Extrahepatic delivery

Abstract

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 a carrier. Another aspect of the present invention relates to a method of gene silencing, comprising administering a therapeutically effective amount of a lipophilic monomer-conjugated compound to a cell or a subject in need thereof.

Description

[0001] sequence list

[0002] This application includes a sequence list that has been electronically submitted in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on May 6, 2022, named 29520_1503_SL.txt, and is 52,281 bytes in size. Background Technology

[0003] Effective delivery of iRNA agents to cells in vivo requires specific targeting and substantial protection from the extracellular environment (especially serum proteins). RNAi-based therapies have shown promising clinical data for treating liver-related disorders. However, obstacles remain in delivering siRNA to extrahepatic tissues, which significantly limits the use of siRNA-based therapies.

[0004] One of the limiting factors for in vivo experiments and therapeutic applications of iRNA agents is the ability to efficiently deliver intact siRNA. Specific challenges have been linked to the in vivo transfer of non-viral genes to the retina. One challenge is overcoming the internal limiting membrane, which hinders retinal transfection. Additionally, it has been shown that negatively charged sugars in the vitreous humor interact with positive DNA-transfection reagent complexes, promoting their aggregation, which hinders diffusion and cellular uptake.

[0005] The delivery of oligonucleotides to the central nervous system (CNS) presents specific challenges because free oligonucleotides cannot cross the blood-brain barrier (BBB). One way to deliver oligonucleotides into the CNS is through intrathecal delivery. However, oligonucleotides also need to be effectively internalized into target cells of the CNS to achieve the desired therapeutic effect. Previous studies have typically used delivery agents such as liposomes, cationic lipids, and nanoparticle-based complexes to aid in the intracellular internalization of oligonucleotides into cells of neuronal origin.

[0006] Therefore, there is a continued need for new and improved methods to deliver siRNA molecules in vivo without the use of tissue delivery agents, in order to realize and enhance the therapeutic potential of iRNA agents. Summary of the Invention

[0007] One aspect of the invention provides a compound (e.g., a single-stranded or double-stranded oligonucleotide) comprising one or more lipophilic monomers having one or more lipophilic moieties optionally conjugated to one or more sites on at least one strand of the oligonucleotide via a linker or vector. For example, some embodiments of the invention provide a compound (e.g., a double-stranded iRNA agent) comprising: an antisense strand complementary to a target gene; a sense strand complementary to said antisense strand; and one or more lipophilic monomers having one or more lipophilic moieties optionally conjugated to one or more sites on at least one strand via a linker or vector.

[0008] In some embodiments, the octanol-water partition coefficient logK is used. ow The measured lipophilicity of the lipophilic fraction exceeds 0. The logK of the lipophilic fraction... ow It can exceed 1, 1.5, 2, 3, 4, 5 or 10.

[0009] In some embodiments, the hydrophobicity of the compound, as measured by the unbound fraction in a plasma protein binding assay, is greater than 0.2. In one embodiment, the plasma protein binding assay is performed using electrophoretic mobility shift assay (EMSA) of human serum albumin. Hydrophobicity of the compound, 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 to enhance in vivo delivery of siRNA.

[0010] In some embodiments, the lipophilic portion is aliphatic, cyclic (e.g., alicyclic), or polycyclic, such as alicyclic compounds, such as steroids (e.g., sterols), or straight-chain or branched aliphatic hydrocarbons. Exemplary lipophilic portions are lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0011] Suitable lipophilic fractions also include those containing saturated or unsaturated C4-C. 30 Hydrocarbon chains (e.g., C4-C) 30 A lipophilic moiety comprising an alkyl or alkenyl group and optional functional groups selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. These functional groups can be used to attach the lipophilic moiety to an iRNA agent. In some embodiments, the lipophilic moiety contains saturated or unsaturated C6-C6 groups. 18Hydrocarbon chains (e.g., straight-chain C6-C) 18 (alkyl or alkenyl). In one embodiment, the lipophilic moiety contains saturated or unsaturated C. 16 Hydrocarbon chains (e.g., straight-chain C) 16 Alkyl or alkenyl groups). In some embodiments, the lipophilic moiety contains two or more carbon-carbon double bonds.

[0012] In some embodiments, the lipophilic portion is a C6-C moiety 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). 30 part.

[0013] In some embodiments, the lipophilic portion is C6-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-C 30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, retinol, vitamin E, cholesterol, etc.).

[0014] A lipophilic monomer may contain a lipophilic moiety conjugated to any part of the iRNA agent (e.g., a nucleobase, sugar moiety, or nucleoside bond). When the lipophilic moiety is conjugated to the iRNA agent via a nucleobase, ribose, or nucleoside bond directly attached to the iRNA agent, the lipophilic monomer contains a nucleobase, ribose, or nucleoside bond, and a lipophilic moiety. Alternatively, a lipophilic monomer may contain a lipophilic moiety conjugated to a non-ribose substitution unit, such as a linker or vector. When the lipophilic moiety is conjugated to the iRNA agent via a non-ribose substitution unit (such as a linker or vector), the lipophilic monomer contains a non-ribose substitution unit (such as a linker or vector) and a lipophilic moiety.

[0015] In some embodiments, the lipophilic monomer does not contain nucleobases.

[0016] In some embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via one or more linkers (chain links).

[0017] In some embodiments, 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, phosphate diester, sulfonamide bond, product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or carbamate.

[0018] In some embodiments, at least one linker (chain linker) is a redox-cleavable linker (such as a reductant linker, e.g., a disulfide group), an acid-cleavable linker (e.g., an 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 ester), or a peptidase-cleavable linker (e.g., a peptide bond).

[0019] In other embodiments, at least one linker (tether) is a biolytic linker selected from the group consisting of: DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, or combinations thereof.

[0020] In some embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via a non-ribose substitution unit (i.e., a carrier) that replaces one or more nucleotides. The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxalinyl, pyridazinoneyl, tetrahydrofuranyl, and decahydronaphthalene. In one embodiment, the acyclic group is a portion based on a serine backbone, a glycerol backbone, or a diethanolamine backbone.

[0021] 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 internal positions of the double-stranded iRNA agent. In other embodiments, the carrier replaces the nucleotides at the ends of the sense or antisense strands. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby acting as a cap protecting the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for example, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinoneyl, tetrahydrofuranyl, or decahydronaphthyl.

[0022] In some embodiments, the lipophilic monomer may be represented by one of the following formulas:

[0023]

[0024] in:

[0025] J1 and J2 are independently O, S, and NR. N Optionally substituted alkyl groups, 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)NH P )2)O, or OP(N(R) P )2);

[0026] It can be a cyclic group or an acyclic group;

[0027] R N It is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroaryl, or amino protecting group.

[0028] R P Each time it appears, it is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, or optionally substituted heteroaryl.

[0029] L 10 It is a substituted or unsubstituted, saturated or unsaturated C3-C8 hydrocarbon (e.g., C3-C8 alkyl, alkenyl or ynyl, or C3-C8 hydrocarbon containing two or more double bonds); the substituted group includes those already described herein as “substituted” hydrocarbon, alkyl, alkenyl or ynyl.

[0030] L 11 It is a substituted or unsubstituted, saturated or unsaturated C6-C26 hydrocarbon (e.g., C6-C26 alkyl, alkenyl, or ynyl, or C3-C8 hydrocarbon containing two or more double bonds); the substituted group includes those already described herein as “substituted” hydrocarbons, alkyl, alkenyl, or ynyl; and

[0031] When the carrier lacks nucleobases, Q is absent, or Q will carry L in vivo. 10 From L 11 At least 10% of the cleavable groups are cleaved. For example, Q can be a group that can be cleaved in vivo to release L. 11The cleavable groups are cleaved from lipophilic monomers, comprising approximately 10%-70%, approximately 15%-50%, approximately 20%-40%, or approximately 20%-30%. Exemplary cleavable groups include -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, and -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)-、 Or a combination thereof, R 11 It is a C2-C8 alkyl or alkenyl group. In each occurrence, R... 3 R 4 and R 5 Each is independently an H or C1-C4 alkyl group.

[0032] In one embodiment, the cleavage of Q is assessed by the stability of the ligand in cerebrospinal fluid (CSF), the stability of the ligand in plasma, the stability of the ligand in brain homogenate or tissue homogenate (liver, eye, etc.), or the stability of the ligand in vitreous fluid.

[0033] Cyclic and acyclic groups include those already described herein.

[0034] In one embodiment, the acyclic group is a serine alcohol, glycerol, or diethanolamine backbone.

[0035] In one embodiment, the cyclic group is selected from pyrrolidinyl, hydroxyprolinyl, cyclopentyl, cyclohexyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolanecycloyl, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinoneyl, tetrahydrofuranyl, and decahydronaphthyl.

[0036] 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.

[0037] In some embodiments, the lipophilic monomer conjugated to one or more positions on the chain of the compound has the following structure:

[0038]

[0039]

[0040]

[0041] In the above structures of lipophilic monomers, the monomers may also contain one or more asymmetric centers, and thus exist as racemates and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of the monomer are explicitly included.

[0042] In the structure of the lipophilic monomers described above, the alkylene chain may contain one or more unsaturated bonds.

[0043] Integer m is 0-8. Integer n is 1-21. R2' can be any functional group (that is, a 2' modification acceptable to ribose), such as 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropane), 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F. 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 a C1-C4 alkyl group (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl). R, R', and R" are each independently H or an alkyl group, such as a C1-C4 alkyl group (e.g., methyl, ethyl, propyl, isopropyl, tert-butyl).

[0044] In some embodiments, the lipophilic monomer conjugated to one or more positions on the chain of the compound has the following structure:

[0045] (R2' is 2'-F, 2'-OMe, 2'-NMA, 2'-deoxy, or 2'-OH); In these structures, B is a modified or unmodified nucleobase.

[0046] Specific examples of lipophilic monomers include:

[0047]

[0048]

[0049] In these structures, B is a modified or unmodified nucleobase; and R and R' are each independently H, methyl, ethyl, isopropyl, or tert-butyl.

[0050] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the chain of the compound (single-stranded oligonucleotide; or sense and / or antisense strand of double-stranded oligonucleotide) via a carrier:

[0051] In these embodiments, R is a lipophilic moiety as defined herein. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0052] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the internal position of the chain of the compound (single-stranded oligonucleotide; or sense and / or antisense strand of double-stranded oligonucleotide) via a carrier:

[0053] In these embodiments, R is the lipophilic moiety as defined herein. n is an integer from 1 to 21. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0054] Additional examples of lipophilic monomers can be found in the examples.

[0055] In some embodiments, the length of the sense strand and the antisense strand of the compound is each 15 to 30 nucleotides.

[0056] In one embodiment, the length of the sense strand and the antisense strand of the compound is each 19 to 25 nucleotides.

[0057] In one embodiment, the length of the sense strand and the antisense strand of the compound is each 21 to 23 nucleotides.

[0058] In some embodiments, the compound includes single-stranded overhangs at at least one of a plurality of ends, such as 3' and / or 5' overhangs of 1-10 nucleotides in length, for example, overhangs of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, both strands have at least one segment of 1-5 (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 blunt ends 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 includes 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 includes 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 at both ends of the iRNA double helix.

[0059] In one embodiment, the sense strand of the compound is 21 nucleotides long and the antisense strand is 23 nucleotides long, wherein these strands form a double-stranded region of 21 consecutive base pairs (with a single-stranded overhang of 2 nucleotides at the 3' end).

[0060] In some embodiments, the sense chain further comprises at least one phosphate thioester bond at its 3' end. In some embodiments, the sense chain further comprises at least two phosphate thioester bonds at its 3' end. In some embodiments, one or more lipophilic monomers are located at the 3' end of the sense chain. In one embodiment, one of these phosphate thioester bonds is located between the lipophilic monomer and the first nucleotide at the 3' end of the sense chain.

[0061] In some embodiments, the sense chain further comprises at least one phosphate thioester bond at its 5' end. In some embodiments, the sense chain further comprises at least two phosphate thioester bonds at its 5' end. In some embodiments, one or more lipophilic monomers are located at the 5' end of the sense chain. In one embodiment, one of these phosphate thioester bonds is located between the lipophilic monomer and the first nucleotide at the 5' end of the sense chain.

[0062] In some embodiments, the antisense strand further comprises at least one phosphate thioester bond at its 3' end. In some embodiments, the antisense strand further comprises at least two phosphate thioester 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 these phosphate thioester bonds is located between the lipophilic monomer and the first nucleotide at the 3' end of the antisense strand.

[0063] In some embodiments, the compound further comprises a phosphate ester or phosphate ester mimic at the 5' end of the antisense chain. In one embodiment, the phosphate ester mimic is 5'-vinylphosphonate (VP).

[0064] In some embodiments, the 5' end of the antisense chain of the compound does not contain 5'-vinylphosphonate (VP).

[0065] In some embodiments, the compound further comprises at least one terminal chiral phosphorus atom.

[0066] Site-specific chiral modifications of nucleotide bonds can occur at the 5' end, 3' end, or both 5' and 3' ends of the strand. This is referred to herein as "terminal" chiral modification. Terminal modifications can occur at the 3' or 5' end of the terminal region, for example, on the terminal nucleotide of the strand or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Chiral modifications can occur in the sense strand, antisense strand, or both sense and antisense strands. Each chiral pure phosphorus atom can be in the Rp or Sp configuration or a combination thereof. Further details regarding chiral modifications and chiral-modified double-stranded RNA agents can be found in PCT / US 18 / 67103, filed December 21, 2018, entitled "Chirally-Modified Double-Stranded RNA Agents," which is incorporated herein by reference in its entirety.

[0067] In some embodiments, the compound further includes terminal chiral modification of a phosphate atom with Sp configuration at the first nucleotide inter-bond at the 3' end of the antisense strand; terminal chiral modification of a phosphate atom with Rp configuration at the first nucleotide inter-bond at the 5' end of the antisense strand; and terminal chiral modification of a phosphate atom with Rp or Sp configuration at the first nucleotide inter-bond at the 5' end of the sense strand.

[0068] In one embodiment, the compound further includes terminal chiral modification of a phosphate atom with Sp configuration at the first and second nucleotide inter-bonds at the 3' end of the antisense strand; terminal chiral modification of a phosphate atom with Rp configuration at the first nucleotide inter-bond at the 5' end of the antisense strand; and terminal chiral modification of a phosphate atom with Rp or Sp configuration at the first nucleotide inter-bond at the 5' end of the sense strand.

[0069] In one embodiment, the compound further comprises terminal chiral modifications of a phosphate atom with Sp configuration occurring at the first, second, and third nucleotide inter-bonds at the 3' end of the antisense strand; terminal chiral modifications of a phosphate atom with Rp configuration occurring at the first nucleotide inter-bond at the 5' end of the antisense strand; and terminal chiral modifications of a phosphate atom with Rp or Sp configuration occurring at the first nucleotide inter-bond at the 5' end of the sense strand.

[0070] In one embodiment, the compound further comprises: terminal chiral modification of a phosphate atom with Sp configuration at the first and second nucleotide inter-bonds at the 3' end of the antisense strand; terminal chiral modification of a phosphate atom with Rp configuration at the third nucleotide inter-bond at the 3' end of the antisense strand; terminal chiral modification of a phosphate atom with Rp configuration at the first nucleotide inter-bond at the 5' end of the antisense strand; and terminal chiral modification of a phosphate atom with Rp or Sp configuration at the first nucleotide inter-bond at the 5' end of the sense strand.

[0071] In one embodiment, the compound further includes terminal chiral modification of a phosphate atom with Sp configuration at the first and second nucleotide inter-bonds at the 3' end of the antisense strand; terminal chiral modification of a phosphate atom with Rp configuration at the first and second nucleotide inter-bonds at the 5' end of the antisense strand; and terminal chiral modification of a phosphate atom with Rp or Sp configuration at the first nucleotide inter-bond at the 5' end of the sense strand.

[0072] In some embodiments, the compound has at least two phosphate thionucleotide internucleotide bonds (counting from the 5' end) at the first five nucleotides of the antisense strand.

[0073] In some embodiments, the antisense strand comprises two blocks having one, two, or three thiophosphate nucleotide interbonds, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate ester nucleotide interbonds.

[0074] In some embodiments, the compound further comprises a targeting ligand that targets and mediates delivery to a receptor in a specific CNS tissue. In one embodiment, the targeting ligand is selected from the group consisting of: angiopep-2, lipoprotein receptor-associated protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter, and LDL receptor ligand.

[0075] In some embodiments, the compound further comprises a targeting ligand that targets and mediates delivery to a receptor in ocular tissue. In one embodiment, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptides, LDL receptor ligands, and carbohydrate-based ligands. In one embodiment, the targeting ligand is an RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO:1) or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

[0076] 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.

[0077] 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 the compound are modified. For example, when 50% of the compound is modified, 50% of all nucleotides present in the compound contain the modifications as described herein.

[0078] 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 almost 100% of nucleotides modified with 2'-O-methyl.

[0079] In one embodiment, the compound is an oligonucleotide, such as a double-stranded dsRNA agent, and 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'-fluorine.

[0080] In one embodiment, the oligonucleotide is antisense, and at least 50% of the antisense nucleotides are independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0081] 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 nucleotides modified with 2'-F. 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 modification 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 modification on the antisense strand.

