will be understood that these formulations, compositions, and methods can be implemented with other siRNA compounds, such as unmodified siRNA compounds, and such implementations are within the scope of the present invention. Compositions containing iRNA can be delivered to a subject by various routes. Exemplary routes include intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and intraocular.

[0485] The iRNA molecules of the present invention can be included in pharmaceutical compositions suitable for administration. Such compositions typically contain one or more iRNAs and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds may also be included in the compositions.

[0486] The pharmaceutical compositions of the present invention can be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, nasal, transdermal), oral, or parenteral. Parenteral administration includes infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrabronchial, intraventricular, or intracerebroventricular administration.

[0487] The route and site of administration can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be a logical choice. Lung cells can be targeted by administering iRNA in a nebulized form. Vascular endothelial cells can be targeted by coating a balloon catheter with iRNA and mechanically introducing the DNA.

[0488] Formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

[0489] Compositions for oral administration include powder or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous solvents, tablets, capsules, lozenges, or troches.For tablets, carriers that can be used include lactose, sodium citrate, and phosphates.Various disintegrants such as starch, and lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are commonly used in tablets.For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycol.When aqueous suspensions are required for oral use, the nucleic acid composition can be combined with emulsifiers and suspending agents.If necessary, certain sweeteners and / or flavoring agents can be added.

[0490] Compositions for intrathecal or intraventricular or intracerebroventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0491] Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Intraventricular infusion may be facilitated, for example, by an intraventricular catheter attached to a re...

Claims

1. an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers wherein the lipophilic monomer is 【Chemistry 1】 (In the formula: m is an integer from 0 to 8; n is an integer from 1 to 21; B is a modified or unmodified nucleobase; W is an alkyl group; R, R', and R" are each independently H or an alkyl group. A compound selected from the group consisting of:

2. 2. The compound of claim 1, wherein the sense strand and the antisense strand are each 15 to 30 nucleotides in length.

3. 2. The compound of claim 1, wherein the sense strand and the antisense strand are each 19 to 25 nucleotides in length.

4. 2. The compound of claim 1, wherein the sense strand and the antisense strand are each 21 to 23 nucleotides in length.

5. 5. The compound of claim 4, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, and the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

6. The compound of claim 1 , wherein the compound comprises a single-stranded overhang on at least one of its termini.

7. 7. The compound of claim 6, wherein the single-stranded overhang is 1, 2, or 3 nucleotides in length.

8. The compound of any one of claims 1 to 7, wherein the sense strand and the antisense strand comprise less than 10 2'-fluoro modified nucleotides.

9. The compound of any one of claims 1 to 7, wherein the sense and antisense strands comprise at least 50%, at least 60%, or at least 70% 2'-OMe modified nucleotides.

10. The lipophilic monomer is 【Chemistry 2】 【Transformation 3】 wherein R and R′ are each independently H, methyl, ethyl, isopropyl, or t-butyl.

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

11. The compound according to any one of claims 1 to 10, wherein the sense strand comprises at least one phosphorothioate bond at the 3' end.

12. The compound according to any one of claims 1 to 10, wherein the sense strand comprises at least two phosphorothioate bonds at the 3' end.

13. 13. The compound of claim 11 or 12, wherein one of the phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide from the 3' end of the sense strand.

14. The compound of any one of claims 1 to 13, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand.

15. The compound of claim 14, wherein the phosphate mimic is 5'-vinylphosphonate (VP).

16. The compound according to any one of claims 1 to 15, wherein the antisense strand comprises at least one GNA in the seed region.

17. 17. The compound of claim 16, wherein the seed region is located at positions 5 to 7 from the 5' end of the antisense strand.

18. 18. The compound of any one of claims 1 to 17, further comprising a targeting ligand that targets a receptor that mediates delivery to central nervous system tissue.

19. 19. The compound of claim 18, wherein the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

20. 18. The compound of any one of claims 1 to 17, further comprising a targeting ligand that targets a receptor that mediates delivery to ocular tissue.

21. 21. The compound of claim 20, wherein the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligand.

22. 22. A composition for reducing expression of a target gene in a cell or a subject, comprising a compound according to any one of claims 1 to 21, wherein the compound is contacted with the cell or administered to the subject.

23. 23. The composition of claim 22, wherein the compound is administered extrahepatically.

24. 24. The composition of claim 23, wherein the compound is administered intrathecally, intracerebroventricularly, or directly to the eye of the subject.

25. 23. The composition of claim 22, which reduces expression of a target gene in brain or spinal tissue.

26. 26. The composition of claim 25, wherein the brain or spinal tissue is selected from the group consisting of the cerebral cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

27. 26. The composition of claim 25, wherein the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT (tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, and TTR.

28. 24. The composition of claim 23, for use in treating a subject suffering from a central nervous system disorder.

29. 29. The composition of claim 28, wherein the central nervous system disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disorders, prion diseases, and Lafora's disease.

30. 23. The composition of claim 22, which reduces expression of a target gene in ocular tissue.

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