[0082] In some embodiments, the compound has one or more 2'-F modifications at any position of the sense or antisense strand.

[0083] In some embodiments, the compound contains less than 20%, less than 15%, less than 10%, less than 5% of non-natural nucleotides, or is 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-methoxypropyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (such as 2'-modified L-nucleosides, e.g., 2'-deoxy-L-nucleosides), BNA without basic sugars, and without basic cyclic and open-chain alkyl groups.

[0084] In some embodiments, the compound has more than 80%, more than 85%, more than 90%, more than 95%, or almost 100% of natural nucleotides. For the purposes of these embodiments, the natural nucleotides may include natural nucleotides having 2'-OH, 2'-deoxy, and 2'-OMe.

[0085] In some embodiments, the antisense strand (e.g., in the seed region of the antisense strand) contains at least one unlocking nucleic acid (UNA) or glycerol nucleic acid (GNA) modification. In one embodiment, the seed region is located at positions 2-8 (or positions 5-7) at the 5' end of the antisense strand.

[0086] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand having a length of 15-30 nucleotides; at least two phosphate thioester nucleotide bonds (counted from the 5' end) at the first five nucleotides of the antisense strand; wherein the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the compound contains less than 20%, less than 15%, less than 10%, less than 5% of non-natural nucleotides, or is substantially free of non-natural nucleotides.

[0087] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand having a length of 15-30 nucleotides; at least two phosphate thioester nucleotide bonds (counted from the 5' end) at the first five nucleotides of the antisense strand; wherein the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the compound has more than 80%, more than 85%, more than 95% or almost 100% of natural nucleotides, such as natural nucleotides having 2'-OH, 2'-deoxy or 2'-OMe.

[0088] One aspect of the 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 phosphate thioester nucleotide inter-bonds 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 (double-stranded) region between 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand does not contain a diol nucleic acid (GNA).

[0089] It should be understood that the antisense strand is sufficiently complementary to the target sequence to mediate RNA interference. In other words, the compound can inhibit the expression of the target gene.

[0090] In one embodiment, the compound comprises at least three 2'-deoxy modifications. The 2'-deoxy modifications are located at positions 2 and 14 (counting from the 5' end of the antisense chain) on the antisense chain and at position 11 (counting from the 5' end of the sense chain) on the sense chain.

[0091] In one embodiment, the compound comprises at least five 2'-deoxy modifications. The 2'-deoxy modifications are located at positions 2, 12, and 14 of the antisense chain (counting from the 5' end of the antisense chain) and positions 9 and 11 of the sense chain (counting from the 5' end of the sense chain).

[0092] In one embodiment, the compound comprises at least seven 2'-deoxy modifications. The 2'-deoxy modifications are located at positions 2, 5, 7, 12, and 14 on the antisense chain (counting from the 5' end of the antisense chain) and positions 9 and 11 on the sense chain (counting from the 5' end of the sense chain).

[0093] In one embodiment, the antisense strand comprises at least five 2'-deoxy modifications located at positions 2, 5, 7, 12, and 14 (counting from the 5' end of the antisense strand). The antisense strand has a length of 18-25 nucleotides or 18-23 nucleotides.

[0094] In one embodiment, the compound may contain one or more non-natural nucleotides. For example, the compound may contain less than 20%, such as less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound may not contain any non-natural nucleotides. For example, the compound may contain all natural nucleotides. Some exemplary non-natural nucleotides include, but are not limited to, acyclic nucleotides, locked nucleic acids (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0095] 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 phosphate thioester nucleotide inter-bonds 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 antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; wherein the sense strand does not contain glyconucleotides (GNA); and wherein the compound contains less than 20%, for example less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound contains all natural nucleotides.

[0096] In one embodiment, at least one of the sense strand and antisense strand contains at least one, such as 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 a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphate thioester nucleotide inter-bonds between the first five nucleotides (counting from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxy nucleotides on the sense strand and / or antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand and / or antisense strand contains at least one, such as 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 antisense strand.

[0097] In some embodiments, the sense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the sense strand. For example, the sense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in positions 7, 8, 9, 10, 11, 12, and 13 (counting from the 5' end of the sense strand).

[0098] In one embodiment, the antisense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the antisense strand. For example, the antisense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in positions 10, 11, 12, 13, 14, 15, and 16 (counting from the 5' end of the antisense strand).

[0099] In one embodiment, the compound comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17-30 nucleotides and contains at least one 2'-deoxy modification in the central region of the sense strand; and wherein the antisense strand independently has a length of 17-30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the antisense strand.

[0100] In one embodiment, the compound comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17-30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the sense strand; and wherein the antisense strand independently has a length of 17-30 nucleotides and contains at least one 2'-deoxy modification in the central region of the antisense strand.

[0101] 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 phosphate thioester nucleotide inter-bonds 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 antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand contains at least one, for example 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.

[0102] 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 phosphate thioester nucleotide bonds 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 antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; and wherein the antisense strand contains at least one, for example 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.

[0103] 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 phosphate thionucleotide internucleotide bonds 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 antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, such as less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the sense strand and / or antisense strand contains at least one, such as 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 and / or antisense strand.

[0104] 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 phosphate thioester nucleotide bonds 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 antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, such as less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the sense strand contains at least one, such as 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.

[0105] 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 phosphate thioester nucleotide bonds 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 antisense strand; and wherein the compound has a double-stranded region between 19 to 25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, such as 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 contains at least one, such as 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.

[0106] In one embodiment, when the compound contains fewer than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA. For example, in any embodiment of the invention, when the compound contains fewer than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA.

[0107] In one embodiment, when the antisense comprises two deoxynucleotides and said nucleotides are located at positions 2 and 14 (counting from the 5' end of the antisense strand), the compound comprises eight or fewer (e.g., eight, seven, six, five, four, three, two, one, or zero) non-2'OMe nucleotides. For example, in any embodiment of the invention, when the antisense comprises two deoxynucleotides and said nucleotides are located at positions 2 and 14 (counting from the 5' end of the antisense strand), the compound comprises zero, one, two, three, four, five, six, seven, or eight non-2'OMe nucleotides.

[0108] In another aspect, the present invention further provides a method for delivering the compound of the present invention to a specific target in a subject via subcutaneous or intravenous administration. The present invention further provides a method for delivering the compound of the present invention to a specific target in a subject via subcutaneous or intravenous administration.

[0109] Another aspect of the invention relates to a method for reducing the expression of a target gene in a cell, the method 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 having one or more lipophilic moieties optionally conjugated to one or more sites on at least one strand via a linker or vector.

[0110] In the first aspect of the invention relating to compounds, all the above-described embodiments relating to lipophilic monomers, lipophilic moieties, and their conjugation with compounds are suitable for this aspect of the invention in relation to methods for reducing the expression of target genes in cells.

[0111] In one embodiment, the cells are extrahepatic cells.

[0112] In one embodiment, the cell is not a liver cell.

[0113] Another aspect of the invention relates to a method for reducing the expression of a target gene in a subject, the method comprising administering a compound to the subject, including contacting the cells with the compound, the 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 optionally conjugated to one or more internal sites on at least one strand via a linker or vector.

[0114] In the first aspect of the invention relating to compounds, all the above-described embodiments relating to lipophilic monomers, lipophilic moieties, and their conjugation with compounds are suitable for this aspect of the invention in relation to methods for reducing target gene expression in subjects.

[0115] In some embodiments, the compound is administered extrahepatically.

[0116] In one embodiment, the compound is administered intrathecally or intraventricularly. By administering the compound intrathecally or intraventricularly, this method can reduce the expression of target genes in brain or spinal tissues (e.g., cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae).

[0117] 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, the compound can be applied directly (e.g., intravitreally) to one or more eyes. By applying the compound intravitreally, this method can reduce the expression of target genes in ocular tissues.

[0118] Another aspect of the invention relates to a method of treating a subject suffering from a CNS disorder, the method comprising administering a therapeutically effective amount of a double-stranded RNAi agent to the subject, 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 optionally conjugated to one or more internal sites on at least one strand via a linker or vector.

[0119] In the first aspect of the invention relating to compounds, all the above-described embodiments relating to lipophilic monomers, lipophilic moieties, and their conjugation with compounds are suitable for this aspect of the invention in relation to methods of treating subjects suffering from CNS disorders. Exemplary CNS disorders treatable by the methods of the invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prions, and Lafra disease. Attached Figure Description

[0120] Figure 1 This is a schematic diagram showing the general structure of ceramides.

[0121] Figure 2 This is a diagram depicting the stability of the siRNA conjugate in rat CSF after incubating the siRNA duplex for 24 hours.

[0122] Figure 3 This is a graph depicting the stability of siRNA conjugates in the vitreous humor (NHP) of rabbits and cynomolgus monkeys over 24 hours. The remaining amount of the ligand-conjugated double strand is plotted.

[0123] Figure 4 This is a graph depicting the stability of siRNA conjugates in the vitreous humor (NHP) of rabbits and cynomolgus monkeys over 24 hours. The remaining amount of the ligand-conjugated double strand is plotted.

[0124] Figure 5A and 5B This is a graph depicting the stability of siRNA conjugates in rat brain homogenate after 4 hours. Figure 5AThe remaining amount of the ligand-conjugated double strand was plotted, and Figure 5B The stability of the PS key is illustrated in the figure.

[0125] Figure 6 This is a graph depicting the stability of siRNA conjugates with esterase-cleavable conjugates in the vitreous humor (NHP) of rabbits and cynomolgus monkeys over 24 hours. The percentage of hydrolyzed ligand-conjugated double strands is plotted.

[0126] Figure 7 This is a graph depicting the stability of siRNA conjugates with esterase-cleavable conjugates in rat plasma, CSF, and brain homogenate over 24 hours. The percentage of hydrolyzed ligand-conjugated double strands is plotted.

[0127] Figure 8 This is a graph depicting the binding of siRNA conjugates to human serum albumin at different HSA concentrations. The fraction of bound siRNA is plotted against human serum albumin concentration.

[0128] Figure 9 This is a graph depicting the binding of siRNA conjugates with exposed carboxylic acids to human serum albumin at different HSA concentrations. The fraction of bound siRNA is plotted against human serum albumin concentration.

[0129] Figure 10 This figure illustrates the inhibition of ocular TTR expression in mice after intravitreal administration of a single dose of 7.5 μg of siRNA duplexes (by qPCR) compared to the PBS control.

[0130] Figure 11 This figure illustrates the inhibition of ocular TTR expression in rat eyes after intravitreal administration of a single dose of 1 μg of siRNA duplexes, compared to the PBS control (by qPCR).

[0131] Figure 12 This figure illustrates the inhibition of ocular TTR expression in mice after intravitreal administration of a single dose of 7.5 μg of siRNA duplexes (by qPCR) compared to the PBS control.

[0132] Figure 13 This figure illustrates the inhibition of ocular TTR expression in rat eyes after intravitreal administration of a single dose of 1 μg of siRNA duplexes, compared to the PBS control (by qPCR).

[0133] Figure 14 This figure illustrates the inhibition of ocular TTR expression in mice after intravitreal administration of a single dose of 7.5 μg of siRNA duplexes (by qPCR) compared to the PBS control.

[0134] Figure 15This figure illustrates the inhibition of ocular TTR expression in rat eyes after intravitreal administration of a single dose of 1 μg of siRNA duplexes, compared to the PBS control (by qPCR).

[0135] Figure 16 This graph depicts the suppression of TTR gene expression in primary mouse hepatocytes 24 hours after transfection with Q367-modified siRNA duplexes, compared to the control duplex AD-900954, at three different concentrations. Each nucleotide is modified across the sense strand by Q367.

[0136] Figure 17 This graph depicts the suppression of SOD1 gene expression in primary mouse hepatocytes 24 hours after transfection with Q367-modified siRNA duplexes, compared to the control duplex AD-900954, at three different concentrations. Each nucleotide is modified across the sense strand by Q367.

[0137] Figures 18A-18D This describes the effects of a single 0.9 mg siRNA double strand / rat administration to the rat spinal cord 14 days later, compared to the artificial CSF-treated control group. Figure 18A ), cerebellum ( Figure 18B ), frontal cortex ( Figure 18C ) and heart ( Figure 18D A diagram showing the inhibition of SOD1 expression (via qPCR) in [a specific region / organization].

[0138] Figures 19A-19E This describes the effects of a single 0.9 mg siRNA double strand / rat administration to the rat spinal cord 14 days later, compared to the artificial CSF-treated control group. Figure 19A ), brainstem ( Figure 19B ), cerebellum ( Figure 19C ), frontal cortex ( Figure 19D ) and heart ( Figure 19E A diagram showing the inhibition of SOD1 expression (via qPCR) in [a specific region / organization].

[0139] Figure 20 This is a graph depicting the inhibition (by qPCR) of SOD1 expression in the rat brain (cerebellum and frontal cortex) and spinal cord (thoracic spinal cord) after a single administration of 0.9 mg of siRNA double strand / rat at IT 14 days later, compared with the artificial CSF-treated control group.

[0140] Figure 21A and 21B It describes 14 days ( Figure 21A ) and 7 days ( Figure 21B After administration of 50 μg of ICV, compared with the control group treated with artificial CSF, the effect of a single dose of ICV was significantly better. Figure 21A ) and 110μg ( Figure 21BThe graph shows the inhibition of SOD1 expression in the mouse brain (right hemisphere) and heart (by qPCR) after siRNA double strands / mouse. Detailed Implementation

[0141] The inventors of this invention have particularly discovered that conjugating a lipophilic monomer containing a lipophilic moiety to one or more sites on at least one chain of a compound for in vivo ocular delivery (e.g., intravitreal delivery) and intrathecal or intraventricular delivery of double-stranded iRNA provides surprisingly good results, enabling efficient entry into CNS and ocular tissues and efficient internalization into cells of the CNS and ocular systems.

[0142] One aspect of the present invention provides a compound comprising:

[0143] 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 optionally conjugated to one or more sites on at least one strand via a linker or vector.

[0144] The term "lipophile" or "lipophilic moiety" broadly refers to any compound or chemical fraction that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient logK. ow K ow The octanol-water partition coefficient is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. It is a laboratory-measured property of the substance. However, it can also be predicted using coefficients attributed to the structural components of the chemical substance, calculated using first-principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. [Journal of Chemical Information and Computer Science] 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments over water (i.e., its hydrophilic / lipophilic balance). In principle, when logK... ow When the value exceeds 0, the chemical substance exhibits lipophilicity. Typically, the logK of the lipophilic portion... ow Exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For example, predicting the logK of, for instance, 6-aminohexanol. ow It is approximately 0.7. Using the same method, the logK of cholesterol-based N-(hexyl-6-ol)carbamate was predicted. ow It is 10.7.

[0145] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, adding a hydroxyl or amino group to the end of the lipophilic moiety can increase or decrease the partition coefficient (e.g., logK) of the lipophilic moiety. ow )value.

[0146] 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 characteristics. For example, it can be determined that the unbound fraction in a plasma protein binding assay of a compound is positively correlated with the relative hydrophobicity of the double-stranded iRNA agent, which may be positively correlated with the silencing activity of the double-stranded iRNA agent.

[0147] In one embodiment, the plasma protein binding assay is performed using the electrophoretic mobility shift assay (EMSA) of human serum albumin. The hydrophobicity of the double-stranded iRNA agent, 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 to enhance in vivo delivery of the siRNA.

[0148] Therefore, conjugating lipophilic monomers (containing lipophilic moieties) with compounds provides optimal hydrophobicity for enhancing in vivo delivery of siRNA.

[0149] In some embodiments, the lipophilic moiety is aliphatic, cyclic (e.g., alicyclic), or polycyclic (e.g., polycyclic) compounds, such as steroids (e.g., sterols) or straight-chain or branched aliphatic hydrocarbons. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moietyes include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C6-C) 18 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic aromatic hydrocarbons. For example, the lipophilic moiety may contain C4-C64. 30 Hydrocarbon chains (e.g., C4-C) 30 Alkyl or alkenyl groups). In some embodiments, the lipophilic moiety contains saturated or unsaturated C6-C6 groups. 18 Hydrocarbon chains (e.g., straight-chain C6-C) 18 (alkyl or alkenyl). In one embodiment, the lipophilic moiety contains saturated or unsaturated C. 16 Hydrocarbon chains (e.g., straight-chain C)16 (alkyl or alkenyl).

[0150] Lipophilic monomers containing a lipophilic moiety can be attached to iRNA agents by any method known in the art, including via functional groups already present in the lipophilic monomer or introduced into the iRNA agent, such as hydroxyl groups (e.g., -CO-CH2-OH). Functional groups already present in the lipophilic monomer or introduced into the iRNA agent include, but are not limited to, hydroxyl groups, amines, carboxylic acids, sulfonates, phosphates, thiols, azides, and alkynes.

[0151] The conjugation of iRNA agents with lipophilic monomers can occur, for example, by forming an ether, carboxyl, or carbamoyl ester bond between a hydroxyl group and an alkyl group (R-), an alkyl 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; and saturated or unsaturated). The alkyl group (R) can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, or octadecyl, etc.

[0152] In some embodiments, a lipophilic monomer comprising a lipophilic moiety is conjugated to a compound via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or carbamate.

[0153] In another embodiment, the lipophilic moiety is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanphenanthrene 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 compounds derived from cholesterol, for example, through substitution, addition, or removal of substituents.

[0154] In another embodiment, the lipophilic moiety is the aromatic moiety. In this context, the term "aromatic" refers to both monoaromatic and polyaromatic hydrocarbons. Aryl groups include, but are not limited to, C6-C hydrocarbons comprising one to three aromatic rings. 14 The aryl moiety may optionally be substituted; "aralkyl" or "arylalkyl" comprising an aryl group covalently linked to an alkyl group, either of which may be optionally substituted or unsubstituted independently; and "heteroaryl". As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; having 6, 10 or 14 π electrons shared in the ring array; and having one to three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O) and sulfur (S) in addition to the carbon atom.

[0155] As used herein, a “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is a group having one to about four, preferably one to about three, more preferably one or two non-hydrogen substituents. Suitable substituents include, but are not limited to, halogen, hydroxy, nitro, haloalkyl, alkyl, alkylaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxyl, hydroxyalkyl, alkylsulfonyl, arylsulfonyl, alkylsulfonamide, arylsulfonamide, arylsulfonamide, alkylcarbonyl, acyloxy, cyano, and urea.

[0156] In some embodiments, the lipophilic moiety is an aralkyl group, such as a 2-arylpropionyl moiety. The structural features of the aralkyl group are chosen such that the lipophilic moiety will bind to at least one protein in vivo. In some embodiments, the structural features of the aralkyl group are chosen such that the lipophilic moiety binds to serum, vascular, or cellular proteins. In some embodiments, the structural features of the aralkyl group facilitate binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulin, or α-1-glycoprotein.

[0157] In some embodiments, the ligand is naproxen or a structural derivative of naproxen. Methods for synthesizing naproxen are available in U.S. Patent Nos. 3,904,682 and 4,009,197, which are incorporated herein by reference in their entirety. The chemical name of naproxen is (S)-6-methoxy-α-methyl-2-naphthacetic acid, and its structure is [insert structure here].

[0158] In some embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. A method for synthesizing ibuprofen is available in U.S. Patent No. 3,228,831, which is incorporated herein by reference in its entirety. The structure of ibuprofen is as follows:

[0159] Additional exemplary aralkyl groups are described in U.S. Patent No. 7,626,014, which is incorporated herein by reference in its entirety.

[0160] In another embodiment, suitable lipophilic fractions include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0161] In some embodiments, the lipophilic portion is C6-C. 30Acids (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-C 30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, retinol, vitamin E, cholesterol, etc.).

[0162] In some embodiments, a lipophilic monomer containing one or more lipophilic moieties may be incorporated into the double-stranded iRNA agent, particularly when the lipophilic moieties are of low lipophilicity or hydrophobicity. In one embodiment, a lipophilic monomer containing two or more lipophilic moieties is incorporated into the same strand of the double-stranded iRNA agent. In one embodiment, each strand of the double-stranded iRNA agent has an incorporated lipophilic monomer containing one or more lipophilic moieties. In one embodiment, a lipophilic monomer containing two or more lipophilic moieties is incorporated into the same position (i.e., the same nucleotide, the same sugar moiety, or the same nucleotide internucleotide bond) of the double-stranded iRNA agent. This can be achieved, for example, by using a lipophilic monomer containing a vector, and / or a direct linker and / or one or more adapters (which can connect two or more lipophilic moieties).

[0163] When the lipophilic moiety is conjugated to the iRNA agent via a nucleotide, ribose, or nucleoside bond directly attached to the iRNA agent, the lipophilic monomer comprises a nucleotide, a ribose, or nucleoside bond, and the lipophilic moiety. Alternatively, the lipophilic monomer may comprise a lipophilic moiety conjugated to a non-ribose substitution unit, such as a linker or vector. When the lipophilic moiety is conjugated to a double-stranded iRNA agent via a non-ribose substitution unit (such as a linker or vector), the lipophilic monomer comprises a non-ribose substitution unit (such as a linker or vector) and the lipophilic moiety.

[0164] In some embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the iRNA agent via one or more linkers (connecting strands).

[0165] 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, phosphate diester, sulfonamide bond, product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or carbamate.

[0166] Connector / Chain

[0167] The connector / tether connects to the lipophilic portion at the "tether attachment point (TAP)". The connector / tether can include any C1-C... 100 Carbon-containing portion (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 It may contain at least one nitrogen atom. In some embodiments, the nitrogen atom forms part of a terminal amino or amide (NHC(O)-) on the connector / chain linker, which may act as a connection point for the lipophilic portion. Non-limiting examples of connectors / chain links (underlined) include TAP. -(CH2) n NH- TAP- C(O)(CH2) n NH- TAP- NR″″(CH2) n NH- TAP- C(O)-(CH2) n -C (O)- TAP- C(O)-(CH2) n -C(O)O-; TAP- C(O)-O- TAP- C(O)-(CH2) n -NH-C(O)- TAP- C(O)- (CH2) n - TAP- C(O)-NH- TAP- C(O)- TAP- (CH2) n -C(O)- TAP- (CH2) n -C(O)O- TAP- (CH2) n - ; or TAP- (CH2) n -NH-C(O)- 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 group. Preferably, n is 5, 6, or 11. In other embodiments, nitrogen may form part of a terminal oxyamino group, such as -ONH2 or hydrazine-NHNH2. The linker / chain may optionally be substituted, for example, with a hydroxyl, alkoxy, or perhaloalkyl group, and / or optionally have one or more additional heteroatoms, such as N, O, or S. Preferred chain ligands may include, for example, TAP. -(CH2) n NH (ligand) TAP- C(O)(CH2) n NH (ligand) TAP- NR″″(CH2) n NH (ligand) TAP - (CH2) n ONH (ligand) TAP- C(O)(CH2) n ONH (ligand) TAP- NR″″(CH2) n ONH (ligand) TAP- (CH2) n NHNH2 (ligand) TAP- C(O)(CH2) n NHNH2 (ligand) TAP- NR″″(CH2) n NHNH2 (ligand) TAP- C(O)-(CH2) n -C(O) (ligand) TAP-C(O)-(CH2) n -C(O)O (ligand); TAP- C(O)-O (ligand) TAP- C(O)-(CH2) n -NH-C(O) (combined with) body) TAP- C(O)-(CH2) n (ligand) TAP- C(O)-NH (ligand) TAP- C(O)(ligand) TAP- (CH2) n -C(O)(combined with) body) TAP- (CH2) n -C(O)O(ligand) TAP- (CH2) n (ligand) ; or TAP- (CH2) n -NH-C(O) (ligand). In some embodiments, the amino-terminated linker / chain (e.g., NH2, ONH2, NH2NH2) may form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / chain (e.g., NH2, ONH2, NH2NH2) may be acylated, for example, with C(O)CF3.

[0168] In some embodiments, the connector / chain may be end-capped with a thiol group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the chain may be TAP. -(CH2) n -SH TAP- C(O)(CH2) n SH TAP -(CH2) n -(CH=CH2 ) or TAP- C(O)(CH2) n (CH= CH2) , where n may be as described elsewhere. The chain may optionally be substituted with, for example, hydroxyl, alkoxy, or perhaloalkyl, and / or optionally have one or more additional heteroatoms, such as N, O, or S. The double bond may be cis or trans or E or Z.

[0169] In other embodiments, the connector / tether may include an electrophilic portion, preferably at the end of the connector / tether. Exemplary electrophilic portions include, for example, aldehydes, alkyl halides, methanesulfonates, toluenesulfonates, nitrobenzenesulfonates, or bromobenzenesulfonates, or activated carboxylic acid esters, such as NHS esters or pentafluorophenyl esters. Preferred connectors / tethers (underlined) include TAP. - (CH2) n GIVE TAP- C(O)(CH2) n GIVE ; or TAP- NR″″(CH2) n GIVE Where n is 1-6 and R″″ is a C1-C6 alkyl group; or TAP -(CH2) n C(O)ONHS TAP- C(O)(CH2) n C(O)ONHS ; or TAP- NR″″(CH2) n C(O)ONHS Where n is 1-6 and R″″ is a C1-C6 alkyl group; TAP- (CH2) n C(O)OC6F5 TAP- C(O)(CH2) n C(O)OC6F5 ; or TAP- NR″″(CH2) n C(O) OC6F5 Where n is 1-11 and R″″ is a C1-C6 alkyl group; or -(CH2) n CH2LG TAP- C(O)(CH2) n CH2LG ; or TAP- NR″″ (CH2) n CH2LG, where n may be as described elsewhere and R″″ is a C1-C6 alkyl group (LG may be a leaving group, such as a halide, methanesulfonate, toluenesulfonate, nitrobenzenesulfonate, or bromobenzenesulfonate). Chain linking can be achieved by coupling the nucleophilic group (e.g., a thiol or amino group) of the ligand to an electrophilic group on the chain.

[0170] In other embodiments, it may be necessary for the monomer to include phthalimide (K) at the end of the connector / chain linker.

[0171] In other embodiments, other protected amino groups may be located at the end of the connector / chain chain, such as allyloxycarbonyl, monomethoxytriphenylmethyl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety may be o-nitrophenyl or o-, p-dinitrophenyl).

[0172] Any connector / chain described herein may further include one or more additional linking groups, such as -O-(CH2). n -、-(CH2) n -SS-、-(CH2) n - or -(CH=CH)-.

[0173] Disintegral connectors / chains

[0174] In some embodiments, at least one of the connectors / chains may be a redox-cleavable connector, an acid-cleavable connector, an esterase-cleavable connector, a phosphatase-cleavable connector, or a peptidase-cleavable connector.

[0175] In one embodiment, at least one of the joints / chains may be a reducible pyrolytic joint (e.g., a disulfide group).

[0176] In one embodiment, at least one of the connectors / chains may be an acid-degradable connector (e.g., hydrazone, ester, acetal, or ketal).

[0177] In one embodiment, at least one of the linkers / connectors may be an esterase-cleavable linker (e.g., an ester group).

[0178] In one embodiment, at least one of the linkers / tethers may be a phosphatase-cleavable linker (e.g., a phosphate ester).

[0179] In one embodiment, at least one of the linkers / connectors may be a peptidase-cleavable linker (e.g., a peptide bond).

[0180] Cleavable linker groups are susceptible to the effects of cleaving agents (e.g., pH, redox potential, or the presence of degrading molecules). Generally, cleaving agents are more prevalent or found at higher levels or with higher activity within cells than in serum or blood. Examples of such degrading agents include: substrate-selective or non-substrate-specific redox agents, including, for example, oxidative or reductive enzymes or reducing agents such as thiols present in cells, which can degrade redox-cleavable linker groups through reduction; esterases; endosomes or agents that can produce an acidic environment, such as endosomes or agents that result in a pH of 5 or lower; and enzymes that can hydrolyze or degrade acid-cleavable linker groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0181] Cleavable linker groups (such as disulfide bonds) can be pH-sensitive. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have a pH of about 5.0 or even more. Some lineages will have linker groups that cleave at a preferred pH, thereby releasing iRNA agents from intracellular ligands (e.g., targeting or cell-permeable ligands, such as cholesterol) or into desired compartments of the cell.

[0182] The chemical junction (e.g., the linker group) connecting the ligand to the iRNA agent can include a disulfide bond. When the iRNA / ligand complex is absorbed into the cell via endocytosis, the acidic environment of the endosome causes the disulfide bond to cleave, 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 targeting ligand or a second therapeutic agent that complements the therapeutic effect of the iRNA agent.

[0183] The ligand may include a linker group that can be cleaved by a specific enzyme. The type of linker group incorporated into the ligand depends on the cell type targeted by the iRNA agent. For example, an iRNA agent targeting mRNA in hepatocytes may be conjugated to a ligand including an ester group. Hepatocytes are rich in esterases, and therefore the ligand will cleave more efficiently in hepatocytes than in cell types that are not rich in esterases. Cleavage of the ligand releases the iRNA agent from the ligand attached to the distal end of the ligand, potentially enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include cells of the lung, renal cortex, and testes.

[0184] Peptide-containing chains can be conjugated to iRNA agents that target peptidase-rich cell types, such as hepatocytes and synovial cells. For example, iRNA agents targeting synovial cells, such as those used to treat inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to peptide-containing chains.

[0185] Generally, the suitability of a candidate cleavable linker can be assessed by testing its ability to cleave the candidate linker group by a degrading agent (or condition). It will also be desirable to additionally test the candidate cleavable linker group's resistance to cleavage in blood or when exposed to other non-target tissues (e.g., tissues to which the iRNA agent will be exposed when administered to a subject). Thus, a relative susceptibility to cleavage can be determined between a first and a second condition, wherein the first condition is selected to indicate cleavage in target cells, and the second condition is selected to indicate cleavage in other tissues or biological fluids (e.g., blood or serum). Evaluation can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. Initial evaluation in cell-free or culture conditions followed by confirmation through further evaluation in whole animals can be useful. In preferred embodiments, the useful candidate compound cleaves at least 2, 4, 10, or 100 times faster in cells (or in vitro conditions selected to simulate intracellular conditions) than in blood or serum (or in vitro conditions selected to simulate extracellular conditions).

[0186] Redox cleavable linker groups

[0187] One class of cleavable linkers is the redox-cleavable linker, which cleaves upon reduction or oxidation. An example of a redox-cleavable linker is a disulfide linker (-SS-). The methods described herein can be considered to determine whether a candidate cleavable linker is a suitable “redox-cleavable linker” or, for example, whether it is suitable for use with a specific iRNA moiety and a specific target agent. For example, a candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which simulates the cleavage rate that would be observed in cells (e.g., target cells). Candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In a preferred embodiment, the candidate compound cleaves in blood by up to 10%. In a preferred embodiment, the useful candidate compound degrades in cells (or under in vitro conditions selected to simulate intracellular conditions) at least 2, 4, 10, or 100 times faster than in blood (or under in vitro conditions selected to simulate extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to simulate intracellular media and compared with conditions selected to simulate extracellular media.

[0188] Phosphate-based cleavable linker groups

[0189] Phosphate-based linker groups are cleaved by agents that degrade or hydrolyze phosphate esters. Examples of agents that cleave phosphate ester groups in cells are cellular enzymes, such as phosphatases. Examples of phosphate-based linker groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —O—P(S)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods similar to those described above.

[0190] Acids can cleave linker groups

[0191] Acid-cleavable linkers are linkers that cleave under acidic conditions. In preferred embodiments, acid-cleavable linkers are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or by agents such as enzymes that can act as general acids. In cells, specific low-pH organelles, such as endosomes and lysosomes, can provide a cleavable environment for acid-cleavable linkers. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acid-cleavable linkers may have the general formula -C═NN-, C(O)O, or -OC(O). Preferred embodiments are when the carbon atom (alkoxy group) attached to the ester is an aryl, substituted alkyl, or tertiary alkyl group, such as dimethylpentyl or tert-butyl. These candidates can be evaluated using methods similar to those described above.

[0192] Based on ester linking groups

[0193] Ester-based linker groups are cleaved by enzymes in the cell, such as esterases and amidases. Examples of cleavable ester-based linker groups include, but are not limited to, alkylene, alkenene, and ynylene esters. Ester-cleavable linker groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0194] Based on peptide cleavage groups

[0195] Peptide-based linkers are cleaved by enzymes in the cell, such as peptidases and proteases. Cleavable peptide-based linkers are peptide bonds that form between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Cleavable peptide-based linkers do not include amide groups (-C(O)NH-). Amide groups can form between any alkylene, alkenylene, or alkyne groups. Peptide bonds are a special type of amide bond that forms between amino acids to produce peptides and proteins. Cleavable peptide-based linkers are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins, and do not include the entire amide functional group. Cleavable peptide linkers have the general formula -NHCHR. 1 C(O)NHCHR 2 C(O)-, where R 1 and R 2 These are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0196] Biodegradable connectors / chains

[0197] Linkers may also include biolytic linkers, which are nucleotide and non-nucleotide linkers or combinations thereof, that connect two parts of a molecule, such as one or both strands of two separate siRNA molecules to produce a bi(siRNA). In some embodiments, only the electrostatic or stacking interaction between two separate siRNAs may represent a linker. Non-nucleotide linkers include chainlets or linkers derived from monosaccharides, disaccharides, oligosaccharides and their derivatives, aliphatic, alicyclic, heterocyclic and combinations thereof.

[0198] In some embodiments, at least one of the linkers (tethers) is a biolytic linker selected from the group consisting of: DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose and mannose, and combinations thereof.

[0199] In one embodiment, the biolytically cleavable carbohydrate linker may have 1 to 10 sugar units having at least one mutarotation isomerate linker capable of connecting two siRNA units. When two or more sugars are present, these units may be linked via 1-3, 1-4, or 1-6 sugar bonds or via an alkyl chain.

[0200] Exemplary biodegradable connectors include:

[0201]

[0202]

[0203]

[0204] Further discussion on biodegradable linkers can be found in PCT application No. PCT / US 18 / 14213, filed January 18, 2018, entitled “Endosomal Cleavable Linkers,” the contents of which are incorporated herein by reference in their entirety.

[0205] carrier

[0206] In some embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to an iRNA agent via a non-ribose substitution unit (i.e., a vector) that replaces one or more nucleotides.

[0207] The carrier can be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinoneyl, tetrahydrofuranyl, and decahydronaphthalene. In one embodiment, the acyclic group is a portion based on the serine alcohol backbone or the diethanolamine backbone.

[0208] The vector can replace one or more nucleotides of the double-stranded iRNA agent.

[0209] In some embodiments, the vector replaces one or more nucleotides at internal positions of the double-stranded iRNA agent.

[0210] In other embodiments, the carrier replaces the nucleotide at the end of the sense or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby acting as a cap protecting the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for example, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinoneyl, tetrahydrofuranyl, or decahydronaphthyl.

[0211] The ribonucleotide subunit in which the ribose has been replaced is referred to herein as a ribose substitution modified subunit (RRMS). The carrier can be a cyclic or acyclic moiety and includes two “main chain attachment sites” (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). The lipophilic moiety can be directly attached to the carrier or indirectly attached to the carrier via an inserted linker / tether, as described above.

[0212]

[0213] The ligand-conjugated monomeric subunit can be the 5' or 3' terminal subunit of the iRNA molecule, meaning 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 monomeric subunit can occupy an internal position, and both "W" groups can be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomeric subunit may be present in the iRNA.

[0214] Monomers based on sugar substitution, such as ligand-conjugated monomers (cyclic).

[0215] Sugar-substituted cyclic monomers, such as sugar-substituted ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The support may have the general formula (LCM-2) provided below (in this structure, the preferred main chain attachment site may be selected from R...). 1 or R 2 ;R 3 or R 4 Or if Y is CR 9 R 10 Then R 9 and R 10 (Choose two locations to give two main chain attachment points, for example, R) 1 and R 4 or R 4 and R 9 Preferred tether attachment points include R. 7 When X is CH2, R 5 or R 6 The carrier is described below as an entity that can be incorporated into the chain. Therefore, it should be understood that this structure also covers cases where one (in the case of an end position) or two (in the case of an internal position) attachment points, such as R. 1 or R 2 ;R 3 or R 4 ; or R 9 or R 10 (when Y is CR) 9 R 10(At the time), it is linked to, for example, a sulfur-containing phosphate ester or a modified phosphate ester. For example, one of the above R groups can be -CH2-, with one bond linked to the support and one bond linked to a main chain atom (e.g., linking an oxygen or central phosphorus atom).

[0216]

[0217] in:

[0218] X is N(CO)R 7 NR 7 Or CH2;

[0219] Y is NR 8 O, S, CR 9 R 10 ;

[0220] Z is CR 11 R 12 or does not exist;

[0221] R 1 R 2 R 3 R 4 R 9 and R 10 Each of them is independently H, OR a or (CH2) n OR b The condition is R 1 R 2 R 3 R 4 R 9 and R 10 At least two of them are OR a And / or (CH2) n OR b ;

[0222] R 5 R 6 R 11 and R 12 Each of these components is independently a ligand, H, and optionally via 1-3 R... 13 Substituted C1-C6 alkyl groups, or C(O)NHR 7 ; or R 5 With R 11 Together for optional land R 14 Substituted C3-C8 cycloalkyl groups;

[0223] R 7 It can be a ligand, such as R 7 It can be R d , or R 7This could be, for example, via a link (e.g., via NR). c R d Replacement C1-C 20 Alkyl groups) are indirectly attached to the support as ligands; or via NHC(O)R d Replacement C1-C 20 alkyl;

[0224] R 8 It is H or C1-C6 alkyl;

[0225] R 13 It is a hydroxyl, C1-C4 alkoxy, or halogen group;

[0226] R 14 NR c R 7 ;

[0227] R 15 It is optionally a C1-C6 alkyl group substituted with a cyano group, or a C2-C6 alkenyl group;

[0228] R 16 For C1-C 10 alkyl;

[0229] R 17 It is a liquid-phase or solid-phase supporting reagent;

[0230] L stands for -C(O)(CH2). q C(O)- or -C(O)(CH2) q S-;

[0231] R a Protecting groups, such as CAr3; (e.g., dimethoxytriphenylmethyl) or Si(X) 5' (X) 5″ (X) 5″′ ), where (X) 5' ), (X 5″ ) and (X 5″′ As described elsewhere.

[0232] R b For P(O)(O) - H, P(OR) 15 )N(R 16 )2 or LR 17 ;

[0233] R c It is H or C1-C6 alkyl;

[0234] R d For H or ligands;

[0235] Each Ar is independently a C6-C alkoxy-substituted C1-C4 alkoxy group. 10 aryl;

[0236] n is 1-4; and q is 0-4.

[0237] Exemplary carriers include those where, for example, X is N(CO)R 7 or NR 7 Y is CR 9 R 10 And Z does not exist; or X is N(CO)R 7 or NR 7 Y is CR 9 R 10 And Z is CR 11 R 12 ; or X is N(CO)R 7 or NR 7 Y is NR 8 And Z is CR 11 R 12 ; or X is N(CO)R 7 or NR 7 Y is O and Z is CR 11 R 12 Or X is CH2; Y is CR 9 R 10 Z is CR 11 R 12 And R 5 With R 11 Together they form a C6 cycloalkyl (H, z=2) or indene ring system, for example, X is CH2; Y is CR 9 R 10 Z is CR 11 R 12 And R 5 With R 11 Together they form a C5 cycloalkyl group (H, z=1).

[0238] In some embodiments, the carrier may be based on a pyrrolidone ring system or a 4-hydroxyproline ring system, for example, X is N(CO)R 7 or NR 7 Y is CR 9 R 10 And Z does not exist (D). OFG 1 Preferably attached to a primary carbon bonded to one of the carbons in a five-membered ring, such as an exocyclic alkylene group, for example, methylene (in the form of -CH2OFG). 1 OFG 2 Preferably, it is directly attached to one carbon in the five-membered ring (-OFG in D). 2For pyrrolidine-based supports, -CH2OFG 1 It can be attached to C-2 and OFG 2 It can be attached to C-3; or -CH2OFG 1 It can be attached to C-3 and OFG 2 It can be attached to C-4. In some embodiments, CH2OFG 1 and OFG 2 It can be replaced by a carbon atom mentioned above via a homologous substitution. For 3-hydroxyproline-based supports, -CH2OFG 1 It can be attached to C-2 and OFG 2 It can attach to C-4. Monomers based on pyrroline and 4-hydroxyproline can therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation around that particular bond is restricted, for example, due to the restriction arising from the presence of a ring. Therefore, CH2OFG 1 and OFG 2 In any pairing described above, they may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more asymmetric centers and thus exist as racemic mixtures and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of the monomer (e.g., those with CH2OFG) are explicitly included. 1 and OFG 2 All centers may have an R configuration; or all may have an S configuration; or one center may have an R configuration and the other center may have an S configuration, or vice versa. The chain attachment site is preferably nitrogen. Preferred examples of the support D include the following:

[0239]

[0240] In some embodiments, the carrier may be based on a piperidine ring system (E), for example, X is N(CO)R 7 or NR 7 Y is CR 9 R 10 And Z is CR 11 R 12 . OFG 1 Preferably, it is attached to a primary carbon bonded to one carbon in a six-membered ring, such as an exocyclic alkylene group, for example, methylene (n=1) or ethylene (n=2) [in E-(CH2)]. n OFG 1 ]. OFG 2 Preferably, it is directly attached to one carbon in a six-membered ring (-OFG in E). 2 ). -(CH2) n OFG 1 and OFG2 They can be arranged in a homologous manner on the ring, meaning that two groups can be attached to the same carbon atom, such as C-2, C-3, or C-4. Alternatively, -(CH2) n OFG 1 and OFG 2 They can be positioned in an adjacent manner on the ring, meaning that two groups can be attached to adjacent ring carbon atoms, for example, -(CH2). n OFG 1 It can be attached to C-2 and OFG 2 It can be attached to C-3; -(CH2) n OFG 1 It can be attached to C-3 and OFG 2 It can be attached to C-2; -(CH2) n OFG 1 It can be attached to C-3 and OFG 2 It can be attached to C-4; or -(CH2). n OFG 1 It can be attached to C-4 and OFG 2 It can attach to C-3. Piperidine-based monomers can therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation around that particular bond is restricted, for example, due to the restriction arising from the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 In any pairing described above, they may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more asymmetric centers and thus exist as racemic mixtures and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of the monomer (e.g., those with CH2OFG) are explicitly included. 1 and OFG 2 All centers may have an R configuration; or all may have an S configuration; or one center may have an R configuration and the other center may have an S configuration, or vice versa. The chain attachment point is preferably nitrogen.

[0241] In some embodiments, the carrier may be based on a piperazine ring system (F), for example, X is N(CO)R 7 or NR 7 Y is NR 8 And Z is CR 11 R 12 Or the morpholine ring system (G), for example, X is N(CO)R 7 or NR 7 Y is O and Z is CR 11 R 12 . OFG1 Preferably attached to a primary carbon bonded to a carbon in a six-membered ring, such as an exocyclic alkylene group, for example, a methylene group (-CH2OFG in F or G). 1 OFG 2 Preferably, it is directly attached to one carbon atom in a six-membered ring (-OFG in F or G). 2 For both F and G, -CH2OFG 1 It can be attached to C-2 and OFG 2 It can be attached to C-3; or vice versa. In some embodiments, CH2OFG 1 and OFG 2 It can be replaced by a carbon atom mentioned above via a homologous substitution. Monomers based on piperazine and morpholine can therefore contain bonds (e.g., carbon-carbon bonds) in which bond rotation is restricted around that particular bond, for example, due to the restriction arising from the presence of a ring. Thus, CH2OFG 1 and OFG 2 In any pairing described above, they may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more asymmetric centers and thus exist as racemic mixtures and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of the monomer (e.g., those with CH2OFG) are explicitly included. 1 and OFG 2 All centers may have an R configuration; or all may have an S configuration; or one center may have an R configuration and the other center may have an S configuration, or vice versa. R″′ may be, for example, a C1-C6 alkyl group, preferably CH3. The chain attachment point is preferably nitrogen from F and G.

[0242] In some embodiments, the support may be based on a decahydronaphthalene ring system, for example, X is CH2; Y is CR 9 R 10 Z represents CR 11 R 12 , and R 5 With R 11 Together they form a C6 cycloalkyl (H, z=2) or indene ring system, for example, X is CH2; Y is CR 9 R 10 Z represents CR 11 R 12 , and R 5 With R 11 Together they form a C5 cycloalkyl group (H, z=1). OFG 1 Preferably attached to a primary carbon, such as an exocyclic methylene (n=1) or ethylene (n=2) [H in -(CH2)] linked to one of C-2, C-3, C-4, or C-5.n OFG 1 OFG 2 Preferably, it is directly attached to one of C-2, C-3, C-4 or C-5 (H in -OFG). 2 ). -(CH2) n OFG 1 and OFG 2 They can be arranged in a homologous manner on the ring, meaning that two groups can be attached to the same carbon atom, such as C-2, C-3, C-4, or C-5. Alternatively, -(CH2) n OFG 1 and OFG 2 They can be positioned in an adjacent manner on the ring, meaning that two groups can be attached to adjacent ring carbon atoms, for example, -(CH2). n OFG 1 It can be attached to C-2 and OFG 2 It can be attached to C-3; -(CH2) n OFG 1 It can be attached to C-3 and OFG 2 It can be attached to C-2; -(CH2) n OFG 1 It can be attached to C-3 and OFG 2 It can be attached to C-4; or -(CH2). n OFG 1 It can be attached to C-4 and OFG 2 It can be attached to C-3; -(CH2) n OFG 1 It can be attached to C-4 and OFG 2 It can be attached to C-5; or -(CH2). n OFG 1 It can be attached to C-5 and OFG 2 It can attach to C-4. Monomers based on decahydronaphthalene or indene may therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation around that particular bond is restricted, for example, due to the restriction arising from the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 In any pairing described above, they may be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more asymmetric centers and thus exist as racemic mixtures and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of the monomer (e.g., those with CH2OFG) are explicitly included. 1 and OFG 2All centers may have an R configuration; or all may have an S configuration; or one center may have an R configuration and the other center may have an S configuration, or vice versa. In a preferred embodiment, the substituents at C-1 and C-6 are trans-represented relative to each other. The chain attachment point is preferably C-6 or C-7.

[0243] Other vectors may include 3-hydroxyproline-based vectors (J). Therefore, -(CH2) n OFG 1 and OFG 2 They can be cis or trans relative to each other. Therefore, all cis / trans isomers are explicitly included. Monomers may also contain one or more asymmetric centers and thus exist as racemic mixtures and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of the monomer (e.g., those with CH2OFG) are explicitly included. 1 and OFG 2 All centers may have an R configuration; or all may have an S configuration; or one center may have an R configuration and the other center may have an S configuration, or vice versa. The chain attachment point is preferably nitrogen.

[0244] Details of more representative sugar-replacement-based cyclic vectors can be found in U.S. Patent Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0245] Monomers (acyclic) based on sugar substitution

[0246] Sugar-substituted acyclic monomers, such as ligand-conjugated monomers based on sugar substitution, are also referred to herein as ribose-substituted monomer subunits (RRMS) monomer compounds. Preferred acyclic supports may have the formula LCM-3 or LCM-4:

[0247]

[0248] In some embodiments, each of x, y, and z can be independently 0, 1, 2, or 3. In formula LCM-3, when y differs from z, the tertiary carbon can have an R or S configuration. In preferred embodiments, x in formula LCM-3 is zero and y and z are each 1 (e.g., based on serinel), and y and z in formula LCM-3 are each 1. Each of the following formulas LCM-3 or LCM-4 can optionally be substituted, for example, with a hydroxyl, alkoxy, or perhaloalkyl group.

[0249] Details of more representative sugar-substituted acyclic vectors can be found in U.S. Patent Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0250] In some embodiments, the compound comprises one or more lipophilic monomers having a lipophilic moiety conjugated to the 5' end of the sense chain or the 5' end of the antisense chain.

[0251] In some embodiments, the lipophilic monomer comprises a lipophilic portion conjugated to the 5' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic monomer comprises a lipophilic portion conjugated to the 5' end of the chain via a carrier having the following formula: R represents a ligand, such as the lipophilic moiety.

[0252] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the 3' end of the sense chain or the 3' end of the antisense chain.

[0253] In some embodiments, the lipophilic monomer contains a lipophilic portion conjugated to the 3' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic monomer contains a lipophilic portion conjugated to the 3' end of the chain via a carrier having the following formula: R represents a ligand, such as the lipophilic moiety.

[0254] In some embodiments, the lipophilic monomer contains a lipophilic portion attached to an internal position of the chain via a carrier and / or a linker. In one embodiment, the lipophilic monomer contains a lipophilic portion attached to an internal position of the chain via a carrier having the following formula: R represents a ligand, such as the lipophilic moiety.

[0255] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the ends of the sense chain.

[0256] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the ends of the antisense chain.

[0257] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to internal positions of the sense or antisense strand. In some embodiments, one or more lipophilic moieties are conjugated to ribose, nucleotides, and / or nucleotides via inter-bonds. In some embodiments, one or more lipophilic moieties are conjugated to ribose at the 2', 3', 4', and / or 5' positions. In some embodiments, one or more lipophilic moieties are conjugated to native nucleotides (such as A, T, G, C, or U) or modified nucleotides as defined herein. In some embodiments, one or more lipophilic moieties are conjugated to phosphate groups or modified phosphate groups as defined herein.

[0258] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the 5' or 3' end of the sense chain, and one or more lipophilic monomers having one or more lipophilic moieties conjugated to the 5' or 3' end of the antisense chain.

[0259] In some embodiments, the lipophilic monomer contains a lipophilic portion attached to the end of the chain via one or more connectors (chain links) and / or a carrier.

[0260] In one embodiment, the lipophilic monomer contains a lipophilic portion attached to the end of the chain via one or more connectors (chain links).

[0261] In one embodiment, the lipophilic monomer contains a lipophilic portion that is attached to the 5' end of the sense or antisense chain via a cyclic carrier, optionally via one or more insert connectors (tethers).

[0262] In some embodiments, at least one lipophilic monomer is located at one or more terminal positions of the sense or antisense strand. In one embodiment, at least one lipophilic monomer is located at the 3' or 5' end of the sense strand. In one embodiment, at least one lipophilic monomer is located at the 3' or 5' end of the antisense strand.

[0263] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to one or more internal positions on at least one chain. An internal position on the chain refers to a nucleotide at any position on the chain, excluding the 3' and 5' end positions (e.g., excluding two positions: position 1 counting from the 3' end and position 1 counting from the 5' end).

[0264] In one embodiment, at least one lipophilic monomer is located at one or more internal locations on at least one chain, including all locations except the two terminal locations at each end of the chain (e.g., excluding four locations: locations 1 and 2 counting from the 3' end and locations 1 and 2 counting from the 5' end). In another embodiment, the lipophilic monomer is located at one or more internal locations on at least one chain, including all locations except the three terminal locations at each end of the chain (e.g., excluding six locations: locations 1, 2, and 3 counting from the 3' end and locations 1, 2, and 3 counting from the 5' end).

[0265] In one embodiment, at least one lipophilic monomer is located at one or more positions at at least one end of the double-stranded region, including all positions within the double-stranded region, but excluding overhanging regions or vectors that replace the terminal nucleotide at the 3' end of the sense strand.

[0266] In one embodiment, at least one lipophilic monomer is located on the sense strand within the first five, four, three, two, or one base pair at the 5' end of the antisense strand in the double-stranded region.

[0267] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, excluding the cleavage site region of the sense chain. For example, the lipophilic monomer is not located at positions 9-12 counting from the 5' end of the sense chain, or positions 9-11 counting from the 5' end of the sense chain. Alternatively, internal positions do not include positions 11-13 counting from the 3' end of the sense chain.

[0268] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, excluding the cleavage site region of the antisense strand. For example, internal positions do not include positions 12-14 counted from the 5' end of the sense strand.

[0269] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, excluding positions 11-13 on the sense chain counting from the 3' end and positions 12-14 on the antisense chain counting from the 5' end.

[0270] In one embodiment, 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 chain, counting from the 5' end of each chain, and positions 6-10 and 15-18 on the antisense chain.

[0271] In one embodiment, 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, counting from the 5' end of each strand, and positions 15 and 17 on the antisense strand.

[0272] definition

[0273] Unless specifically defined, the nomenclature, methods, and techniques used in this document in conjunction with molecular biology, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry are those well-known and commonly used in the field. Standard techniques are applicable to chemical synthesis and chemical analysis. Some of these techniques and methods can be found, for example, in “Carbohydrate Modifications in Antisense Research” edited by Sangvi and Cook, American Chemical Society, Washington, 1994; “Remington’s Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., 18th ed., 1990; and “Antisense Drug Technology, Principles, Strategies, and Applications” edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla., Boca Raton, Florida; and Sambrook et al., “Molecular Cloning, A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for any purpose. Where permitted, all patents, applications, publications and other publications and data cited throughout this disclosure are incorporated herein by reference in their entirety.

[0274] 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 target proteins (including, but not limited to, mRNA precursors and mRNAs or portions thereof), cDNA derived from such RNA, and miRNAs. For example, a target nucleic acid can be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a specific disorder or disease state. In some embodiments, a target nucleic acid can be a nucleic acid molecule derived from an infectious agent.

[0275] As used herein, the term "iRNA" refers to agents that mediate targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic polyprotein complex called the RNAi-induced silencing complex (RISC). Agents that effectively induce RNA interference are also referred to herein as siRNA, RNAi agents, or iRNA agents. Therefore, these terms are used interchangeably herein. As used herein, the term iRNA includes microRNAs and microRNA precursors. Furthermore, as used herein, the term "compound" of the present invention also refers to iRNA agents and is used interchangeably with iRNA agents.

[0276] iRNA agents should include regions that are sufficiently homologous to the target gene and of sufficient nucleotide length to mediate downregulation of the target gene. (For clarity, the terms nucleotide or ribonucleotide are sometimes used herein to refer to one or more monomeric subunits of the iRNA agent. It should be understood herein that the terms “ribonucleotide” or “nucleotide” can be used in the case of modified RNA or nucleotide substitutions, and also to modified nucleotides or substitutions 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 the target RNA. Perfect complementarity between the iRNA agent and the target is not required, but the correspondence must be sufficient to enable the iRNA agent or its cleavage product to direct sequence-specific silencing, e.g., by cleaving the target RNA, such as mRNA, via RNAi. The degree of complementarity or homology with the target strand is most critical in the antisense strand. While perfect complementarity is generally required, particularly in the antisense strand, some embodiments may include, particularly in the antisense strand, one or more, or for example, six, five, four, three, two, or fewer mismatches (relative to the target RNA). The sense chain only needs to be fully complementary to the antisense chain to maintain the overall double-stranded characteristic of the molecule.

[0277] iRNA agents include: molecules long enough to trigger an interferon response (these molecules can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter the RISC (RNAi-inducible silencing complex)); and molecules short enough not to trigger an interferon response (these molecules can also be cleaved by Dicer and / or enter the RISC), such as molecules of a size that allows entry into the RISC, such as molecules similar to Dicer cleavage products. Molecules short enough not to trigger an interferon response are referred to herein as siRNA agents or shorter iRNA agents. As used herein, “siRNA agent or shorter iRNA agent” means an iRNA agent short enough not to induce a harmful interferon response in human cells, such as a double-stranded RNA agent or a single-stranded agent, for example, having a double-stranded region of fewer than 60, 50, 40, or 30 nucleotide pairs. siRNA agents or their cleavage products can downregulate target genes, for example by inducing RNAi relative to a target RNA, where the target may contain endogenous or pathogenic target RNA.

[0278] As used herein, a “single-stranded iRNA agent” is an iRNA agent consisting of a single molecule. It may include a double-stranded region formed by intrastrand pairing, for example, it may be or include a hairpin or pan-handle structure. The single-stranded iRNA agent may be antisense relative to the target molecule. The single-stranded iRNA agent may be long enough for it to enter RISC and participate in RISC-mediated cleavage of the target mRNA. The length of the single-stranded iRNA agent is at least 14, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides. In some embodiments, its length is less than 200, 100, or 60 nucleotides.

[0279] A loop refers to a region in the iRNA chain where the iRNA strand is not paired with a relative nucleotide in the duplex when a part of the iRNA strand forms a base pair with another strand or another part of the same strand.

[0280] The hairpin iRNA agent will have a double-stranded region of equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the double-stranded region may be equal to or less than 200, 100, or 50. In some embodiments, the length of the double-stranded region ranges from 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs. The hairpin may have a single-stranded overhang or a terminal unpaired region, in some embodiments at 3', and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhang is 2-3 nucleotides long.

[0281] As used in this article, "double-stranded (ds)iRNA agent" refers to an iRNA agent that includes more than one strand, and in some cases two strands, wherein interstrand hybridization can form a double-stranded structural region.

[0282] As used in this article, the terms “siRNA activity” and “RNAi activity” refer to gene silencing via siRNA.

[0283] As used herein, “gene silencing” by RNA interference molecules refers to a reduction in the cellular mRNA level of a target gene that is 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% up to and including 100%, and any integer between therebetween. In a preferred embodiment, the mRNA level is reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% up to and including 100%, and any integer between 5% and 100%.

[0284] As used herein, the term “regulation of gene expression” means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated such that the expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term “regulation” can mean “inhibition,” but the use of the word “regulation” is not limited to this definition.

[0285] As used herein, gene expression regulation occurs when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, 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 in the absence of siRNA (e.g., an RNAi agent). The percentage and / or fold differences can be calculated relative to a control or non-control, for example,

[0286]

[0287] or

[0288]

[0289] As used herein, the terms “inhibition,” “downregulation,” or “reduction” in relation to gene expression mean a reduction in gene expression, or the level of an RNA molecule or equivalent encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, to levels lower than those observed in the absence of a regulator. Gene expression is downregulated when gene expression, or the level of an RNA molecule or equivalent encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced by at least 10% relative to the corresponding unregulated control, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or best 100% (i.e., no gene expression).

[0290] As used herein, the terms “increase” or “upregulation” in relation to gene expression mean an increase in gene expression, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, above what would be observed in the absence of a regulator. Gene expression is upregulated when gene expression, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased by at least 10% relative to the corresponding unregulated control, and preferably by 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.

[0291] As used herein, the term “increased” generally means an increase that is statically significant; for the avoidance of any doubt, “increased” means an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or greater compared to a reference level.

[0292] As used herein, the term “reduced” generally refers to a reduction in a statistically significant amount. However, to avoid ambiguity, “reduction” means a reduction of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% (i.e., no level compared to the reference sample), or any reduction between 10% and 100%.

[0293] Double-stranded iRNA comprises two oligonucleotide chains that are fully complementary to hybridize and form a double-stranded structure. Typically, the length of the double-stranded structure is 15 to 30 base pairs, more typically 18 to 25, even more typically 19 to 24, and most typically 19 to 21 base pairs. In some embodiments, longer double-stranded iRNAs of 25 to 30 base pairs are preferred. In some embodiments, shorter double-stranded iRNAs of 10 to 15 base pairs are preferred. In another embodiment, the double-stranded iRNA is at least 21 nucleotides long.

[0294] In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a complementary region that is complementary to at least a portion of the target sequence, and the length of the double-stranded region is 14-30 nucleotides. Similarly, the length of the complementary region of the target sequence is 14 to 30 nucleotides, more typically 18 to 25 nucleotides, even more typically 19 to 24 nucleotides, and most typically 19 to 21 nucleotides.

[0295] As used herein, the term "compound" refers to an oligomeric compound, which may be an oligonucleotide, antisense, or iRNA agent, such as siRNA.

[0296] As used herein, the phrase “antisense chain” refers to an oligomer that is substantially or 100% complementary to the target sequence of interest. The phrase “antisense chain” includes the antisense regions of two oligomers formed by two separate chains, as well as monomeric oligomers capable of forming hairpin or dumbbell-shaped structures. The terms “antisense chain” and “guide chain” are used interchangeably herein.

[0297] The phrase “sense chain” refers to an oligomeric compound having a nucleoside sequence that is wholly or partially identical to a target sequence (such as a messenger RNA or DNA sequence). The terms “sense chain” and “lackey chain” are used interchangeably herein.

[0298] The terms "specific hybridization" and "complementarity" imply that nucleic acids can form hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-conventional methods. Regarding the nuclear molecule of this invention, the binding free energy of the nucleic acid molecule and its complementary sequence is sufficient to allow for the relevant functions of the nucleic acid, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. [Cold Spring Harbor Quantitative Biology Symposium] LII, pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 83:9373-9377; Turner et al., 1987, / . Am. Chem. Soc. [Journal of the American Chemical Society] 109:3783-3785). Percentage complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementarity for 5, 6, 7, 8, 9, and 10 out of 10). "Perfect complementarity" or 100% complementarity means that all consecutive residues in the nucleic acid sequence will bind to the same number of consecutive residues in the second nucleic acid sequence via hydrogen bonds. Imperfect complementarity refers to a situation where some, but not all, nucleoside units in the two chains can bind to each other via hydrogen bonds. "Significant complementarity" means that the polynucleotide chain exhibits 90% or higher complementarity, excluding regions of the polynucleotide chain that are selected to be non-complementary, such as overhangs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomer to the non-target sequence under conditions requiring specific binding, i.e., under physiological conditions in in vivo assays or therapeutic applications, or under the conditions under which the assay is performed in vitro. Non-target sequences typically differ by at least 5 nucleotides.

[0299] In some embodiments, the length of the double-stranded region of the compound is equal to or at least 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.

[0300] In some embodiments, the length of the antisense chain of the compound is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0301] In some embodiments, the length of the sense chain of the compound is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0302] In one embodiment, the length of the sense strand and the antisense strand of the compound is each 15 to 30 nucleotides.

[0303] In one embodiment, the length of the sense strand and the antisense strand of the compound is each 19 to 25 nucleotides.

[0304] In one embodiment, the length of the sense strand and the antisense strand of the compound is each 21 to 23 nucleotides.

[0305] In some embodiments, one strand has at least one to five single-stranded nucleotides in the double-stranded region. A “single-stranded nucleotide segment in the double-stranded region” means that at least one nucleotide base pair is present at both ends of a monoblock. In some embodiments, both strands have at least one to five (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. When both strands have one to five (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides may be opposite to each other (e.g., a mismatch) or may be positioned such that the second strand does not have single-stranded nucleotides opposite to those of the single-stranded iRNA of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, single-stranded nucleotides are present within eight nucleotides at either end, such as eight, seven, six, five, four, three, or two nucleotides at the 5' or 3' end of the complementary region between the two strands.

[0306] In one embodiment, the compound includes a single-stranded overhang at at least one of the plurality of ends. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0307] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides long and the antisense strand is 23 nucleotides long, wherein these strands form a double-stranded region of 21 consecutive base pairs (with a single-stranded overhang of 2 nucleotides at the 3' end).

[0308] In some embodiments, the strands of the double-stranded iRNA have a ZXY structure, as described in PCT Publication No. 2004080406, which is incorporated herein by reference in its entirety.

[0309] In certain embodiments, the two chains of the double-stranded oligomer can be linked together. The two chains can be linked to each other at both ends, or only at one end. Linking at one end means that the 5' end of the first chain is linked to the 3' end of the second chain, or the 3' end of the first chain is linked to the 5' end of the second chain. When the two chains are linked to each other at both ends, the 5' end of the first chain is linked to the 3' end of the second chain, and the 3' end of the first chain is linked to the 5' end of the second chain. The two chains can be linked together by an oligonucleotide linker, which includes, but is not limited to, (N) n N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiments, n is 3-10, such as 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 nucleotides in the linker may be involved in base pair interactions with other nucleotides in the linker. Two chains can also be linked together by non-nucleoside linkers, such as those described herein. Those skilled in the art will understand that any oligonucleotide chemical modifications or variations described herein can be used for oligonucleotide linkers.

[0310] The hairpin and dumbbell-shaped oligomers have a double-stranded region of 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the double-stranded region may be equal to or less than 200, 100, or 50. In some embodiments, the length of the double-stranded region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs.

[0311] Hairpin oligomers may have single-stranded overhangs or terminal unpaired regions, in some embodiments at 3', and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhang is 1-4, more typically 2-3 nucleotides long. Hairpin oligomers that can induce RNA interference are also referred to herein as "shRNA".

[0312] In some embodiments, the two oligomeric chains specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to the non-target nucleic acid sequence under conditions requiring specific binding, i.e., under physiological conditions in vivo assays or therapeutic treatments, and under conditions for assays performed in vitro.

[0313] As used herein, “strict hybridization conditions” or “strict conditions” refer to the conditions under which an antisense compound will hybridize with its target sequence but with a minimum number of other sequences. Strict conditions are sequence-dependent and vary under different conditions, and the “strict conditions” for an antisense compound to hybridize with its target sequence are determined by the properties and composition of the antisense compound and the measurements performed on it.

[0314] It should be understood in the art that incorporation of nucleotide affinity modifications allows for a greater number of mismatches compared to unmodified compounds. Similarly, some oligonucleotide sequences may be more tolerant of mismatches than others. Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, such as by measuring the melting temperature (Tm). Tm or ΔTm can be calculated using techniques familiar to those skilled in the art. For example, the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to assess the ability of nucleotide modifications to increase the melting temperature of the RNA:DNA duplex.

[0315] siRNA design

[0316] In one embodiment, the iRNA agent is a diterminate having a length of 19 nt, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 starting from the 5' end. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 starting from the 5' end.

[0317] In one embodiment, the iRNA agent is a double-ended blunt strand having a length of 20 nt, wherein the sense strand contains at least one motif at positions 8, 9, and 10 from the 5' end, having three 2'-F modifications on three consecutive nucleotides. The antisense strand contains at least one motif at positions 11, 12, and 13 from the 5' end, having three 2'-O-methyl modifications on three consecutive nucleotides.

[0318] In one embodiment, the iRNA agent is a double-ended blunt strand having a length of 21 nt, wherein the sense strand contains at least one motif at positions 9, 10, and 11 from the 5' end, having three 2'-F modifications on three consecutive nucleotides. The antisense strand contains at least one motif at positions 11, 12, and 13 from the 5' end, having three 2'-O-methyl modifications on three consecutive nucleotides.

[0319] In one embodiment, the iRNA agent comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; the antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the iRNA is blunt, and the other end contains a 2nt overhang. Preferably, the 2nt overhang is located at the 3' end of the antisense. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0320] In one embodiment, the iRNA agent comprises a sense strand and an antisense strand, wherein: the sense strand is 25-30 nucleotide residues in length, wherein it contains at least 8 ribonucleotides starting from positions 1 to 23 of the 5' terminal nucleotide (position 1) of the first strand; the antisense strand is 36-66 nucleotide residues in length, and, starting from the 3' terminal nucleotide, contains at least 8 ribonucleotides at positions 1-23 of the sense strand to form a double strand; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and at most 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a 1-6 nucleotide 3' single-stranded overhang; wherein the 5' terminal of the antisense strand... It comprises 10-30 consecutive nucleotides unpaired with the sense strand, forming a 10-30 nucleotide single-stranded 5' overhang; wherein, when comparing the maximum complementarity of the sense and antisense strands, at least the 5' and 3' terminal nucleotides of the sense strand pair with the nucleotide bases of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; and when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand is sufficiently complementary to the target RNA along the antisense strand length of at least 19 ribonucleotides to reduce target gene expression; and wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides, wherein at least one motif appears at or near a cleavage site. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at or near a cleavage site.

[0321] In one embodiment, the iRNA agent comprises a sense strand and an antisense strand, wherein the iRNA agent comprises a first strand and a second strand, the first strand having a length of at least 25 and at most 29 nucleotides, and the second strand having a length of at most 30 nucleotides, wherein at positions 11, 12, and 13 from the 5' end, at least one 2'-O-methyl modified motif is present on three consecutive nucleotides; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer than the first strand at its 3' end, wherein the double-stranded region is at least 25 nucleotides long, and when the iRNA agent is introduced into mammalian cells, the second strand is sufficiently complementary to the target mRNA along a length of at least 19 nt to reduce target gene expression, and wherein dicer cleavage of the iRNA preferably produces siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in mammals. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0322] In one embodiment, the sense strand of the iRNA agent contains at least one motif with three identical modifications on three consecutive nucleotides, one of which appears at a cleavage site of the sense strand. For example, the sense strand may contain at least one motif with three 2'-F modifications on three consecutive nucleotides within 7-15 positions at the 5' end.

[0323] In one embodiment, the antisense strand of the iRNA agent may also contain at least one motif with three identical modifications on three consecutive nucleotides, one of which appears at or near the cleavage site of the antisense strand. For example, the antisense strand may contain at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides within 9-15 positions at the 5' end.

[0324] For iRNAs with a double-stranded region of 17-23 nt in length, the cleavage site of the antisense strand is typically near positions 10, 11, and 12 at the 5' end. Therefore, motifs with the same modification can appear at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counted from the first nucleotide at the 5' end of the antisense strand, or from the first paired nucleotide within the double-stranded region at the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the distance of the double-stranded region of the iRNA from the 5' end.

[0325] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs having three identical modifications on three consecutive nucleotides, wherein at least one motif appears at or near a cleavage site within the strand, and at least one motif appears in another portion of the strand (the motif being separated from the motif at the cleavage site by at least one nucleotide). In one embodiment, the antisense strand also contains at least one motif having three identical modifications on three consecutive nucleotides, wherein at least one motif appears at or near a cleavage site within the strand. The modifications in the motifs appearing at or near the cleavage site in the sense strand differ from the modifications in the motifs appearing at or near the cleavage site in the antisense strand.

[0326] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides, wherein at least one motif appears at or near a cleavage site in the strand. In one embodiment, the antisense strand also contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at or near a cleavage site.

[0327] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 at the 5' end, and wherein the antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 at the 5' end.

[0328] In one embodiment, the iRNA agent contains one or more mismatches, or combinations thereof, with the target within the duplex. Mismatches can be present in salient regions or duplex regions. Base pairs can be sorted based on their tendency to promote dissociation or unwinding (e.g., based on the free energy of association or dissociation of a particular pair; the simplest approach is to examine pairings on a single-pair basis, but next-nearest or similar analyses can also be used). Regarding promoting dissociation: A:U is superior to G:C; G:U is superior to G:C; and I:C is superior to G:C (I = inosine). Mismatches (e.g., atypical or non-typical pairings (as described elsewhere herein)) are superior to typical (A:T, A:U, G:C) pairings; and pairings including universal bases are superior to typical pairings.

[0329] In one embodiment, the iRNA agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the 5' end duplex region of the antisense strand, which may be independently selected from the group consisting of A:U, G:U, I:C, and mismatched pairings, such as atypical pairings including universal bases or other than classical pairings, to promote the dissociation of the antisense strand at the 5' end of the duplex.

[0330] In one embodiment, the nucleotide at position 1 of the 5' end double-stranded region of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the 5' end double-stranded region of the antisense strand is an AU base pair. For example, the first base pair in the 5' end double-stranded region of the antisense strand is an AU base pair.

[0331] 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, when 50% of the dsRNA agent is modified, 50% of all nucleotides present in the dsRNA agent contain the modifications described herein.

[0332] In one embodiment, each of the sense and antisense strands is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluorine, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0333] In one embodiment, each of the sense and antisense strands of the dsRNA agent contains at least two different modifications.

[0334] In one embodiment, the dsRNA agent does not contain any 2'-F modification.

[0335] In one embodiment, the sense and / or antisense strand of the dsRNA agent comprises one or more blocks having inter-nucleotide bonds of phosphate thioester or methylphosphonate. In one example, the sense strand comprises one block having two inter-nucleotide bonds of phosphate thioester or methylphosphonate. In one example, the antisense strand comprises two blocks having two inter-nucleotide bonds of phosphate thioester or methylphosphonate. For example, the two blocks having inter-nucleotide bonds of phosphate thioester or methylphosphonate are separated by 16-18 phosphate ester nucleotide bonds.

[0336] In one embodiment, each of the sense and antisense strands of the dsRNA agent has 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0337] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand of 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.

[0338] In one embodiment, the antisense strand of the dsRNA agent is 100% complementary to the target RNA to hybridize with it and inhibit its expression by RNA interference. In another embodiment, the antisense strand of the 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.

[0339] In one aspect, the present invention relates to dsRNA agents as defined herein capable of inhibiting the expression of target genes. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one heat-destabilizing nucleotide, wherein at least one of said heat-destabilizing nucleotides is present at or near a site opposite to the seed region of the antisense strand (i.e., at position 2-8 of the 5' end of the antisense strand).

[0340] When the sense strand is 21 nucleotides long, the heat-destabilized nucleotides can appear, for example, at positions 14-17 at the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids smaller than the spatially restrictive 2'-OMe modification. Preferably, the two modified nucleic acids smaller than the spatially restrictive 2'-OMe are separated by an 11-nucleotide length. For example, the two modified nucleic acids are located at positions 2 and 14 at the 5' end of the antisense strand.

[0341] In one embodiment, the dsRNA agent comprises:

[0342] (a) A meaningful chain that has:

[0343] (i) The length of 18-23 nucleotides;

[0344] (ii) the three consecutive 2'-F modifications at positions 7-15; and

[0345] (b) Antisense chains, which have:

[0346] (i) The length of 18-23 nucleotides;

[0347] (ii) at least 2'-F modification anywhere on the chain; and

[0348] (iii) At least two phosphate thioester nucleotide bonds in the first five nucleotides (counting from the 5' end);

[0349] The dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or both ends of the double strand are blunt.

[0350] In one embodiment, the dsRNA agent comprises:

[0351] (a) A meaningful chain that has:

[0352] (i) The length of 18-23 nucleotides;

[0353] (ii) Fewer than four 2'-F modifiers;

[0354] (b) Antisense chains, which have:

[0355] (i) The length of 18-23 nucleotides;

[0356] (ii) fewer than twelve 2'-F modifiers; and

[0357] (iii) At least two phosphate thioester nucleotide bonds in the first five nucleotides (counting from the 5' end);

[0358] The dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or both ends of the double strand are blunt.

[0359] In one embodiment, the dsRNA agent comprises:

[0360] (a) A meaningful chain that has:

[0361] (i) The length of 19-35 nucleotides;

[0362] (ii) Fewer than four 2'-F modifiers;

[0363] (b) Antisense chains, which have:

[0364] (i) The length of 19-35 nucleotides;

[0365] (ii) fewer than twelve 2'-F modifiers; and

[0366] (iii) At least two phosphate thioester nucleotide bonds in the first five nucleotides (counting from the 5' end);

[0367] The double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); and the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic portions conjugated to one or more positions on at least one strand; and has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or both ends of the double-stranded region are blunt.

[0368] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphate thioester nucleotide inter-bonds (counted from the 5' end) at the first five nucleotides of the antisense strand; wherein the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more sites on at least one strand; and wherein the dsRNA agent contains less than 20%, less than 15%, and less than 10% of non-natural nucleotides.

[0369] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluorine, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, etc.

[0370] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphate thioester nucleotide inter-bonds (counted from the 5' end) at the first five nucleotides of the antisense strand; wherein the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more sites on at least one strand; and wherein the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% of native nucleotides, such as 2'-OH, 2'-deoxy, and 2'-OMe, which are native nucleotides.

[0371] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphate thioester nucleotide inter-bonds (counted from the 5' end) at the first five nucleotides of the antisense strand; wherein the double-stranded region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent is 100% natural nucleotides, such as 2'-OH, 2'-deoxy, and 2'-OMe.

[0372] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I):

[0373] 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0374] (I)

[0375] in:

[0376] i and j are each independently 0 or 1;

[0377] p and q are each independently 0-6;

[0378] Each N a Independently representing oligonucleotide sequences containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;

[0379] Each N b Independently representing oligonucleotide sequences containing 1, 2, 3, 4, 5, or 6 modified nucleotides;

[0380] Each n p and n q Independently represent the overhanging nucleotide;

[0381] Where N b Y and Y do not have the same modifications;

[0382] XXX, YYY, and ZZZ each independently represent a motif with three identical modifications on three consecutive nucleotides;

[0383] The dsRNA agent comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and

[0384] The antisense strand of the dsRNA contains two blocks separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 phosphate ester nucleotide bonds, each having one, two or three thiophosphate ester nucleotide bonds.

[0385] Various publications describe multimeric siRNAs, all of which can be used with the iRNA of this invention. Such publications include WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, which are incorporated herein by reference in their entirety.

[0386] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agent of the present invention is modified with 2'-OMe.

[0387] In some embodiments, each of the sense and antisense strands of the iRNA agent is independently modified with a noncyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluorine, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0388] In some embodiments, each of the sense and antisense strands of the iRNA agent contains at least two different modifications.

[0389] In some embodiments, the compounds of the present invention do not contain any 2'-F modification.

[0390] In some embodiments, the compounds of the present invention contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve 2'-F modifications. In one example, the compounds of the present invention contain nine or ten 2'-F modifications.

[0391] The iRNA agent of the present invention may further comprise at least one phosphate thioester or methylphosphonate nucleotide internucleotide bond. The phosphate thioester or methylphosphonate nucleotide internucleotide bond modification may appear on any nucleotide of the sense strand or antisense strand, or both, at any position in the strand. For example, the nucleotide internucleotide bond modification may appear on each nucleotide of the sense strand or antisense strand; the nucleotide internucleotide bond modifications may appear in alternating patterns on the sense strand or antisense strand; or the sense strand or antisense strand may contain two nucleotide internucleotide bond modifications in alternating patterns. The alternation pattern of nucleotide internucleotide bond modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternation pattern of nucleotide internucleotide bond modifications on the sense strand may have a migration relative to the alternation pattern of nucleotide internucleotide bond modifications on the antisense strand.

[0392] In one embodiment, the iRNA includes a phosphate thioester or methylphosphonate nucleotide inter-linking modification in the overhang region. For example, the overhang region may contain two nucleotides with a phosphate thioester or methylphosphonate nucleotide inter-linking bond between them. Nucleotide inter-linking modifications may also be made to link the overhang nucleotide to a terminal pairing nucleotide within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked by a phosphate thioester or methylphosphonate nucleotide inter-linking bond, and optionally, additional phosphate thioester or methylphosphonate nucleotide inter-linking bonds may be present to link the overhang nucleotide to a pairing nucleotide immediately adjacent to the overhang nucleotide. For example, at least two phosphate thioester nucleotide inter-linking bonds may be present between the three terminal nucleotides, wherein two of the three nucleotides are overhang nucleotides, and the third is a pairing nucleotide immediately adjacent to the overhang nucleotide. Preferably, these three terminal nucleotides may be located at the 3' end of the antisense strand.

[0393] In some embodiments, the sense and / or antisense strands of the iRNA agent comprise one or more blocks having inter-nucleotide bonds of phosphate thioester or methylphosphonate. In one example, the sense strand comprises one block having two inter-nucleotide bonds of phosphate thioester or methylphosphonate. In one example, the antisense strand comprises two blocks having two inter-nucleotide bonds of phosphate thioester or methylphosphonate. For example, the two blocks having inter-nucleotide bonds of phosphate thioester or methylphosphonate are separated by 16-18 phosphate ester nucleotide bonds.

[0394] In some embodiments, the antisense strand of the iRNA agent is 100% complementary to the target RNA to hybridize with it and inhibit its expression by RNA interference. In another embodiment, the antisense strand of the 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.

[0395] Nucleic acid modification

[0396] In some embodiments, the compound comprises at least one nucleic acid modification described herein. For example, at least one modification is selected from the group consisting of modified nucleoside internucleotides, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modification may be present at any position in the compound. For example, the modification may be present in one of the RNA molecules.

[0397] Nucleic acid modification (nucleobases)

[0398] The naturally occurring base moiety of nucleosides is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. For nucleosides that include furanylpentasyl sugars, the phosphate ester group can be attached to the 2', 3', or 5' hydroxyl portion of the sugar. In the formation of oligonucleotides, these phosphate esters covalently link adjacent nucleosides to each other to form a linear polymer. Within oligonucleotides, the phosphate ester is often referred to as the inter-nucleoside backbone that forms the oligonucleotide. The naturally occurring bonds or backbone of RNA and DNA are 3' to 5' phosphodiester bonds.

[0399] In addition to “unmodified” or “natural” nucleobases (such as purine nucleobases adenine (A) and guanine (G), and pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U)), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for the compounds described herein. Unmodified or natural nucleobases may be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides may be prepared using these bases or with any of the synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and the oligomers described herein. Alternatively, substituted or modified analogs of any of the above-described bases and “universal bases” may be used. When a natural base is replaced by a non-natural and / or universal base, the nucleotide is referred to as containing modified nucleosides and / or nucleobase modifications as described herein. Modified nucleosides and / or nucleobase modifications also include natural, non-natural, and universal bases that contain conjugated moieties, such as the ligands described herein. Preferred conjugated moieties for conjugation with nucleosides include cationic amino groups, which may be conjugated to nucleosides via suitable alkyl, alkenyl, or amide linkers.

[0400] The oligomers described herein may also include nucleobase (generally referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and 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, muscarin, isoguanosine, tuberculin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, and 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(denitro)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxy)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6 2-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(denitro)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deazo)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-( (aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2-(thio)pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil ) pseudouracil, 1-(aminocarbonylvinyl)-pseudouracil, 1-(aminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3 ...2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2- (Aza)-Phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(guanidinyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, muscarin, tuberculin, isoguanosine, inosine, 2-aza-inosine, 7-deaza-inosine, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindole, pyrrolopyrimidinyl, 3-(methyl)isoquinolone, 5-(methyl)isoquinolone, 3-(methyl)-7-(propynyl)isoquinolone, 7-(aza)indole, 6-(methyl)-7-(aza)indole, imidazopyridyl, 9-(methyl)imidazopyridyl, pyrrolopyrazinyl, isoquinol Keto, 7-(propynyl)isoquinolone, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthracene, phenanthrene, pyrene, stilbene, tetraphenyl, pentaphenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymidine, 2-pyridone, 5-nitroindol, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N, 2 -substituted purines, N 6 -substituted purines, O 6 -Substituted purines, substituted 1,2,4-triazoles, pyrrolopyrimidin-2-one-3-yl, 6-phenylpyrrolopyrimidin-2-one-3-yl, para-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl, ortho-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl, di-ortho-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl, p-(aminoalkylhydroxy)-6-benzyl The bases are 2-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrrolo-pyrrolo-2-one-3-yl, bis(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrrolo-2-one-3-yl, pyridopyrrolo-3-yl, 2-oxo-7-amino-pyridopyrrolo-3-yl, 2-oxo-pyridopyrrolo-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, any of the above bases and substituted or modified analogs of the "general base" may be used.

[0401] As used in this article, a universal nucleobase is any nucleobase that can pair with all four naturally occurring nucleobases without substantially affecting unwinding behavior, intracellular enzyme recognition, or the activity of the iRNA double strand. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisoquinolone, 5-methylisoquinolone, 3-methyl-7-propynylisoquinolone, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridyl, 9-methyl-imidazopyridyl, pyrrolopyrazinyl, isoquinolone, 7-propynylisoquinolone, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthraceneyl, phenanthryl, pyrene, stilbene, tetraphenyl, pentaphenyl and their structural derivatives (see, for example, Loakes, 2001, Nucleic Acids). Research [Nucleic Acid Research], 29, 2437-2447.

[0402] Other nucleosides include those disclosed in U.S. Patent No. 3,687,808; those disclosed in International Application No. PCT / US 09 / 038425, filed March 26, 2009; those disclosed in *Concise Encyclopedia of Polymer Science and Engineering*, pp. 858-859, edited by Kroschwitz, JI, John Wiley & Sons, 1990; those disclosed in *Angewandte Chemie*, International Edition, 1991, 30, 613; and those disclosed in *Modified Nucleosides in Biochemistry*, *Biotechnology and...* Nucleotides disclosed in *Medicine*, Herdewijin, P. (ed.), Wiley-VCH, 2008; and in Sanghvi, YS, Chapter 15, *dsRNA Research and Applications*, pp. 289-302, Crooke, ST. and Lebleu, B. (eds.), CRC Press, 1993. All of the above are incorporated herein by reference.

[0403] In some embodiments, the modified nucleobase is a nucleobase that is structurally very similar to the parent nucleobase, such as 7-deazine, 5-methylcytosine, or a G-clamp. In some embodiments, the nucleobase mimic includes a more complex structure, such as a tricyclic phenoxazine nucleobase mimic. Methods for preparing the above-described modified nucleobases are well known to those skilled in the art.

[0404] Nucleic acid modification (sugar)

[0405] The compounds of the present invention provided herein may comprise one or more monomers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) including nucleosides or nucleotides having modified sugar moieties. For example, the furanyl glycan ring of the nucleoside may be modified in various ways, including but not limited to adding substituents or bridging two non-homogeneous ring atoms to form locked nucleic acids or bicyclic nucleic acids. In some embodiments, the oligomeric compounds comprise one or more monomers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) that are LNAs.

[0406] In some embodiments of locked nucleic acids, the 2' position of the furanyl group is linked to the 4' position via a linker independently selected from: -[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)-;

[0407] in:

[0408] x is 0, 1, or 2;

[0409] n is 1, 2, 3 or 4;

[0410] Each R1 and R2 is independently H, a protecting group, a hydroxyl group, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 ynyl group, a substituted C2-C12 ynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, a heterocyclic group, a substituted heterocyclic group, a heteroaryl group, a substituted heteroaryl group, a C5-C7 alicyclic group, a substituted C5-C7 alicyclic group, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, an acyl group (C(═O)-H), a substituted acyl group, CN, a sulfonyl group (S(═O)2-J1) or a sulfonyl group (S(═O)-J1); and

[0411] 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, substituted heterocyclic, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or protecting group.

[0412] In some embodiments, each linker of the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In another embodiment, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, wherein each R1 is independently H, a protecting group, or a C1-C12 alkyl group.

[0413] Some LNAs have been prepared and disclosed in patent and scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahllestedt 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., J.Org.Chem., 1998, 63, 10035-10039; Examples of U.S. patents and published applications disclosing LNA include, for example, U.S. Patent Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807).

[0414] This article also provides LNA, in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring to form a methyleneoxy (4′-CH2-O-2′) bond to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs [Recent Opinions on Drug Research], 2001, 2, 558-561; Braasch et al., Chem. Biol. [Biochemistry], 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther. [Recent Opinions on Molecular Therapy], 2001, 3, 239-243; see also US Patent Nos. 6,268,490 and 6,670,461). The bond can be a methylene (-CH2-) bridging the 2' oxygen atom and the 4' carbon atom, where the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; in the case of an ethylene moiety at this position, the term ethyloxy(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 extremely high double-stranded thermal stability (Tm = +3 to +10 °C) with complementary DNA and RNA, stability towards 3'-exonuclease degradation, and good solubility. Efficient and non-toxic antisense oligonucleotides containing BNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0415] The isomer of methyleneoxy(4'-CH2-O-2')LNA, which has also been discussed, is α-L-methyleneoxy(4'-CH2-O-2')LNA, and has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras that exhibit effective antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0416] The synthesis and preparation of methyleneoxy (4'-CH2-O-2')LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation are also described in WO 98 / 39352 and WO99 / 14226.

[0417] Analogs of methyleneoxy (4'-CH2-O-2')LNA, such as phosphate thioester-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 oligodeoxynucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of 2'-amino-LNA, a novel configuration-restricted high-affinity oligonucleotide analog, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-LNA have been prepared, and their thermal stability with duplexes of complementary RNA and DNA strands has been previously reported.

[0418] Modified sugar moieties are well known and can be used to alter, typically increasing the affinity of antisense compounds for their targets and / or increasing nuclease resistance. A preferred representative list of 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' bridging)ENA; substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; and 4'-thio modified sugars. Sugars may also be modified with glycomimetic groups and other substitutions. Methods for preparing modified sugars are well known to those skilled in the art. Representative patents and disclosures teaching the preparation of such modified sugars include, but are not limited to, US patents 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; 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 WO2005 / 121371.

[0419] Examples of "oxygen"-2′ hydroxyl modifications include alkoxy or aryloxy groups (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG); O(CH2CH2O). n CH2CH2OR, n = 1-50; "Locked" nucleic acid (LNA), in which the furanose portion of the nucleoside includes a bridging bond connecting two carbon atoms on the furanose ring, thus forming a bicyclic system; O-amine or O-(CH2) n Amines (n = 1-10, amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0420] "Deoxy" modifications include hydrogen (i.e., deoxyribose, which is particularly relevant to single-chain overhangs); halogen groups (e.g., fluorine); amino groups (e.g., NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); NH (CH2CH2NH). nCH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclic, 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 optionally be substituted with, for example, an amino functional group.

[0421] Other suitable 2'-modifications (e.g., modified MOEs) are described in U.S. Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference in their entirety.

[0422] Modifications at the 2' position can exist in the arabinose configuration. The term "arabinose configuration" refers to the configuration in which the substituent is located at the C2' of the ribose and the 2'-OH group is in the arabinose.

[0423] Sugars can contain two different modifications at the same carbon atom, such as homologous modifications. A glycosyl group can also contain one or more carbons with a stereochemical configuration opposite to that of the corresponding carbon atom in ribose. Therefore, oligomers can include one or more monomers containing, for example, arabinose as a sugar. Monomers can have an α-bond at the 1' position of the sugar, such as an α-nucleoside. Monomers can also have opposite configurations at the 4' position, such as C5' and H4' or their substituents being interchanged. When C5' and H4' or their substituents are interchanged, the sugar is said to be modified at the 4' position.

[0424] The compounds of the present invention disclosed herein may also include abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having other chemical groups replacing the nucleobase at C1'. See, for example, U.S. Patent No. 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars may also be further modified at one or more sites on the constitutive sugar atom. The compounds of the present invention may also contain one or more L-isomer sugars, such as L-nucleosides. Modification of the sugar group may also include replacing 4'-O with a sulfur, optionally substituted nitrogen, or CH2 group. In some embodiments, the bond between C1' and the nucleobase is α-configured.

[0425] Sugar modifications may also include “acyclic nucleotides,” which refer to any nucleotide having a noncyclic ribose, such as in which the C-C bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') are absent and / or at least one of the ribose carbons or oxygen (e.g., C1', C2', C3', C4', or O4') is not present independently or in combination in the nucleotide. In some embodiments, the noncyclic nucleotide is… Where B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3 or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.

[0426] 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), and 2'-OMe / 2'F at the homologous position, having 2'-O-Me in the arabinose configuration.

[0427] It should be understood that when a particular nucleotide is linked to the next nucleotide via its 2'-position, the sugar modification described herein can be placed at the 3'-position of the sugar of that particular nucleotide (e.g., the nucleotide linked via its 2'-position). Modifications at the 3' position can be present in the xylose configuration. The term "xylose configuration" refers to the position of the substituent at C3' of the ribose being identical to the configuration of the 3'-OH group in xylose.

[0428] The hydrogen atom attached to C4' and / or C1' may be replaced by a straight-chain or branched optionally substituted alkyl group, optionally substituted alkenyl group, or optionally substituted alkynyl group, wherein the main chain of the alkyl, alkenyl, or alkynyl group may contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), phosphorus-containing group, optionally substituted aryl group, optionally substituted heteroaryl group, optionally substituted heterocyclic group, or optionally substituted cycloalkyl group, wherein R' is hydrogen, acyl group, or optionally substituted aliphatic group, and Z' is selected from the group consisting of: OR 11 、COR 11 CO2R 11 , NR 21 R 31 、CONR 21 R 31 CON(H)NR 21 R 31 ONR 21 R 31 CON(H)N=CR 41R 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 R 51 SO2R 11 、SOR 11 SR 11 and substituted or unsubstituted heterocycles; R 21 and R 31 Each time it appears, it is independently of hydrogen, acyl, unsubstituted or substituted aliphatic group, aryl, heteroaryl, heterocyclic, or OR. 11 COR 11 CO2R 11 or NR 11 R 11 '; or R 21 and R 31 It forms a heterocycle together with the atoms to which it is attached; R 41 and R 51 Each time it appears, it is independently of hydrogen, acyl, unsubstituted or substituted aliphatic group, aryl, heteroaryl, heterocyclic, or OR. 11 COR 11 or CO2R 11 or NR 11 R 11 '; and R 11 and R 11 Independently, it is hydrogen, an aliphatic group, a substituted aliphatic group, an aryl group, a heteroaryl group, or a heterocyclic group. In some embodiments, the hydrogen at C4' attached to the 5' terminal nucleotide is replaced.

[0429] In some embodiments, C4' and C5' together form an optionally substituted heterocyclic group, which preferably comprises at least one -PX(Y)-, wherein X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, wherein M is independently an alkali metal or transition metal with a total charge of +1 each time it appears; and Y is O, S, or NR', wherein R' is hydrogen or an optionally substituted aliphatic group. Preferably, this modification is located at the 5' end of the iRNA.

[0430] In some embodiments, the compounds of the present invention comprise at least two regions having at least two consecutive monomers of the above formula. In some embodiments, the compounds of the present invention comprise a nicked motif. In some embodiments, the compounds of the present invention comprise at least one region having about 8 to about 14 consecutive β-D-2'-deoxyfuranose ribosyl nucleotides. In some embodiments, the compounds of the present invention comprise at least one region having about 9 to about 12 consecutive β-D-2'-deoxyfuranose ribosyl nucleotides.

[0431] In some embodiments, the compounds of the present invention comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) of at least one (S)-cEt monomer having the following formula:

[0432]

[0433] Bx represents the heterocyclic base portion.

[0434] In some embodiments, the monomer comprises a glycomimetic. In some such embodiments, the glycomimetic is used in place of the sugar or the sugar-nucleoside intergrowth, and the nucleobase is retained for hybridization with a selected target. Representative examples of glycomimetics include, but are not limited to, cyclohexenyl or N-morpholinyl. Representative examples of glycomimetics for sugar-nucleoside intergrowths include, but are not limited to, peptide nucleic acids (PNAs) and N-morpholinyl groups linked by uncharged achiral bonds. In some cases, the glycomimetic is used in place of the nucleobase. Representative nucleobase mimics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. [Nucleic Acid Research] 2000, 28:2911-14, which are incorporated herein by reference). Methods for synthesizing sugars, nucleosides, and nucleobase mimics are well known to those skilled in the art.

[0435] Nucleic acid modification (glycosidic bonds)

[0436] This article describes linking groups that connect monomers (including but not limited to modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, such as oligonucleotides. These linking groups are also called sugar bonds. Two main categories of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing bonds include, but are not limited to, phosphodiester (P═O), phosphotriesters, methylphosphonates, aminophosphates, and thiophosphates (P═S). Representative phosphorus-free linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified bonds can be used to alter, typically increasing, the nuclease resistance of oligonucleotides compared to native phosphodiester bonds. In some embodiments, bonds having chiral atoms can be prepared as racemic mixtures, or as separate enantiomers. Representative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. Methods for preparing phosphorus-containing and phosphorus-free bonds are well known to those skilled in the art.

[0437] Phosphate esters in the linking group can be modified by replacing an oxygen atom with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolysis. Examples of modified phosphate esters include thiophosphates, selenophosphates, boran phosphates, borane phosphates, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the bond can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or (R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate ester is achiral. However, replacing one of the non-bridging oxygen atoms with one of the aforementioned atoms or groups makes the phosphorus atom chiral; in other words, the phosphorus atom in the phosphate ester modified in this way is a stereosymmetry center. The stereosymmetric phosphorus atom can have an "R" configuration (Rp in this document) or an "S" configuration (Sp in this document).

[0438] Dithiophosphates have two unbridged oxygen atoms replaced by sulfur. The phosphorus center in a dithiophosphate is achiral, preventing the formation of diastereomers of oligonucleotides. Therefore, although it is undesirable to be bound by theory, modification of the two unbridged oxygen atoms to eliminate the chiral center (e.g., dithiophosphate formation) can be desirable because it cannot produce a mixture of diastereomers. Thus, the unbridged oxygen atoms can independently be any of O, S, Se, B, C, H, N, or OR (R being alkyl or aryl).

[0439] Phosphate ester linkers can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate ester to the monomer sugar) with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylenephosphonate). Substitution can occur at any or both bridging oxygen sites. Carbon substitution is preferred when the bridging oxygen is the 3'-oxygen of the nucleoside. Nitrogen substitution is preferred when the bridging oxygen is the 5'-oxygen of the nucleoside.

[0440] Modified phosphate ester bonds, in which at least one oxygen atom linked to the phosphate ester has been replaced or the phosphate ester has been replaced by a non-phosphate group, are also known as "non-phosphate diester sugar bonds" or "non-phosphate diester joints".

[0441] In some embodiments, the phosphate ester may be replaced by a phosphorus-free linker, such as a dephosphorylated linker. The dephosphorylated linker is also referred to herein as a non-phosphodiester linker. While not wishing to be bound by theory, it is believed that replacing the charged phosphate diester group with a neutral structural mimic should confer enhanced nuclease stability because the charged phosphate diester group serves as the reaction center for nucleolysis degradation. Similarly, while not wishing to be bound by theory, in some embodiments it may be necessary to introduce a modification in which the charged phosphate ester is partially replaced by a neutral component.

[0442] Examples of substituted phosphate groups include, but are not limited to, amides (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxyamino, siloxanes (dialkylsiloxanes), formamides, carbonates, carboxymethyl, carbamates, carboxylic acid esters, thioethers, ethylene oxide connectors, sulfides, sulfonates, sulfonamides, sulfonates, thiomethyl acetals (3'-S-CH2-O-5'), methyl acetals (3'-O-CH2-O-5'), oximes, methyleneimino, methylene carbonylamino, and methylene methylimino. (MMI, 3'-CH2-N(CH3)-O-5'), methylene hydrazine, methylene dimethyl hydrazine, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5') and nonionic bonds containing mixed N, O, S and CH2 components. See, for example, Carbohydrate Modifications in Antisense Research; YSSanghvi and PDCook, eds., ACS Symposium Series 580; Chapters 3 and 4 (pp. 40-65). Preferred embodiments include methylene methylimino (MMI), methylene carbonylamino, amide, urethane, and ethylene oxide connector.

[0443] Those skilled in the art are well aware that, in some cases, the substitution of non-bridging oxygens can cause enhanced cleavage of the interglycosidic bond by the adjacent 2'-OH. Therefore, in many cases, the modification of non-bridging oxygens may require modification of the 2'-OH, such as modifications that do not participate in the cleavage of the adjacent interglycosidic bond, such as arabinose, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0444] Preferred non-phosphodiester interglycoses include thiophosphates, thiophosphates having at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of an enantiomeric excess of the Sp isomer, thiophosphates having at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of an enantiomeric excess of the Rp isomer, dithiophosphates, triphosphates, aminoalkyl phosphates, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, aminophosphates (e.g., N-alkylaminophosphates), and boron phosphonates.

[0445] In some embodiments, the compounds of the present invention comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and at most all) modified or non-phosphodiester bond. In some embodiments, the compounds of the present invention comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and at most all) thiophosphate bond.

[0446] The compounds of this invention can also be constructed in which the phosphate linker and sugar are replaced with nuclease-resistant nucleosides or nucleotide substitutes. While not wishing to be bound by theory, it is believed that the absence of a repeating charged backbone reduces binding to proteins that recognize polyanions (e.g., nucleases). Similarly, while not wishing to be bound by theory, in some embodiments it may be necessary to introduce changes in backbone binding where bases are neutrally replaced. Examples include N-morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and backbone-extended pyrrolidine PNA (bepPNA) nucleoside substitutes. Preferred substitutes are PNA substitutes.

[0447] The compounds of the present invention described herein may contain one or more asymmetric centers, and thus produce enantiomers, diastereomers, and other stereoisomers, which, in absolute stereochemistry, may be defined as (R) or (S), such as for glycoterminal isomers, or as (D) or (L), such as for amino acids. The compounds of the present invention provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0448] Nucleic acid modification (terminal modification)

[0449] In some embodiments, the compound further comprises a phosphate ester or phosphate ester mimic at the 5' end of the antisense chain. In one embodiment, the phosphate ester mimic is 5'-vinylphosphonate (VP).

[0450] In some embodiments, the 5' end of the antisense chain of the compound does not contain 5'-vinylphosphonate (VP).

[0451] The ends of the iRNA agents of the present invention may be modified. Such modifications may be made at one end or both ends. For example, the 3' and / or 5' ends of the iRNA may be conjugated to other functional molecular entities, such as labeled moieties, for example fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based on, for example, sulfur, silicon, boron or esters). The functional molecular entities may be attached to sugars via phosphate esters and / or linkers. The terminal atoms of the linker may be connected to or replace the linking atoms of the C-3′ or C-5′ O, N, S or C groups of the phosphate ester or sugar. Alternatively, the linker may be connected to or replace the terminal atoms of a nucleotide substitute (e.g., PNA).

[0452] When a connector / phosphate ester-functional molecular entity-connector / phosphate ester array is inserted between the two chains of a double-stranded oligomer, this array can replace the hairpin ring in a hairpin-type oligomer.

[0453] Terminal modifications that can be used to modulate activity include modifying the 5' end of the iRNA with a phosphate ester or a phosphate ester analog. In some embodiments, the 5' end of the iRNA is phosphorylated or includes a phosphoryl ester analog. Exemplary 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Modifications at the 5' end can also be used to stimulate or suppress the immune system of a subject. In some embodiments, the 5' end of the oligomeric compound contains a modification. W, X, and Y are each independently selected from the following groups: O, OR (R is hydrogen, alkyl, or aryl), S, Se, BR3 (R is hydrogen, alkyl, or aryl), and BH3. - C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl, or aryl); A and Z are each independently absent, O, S, CH2, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene groups, wherein the backbone of the alkylene group may contain one or more of O, S, SS, and NR (R is hydrogen, alkyl, aryl) internally and / or at the end; and n is 0-2. In some embodiments, n is 1 or 2. It should be understood that A replaces the oxygen attached to the 5' carbon of the sugar. When n is 0, W and Y together with their attached P can form optionally substituted 5-8 membered heterocycles, wherein W and Y are each independently O, S, NR', or alkylene groups. Preferably, the heterocycle is substituted with an aryl or heteroaryl group. In some embodiments, one or both hydrogens at the C5' of the 5' terminal nucleotide are substituted with a halogen (e.g., F).

[0454] 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 (thiophosphate; (HO)2(S)PO-5'); 5'-monodithiophosphate (dithiophosphate; (HO)(HS)(S)PO-5'), 5'-thiophosphate ((HO)2(O)PS-5'); 5'-α-thiotriphosphate; 5'-β-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-aminophosphate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkylphosphonates (R(OH)(O)PO-5', where R = alkyl, such as methyl, ethyl, isopropyl, propyl, etc.) and 5'-alkyl ether phosphonates (R(OH)(O)PO-5', where R = alkyl ether, such as methoxymethyl (CH2OMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is optionally substituted with an alkyl group at least once, such as ((HO)2(X)PO[-(CH2)] a -OP(X)(OH)-O] b -5'、((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5'、((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl-terminated phosphate esters and phosphate ester analogs: 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 1-10. Other embodiments include using BH3, BH3 - And / or Se replaces oxygen and / or sulfur.

[0455] Terminal modifications can also be used to monitor distribution, and in such cases, preferred groups to be added include fluorophores, such as fluorescein or Alexa dyes, such as Alexa488. Terminal modifications can also be used to enhance uptake; useful modifications for this include targeting ligands. Terminal modifications can also be used to crosslink oligonucleotides with another moiety; useful modifications for this include mitomycin C, psoralen, and their derivatives.

[0456] Thermal destabilization modification

[0457] The compounds of the present invention (such as iRNA or dsRNA agents) can be optimized for RNA interference by introducing a thermal destabilizing modification at a site opposite the seed region of the antisense strand in the sense strand (i.e., at position 2-8 at the 5' end of the antisense strand), which increases the tendency of the iRNA duplex to dissociate or unwind (reducing the free energy of duplex association). This modification increases the tendency of the duplex to dissociate or unwind in the seed region of the antisense strand.

[0458] Thermal destabilization modifications can include baseless modifications; relative nucleotide mismatches with the opposite strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA).

[0459] An example of no base modification is:

[0460]

[0461] Example sugar modifications are:

[0462]

[0463] The term “UNA” refers to an unlocked acyclic nucleic acid in which any bond of the sugar has been removed, forming an unlocked “sugar” residue. In one instance, UNA also encompasses monomers in which the bond between C1’ and C4’ has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1’ and C4’ carbons). In another instance, the C2’-C3’ bond of the sugar has been removed (i.e., the covalent carbon-carbon bond between the C2’ and C3’ carbons) (see Mikhailov et al., Tetrahedron Letters, 26(17):2059(1985); and Fluiter et al., Mol. Biosyst., 10:1039(2009), which are incorporated herein by reference in their entirety. Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2’-5’ or 3’-5’ bonds.

[0464] The term 'GNA' refers to diol nucleic acid, a polymer similar to DNA or RNA but with a different "backbone" composition, consisting of repeating glycerol units linked by phosphodiester bonds.

[0465]

[0466] Thermal destabilization modification can be a mismatch (i.e., non-complementary base pair) between a thermally destabilized nucleotide within the dsRNA duplex and a corresponding nucleotide in the opposing strand. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatched base pairs known in the art are also applicable to this invention. Mismatches can occur between nucleotides (naturally occurring or modified nucleotides), i.e., mismatched base pairs can occur between the nucleobases of the corresponding nucleotides, regardless of modifications to the ribose of the nucleotide. In some embodiments, the compounds of this invention (such as siRNA or iRNA agents) contain at least one nucleobase, i.e., a 2'-deoxynucleobase, in the mismatched pair; for example, the 2'-deoxynucleobase is in the sense strand.

[0467] Further examples of non-base nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0468] Thermal destabilization modifications may also include general bases with the ability to reduce or eliminate hydrogen bonds with relative bases, as well as phosphate ester modifications.

[0469] Nucleotide modifications with impaired or completely eliminated ability to form hydrogen bonds with bases in the opposing strand have been evaluated for destabilization of the central region of the dsRNA duplex, as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleotide modifications are:

[0470]

[0471] Compared to natural phosphodiester bonds, exemplary phosphate modifications that reduce the thermal stability of dsRNA duplexes are:

[0472]

[0473] In some embodiments, the compounds of the present invention may contain 2'-5' bonds (having 2'-H, 2'-OH, and 2'-OMe and having P=O or P=S). For example, 2'-5' bond modification can be used to promote nuclease resistance or inhibit the binding of sense and antisense strands, or it can be used at the 5' end of the sense strand to avoid sense strand activation in RISC.

[0474] In another embodiment, the compounds of the present invention may comprise L-sugars (e.g., L-ribose, L-arabinose having 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to promote nuclease resistance or inhibit the binding of sense and antisense strands, or can be used at the 5' end of the sense strand to avoid sense strand activation in RISC.

[0475] In one embodiment, the iRNA agent of the present invention is conjugated to a ligand via a carrier, wherein the carrier may be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinoneyl, tetrahydrofuranyl, and decahydronaphthalene; preferably, the acyclic group is selected from the serine alcohol backbone or the diethanolamine backbone.

[0476] In some embodiments, at least one strand of the iRNA agent disclosed herein is 5' phosphorylated or includes a phosphoryl analog at the 5' end. 5'-phosphate modification includes those 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 (thiophosphate; (HO)2(S)PO-5'); 5'-monodithiophosphate (dithiophosphate); (HO)(HS)(S)PO-5'), 5'-thiophosphate ((HO)2(O)PS-5'); any combination of additional oxygen / sulfur-substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-aminophosphate ((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'-).

[0477] target gene

[0478] Without restrictions, the target genes of siRNA include, but are not limited to, genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, genes expressing kinases, adhesion protein genes, genes encoding G protein superfamily molecules, genes encoding transcription factors, genes mediating angiogenesis, viral genes, genes required for viral replication, cellular genes mediating viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes mediating unwanted immune responses, genes mediating pain management, genes mediating neurological diseases, and an allele or polymorphic gene found in cells characterized by loss of heterozygosity.

[0479] Specific exemplary target genes of siRNA include, but are not limited to, PCSK-9, ApoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGFβ genes; 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 genes; Cyclin D gene; VEGF gene; EGFR gene; Cyclin A gene; Cyclin E gene; WNT-1 gene; β-linkin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; susceptin gene; Her2 / Neu gene; Topoisomerase I gene; Topoisomerase IIα gene; p73 gene; p21 (WAF1 / CIP1) gene, p27 (KIP1) gene; PPM1D gene; Cavernin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor gene; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, such as MLL-AF9, BCR / ABL fusion gene; TEL / AML1 fusion gene; EWS / FLI1 fusion gene; TLS / FUS1 fusion gene; PAX3 / FKHR fusion gene; AML1 / ETO fusion gene; αv-integrin gene; Flt-1 receptor gene; microtubule gene; human papillomavirus gene, genes required for human papillomavirus replication, human immunodeficiency virus gene, genes required for human immunodeficiency virus replication, hepatitis A virus gene, hepatitis A... Genes required for viral replication, including: Hepatitis B virus gene, Hepatitis C virus gene, Hepatitis D virus gene, Hepatitis E virus gene, Hepatitis E virus gene, Hepatitis E virus gene, Hepatitis F virus gene, Hepatitis F virus gene, Hepatitis G virus gene, Hepatitis G virus gene, Hepatitis H virus gene, Respiratory syncytial virus gene, Respiratory syncytial virus gene, Herpes simplex virus gene, Herpes cytomegalovirus gene, Herpes cytomegalovirus gene, Epstein-Barr virus gene, Kaposi's sarcoma.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 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.Encephalitis virus gene, genes required for the replication of Murray Valley encephalitis virus, dengue virus gene, genes required for the replication of dengue virus, simian virus 40 gene, genes required for the replication of simian virus 40, human T-cell lymphotropic virus gene, genes required for the replication of human T-cell lymphotropic virus, Moloney-Murine leukemia virus gene, genes required for the replication of Moloney-Murine leukemia virus, encephalomyocarditis virus gene, genes required for the replication of encephalomyocarditis virus, measles virus gene, genes required for the replication of measles virus, varicella-zoster virus. Viral 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 gene replication, Mycobacterium ulcerans genes, genes required for Mycobacterium ulcerans replication, Mycobacterium tuberculosis genes, genes required for Mycobacterium tuberculosis replication, Mycobacterium leprae genes, genes required for Mycobacterium leprae replication, Staphylococcus aureus genes, genes required for Staphylococcus aureus replication. Genes, Streptococcus pneumoniae gene, genes required for Streptococcus pneumoniae replication, Streptococcus pyogenes gene, genes required for Streptococcus pyogenes replication, Chlamydia pneumoniae gene, genes required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, genes required for Mycoplasma pneumoniae replication, 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, M CP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene, CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, genes of ion channel components, neurotransmitter receptor genes, neurotransmitter ligand genes, amyloid family genes, presenilin gene, HD gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, alleles found in loss-of-heterozygosity (LOH) cells, one allele of polymorphic genes, and combinations thereof.

[0480] Loss of heterozygosity (LOH) can result in hemizygosity of sequences (e.g., genes) within the LOH region. This can lead to significant genetic differences between normal and diseased cells (e.g., cancer cells) and provides a useful basis for differentiation between normal and diseased cells (e.g., cancer cells). This difference can arise because a gene or other sequence is heterozygous in diploid cells but hemizygous in cells with an LOH. LOH regions typically include genes whose deletion causes unwanted proliferation, such as tumor suppressor genes; and other sequences, including, for example, genes essential for normal function (e.g., growth) in some cases. The method of the present invention relies in part on the specific regulation of an allele of an essential gene using the compositions of the present invention.

[0481] In some embodiments, the present invention provides compounds of the present invention for regulating microRNAs.

[0482] Targeting CNS

[0483] In some embodiments, the present invention provides a compound that targets APP for early-onset familial Alzheimer's disease, spinocerebellar ataxia 2 and ATXN2 for ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia.

[0484] In some embodiments, the present invention provides a compound that targets TARDBP for ALS, MAPT (Tau) for frontotemporal dementia, and HTT for Huntington's disease.

[0485] In some embodiments, the present invention provides a compound that targets 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, latent CNS disorders: PRNP for Lafra disease, DMPK for DM1 (CNS and skeletal muscle), and TTR for hATTR (CNS, eye and systemic).

[0486] Spinocerebellar ataxia is a hereditary brain dysfunction. Dominantly inherited forms of spinocerebellar ataxia (such as SCA1-8) are devastating disorders without disease-modifying therapies. Exemplary targets include SCA2, SCA3, and SCA1.

[0487] ATXN2 targeting SCA2

[0488] Spinocerebellar ataxia 2 (SCA2), a progressive ataxia, is the second most common form of SCA. Another disease associated with this target is amyotrophic lateral sclerosis (ALS). These are debilitating and ultimately fatal diseases with no disease-modifying therapies. The prevalence of SCA is 2–6 per 100,000 people; ATXN2 accounts for 15% of the global SCA population, and the SCA population is much higher in some countries, particularly Cuba (40 per 100,000 people). Targeting ATXN2 via human molecular genetics can be excellent, for example, in familial and sporadic SCA and ALS, where CAG repeats encoding ATXN2 are found amplified in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be that the autosomal dominant CAG amplification of ATXN2 leads to the expression of a toxic, misfolded protein and the death of Purkinje cells and neurons. Efficacy has been demonstrated through 70% knockdown (KD) of ATXN2 mRNA; and KD POC in mATXN2 mice has been confirmed. Regarding safety, mATXN2 knockout (KO) mice have been 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 repeats.

[0489] ATXN3 targeting SCA3

[0490] Spinocerebellar ataxia 3 (SCA3), a progressive ataxia, is the most common form of SCA worldwide. This disease is debilitating and ultimately fatal, with no disease-modifying treatment. It is the most common cause of SCA, with a prevalence of 2–6 per 100,000 people; ATXN3 accounts for 21% of the SCA population in the United States and is much more prevalent in Europe (particularly Portugal). Targeting ATXN3 via human molecular genetics can be excellent, for example, in familial and sporadic SCA, where CAG repeats encoded by ATXN3 are found amplified in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be due to the autosomal dominant CAG amplification of ATXN3 leading to the expression of toxic, misfolded proteins, Purkinje cell and neuronal death. Efficacy has been shown through 70% KD of ATXN3 mRNA; and mATXN3 KD mice have been proven effective in proto-occurrence. Regarding safety, mATXN3 KO mice have been 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 repeats.

[0491] ATXN1 targeting SCA1

[0492] Spinocerebellar ataxia 1 (SCA1), a progressive ataxia, was the first SCA gene discovered in 1993. This disease is debilitating and ultimately fatal, 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 globally, and much higher in some countries (25% in Japan), particularly in Poland (64%) and Siberia (100%). Targeting ATXN1 via human molecular genetics can be excellent, for example, in familial and sporadic SCA, where CAG repeats encoded by ATXN1 are found amplified in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be due to the autosomal dominant CAG amplification of ATXN1 leading to the expression of toxic, misfolded proteins, Purkinje cell and neuronal death. Efficacy has been shown through 70% KD of ATXN1 mRNA; and mouse prognostic studies (POCs) of mATXN1 have been confirmed. Regarding safety, mATXN1 KO mice have been 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 repeats.

[0493] ATXN7 targeting SCA7

[0494] Spinocerebellar ataxia 7 (SCA7) causes progressive ataxia and retinal degeneration. This disease is a debilitating and ultimately fatal retinal and cerebellar disorder with no disease-modifying treatment. The prevalence of SCA is 2–6 per 100,000 people; ATXN7 accounts for 5% of the global SCA population, and is much higher in some countries (particularly South Africa). Targeting ATXN7 via human molecular genetics can be excellent, for example, in familial and sporadic SCA, where CAG repeats encoding ATXN7 are found amplified in tissues such as the spinal cord, brainstem, cerebellum, or retina. The mechanism of this targeting may be due to the expression of a toxic, misfolded protein caused by autosomal dominant CAG amplification of ATXN1, leading to cone and rod degeneration, Purkinje cell and neuronal lethality. Efficacy has been shown via 70% KD of ATXN1 mRNA through 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 repeats.

[0495] ATXN8 targeting SCA8

[0496] Spinocerebellar ataxia 8 (SCA8), a progressive neurodegenerative ataxia, is caused by CTG repeat amplification in ATXN8. This disease is debilitating and ultimately fatal, with no disease-modifying treatment. Prevalence: SCA affects 2-6 people per 100,000; ATXN8 causes 3% of the global SCA population, and is much higher in some countries (especially Finland). Targeting ATXN8 via human molecular genetics can be excellent, for example, in familial and sporadic SCA, where CTG repeat amplification in ATXN8 is found in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be due to the autosomal dominant CTG amplification of ATXN8 leading to the expression of a toxic, misfolded protein, thereby triggering lethality in Purkinje cells and neurons. Efficacy has been shown with 70% KD of ATXN8 mRNA. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF, CTG mRNA, and peptide repeats.

[0497] CACNA1A targeting SCA6

[0498] Spinocerebellar ataxia 6 (SCA6) is a progressive ataxia. This disease is debilitating and ultimately fatal, and there is no disease-modifying therapy. The prevalence of SCA is 2–6 per 100,000 people; and CACNA1A accounts for 15% of the global SCA population. Targeting CACNA1A via human molecular genetics can be excellent, for example, in familial and sporadic SCA, where CAG repeats encoding CACNA1A are found amplified in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be due to the autosomal dominant CAG amplification of CACNA1A leading to the expression of toxic, misfolded proteins and the death of Purkinje cells and neurons. 70% KD has shown efficacy through CACNA1A CAG amplification. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeats.

[0499] Exemplary targets of hereditary polyglutamine disorders include Huntington's disease (HD).

[0500] HTT targeting Huntington's disease

[0501] Huntington's disease (HD) is caused by mutations in HTT, a progressive CNS degenerative disease. This disease is debilitating and ultimately fatal, and there are no disease-modifying therapies. The global prevalence of HD is 5–10 per 100,000 people, and it is more common in certain countries (particularly Venezuela). Targeting HTT via human molecular genetics can be excellent, for example, in familial and sporadic HD, by amplification of the CAG-encoding repeat in HTT found in tissues such as the striatum or cortex. The mechanism of this targeting may be that autosomal dominant CAG amplification in HTT leads to the expression of a toxic, misfolded protein and neuronal death. Efficacy has been shown in 70% KD with HTT CAG amplification alone; and a mouse prognostic value (POC) has been confirmed. Regarding safety, knockout (KO) of HTT in mice can be lethal; a KD in humans has been confirmed. Possible diagnoses include family history; genetic testing; early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0502] ATN1 targeting DRPLA

[0503] Atrophin 1 mutations cause dentate nucleus-globus pallidus Lewy body atrophy (DRPLA), a progressive spinocerebellar disorder similar to HD. This disease is debilitating and ultimately fatal, with no disease-modifying therapies. In Japan, the prevalence of DRPLA is 2–7 per 1,000,000 people. Targeting ATN1 via human molecular genetics can be excellent, for example, in familial and sporadic SCA, where CAG repeat amplification in ATN1 is found in tissues such as the spinal cord, brainstem, cerebellum, or cortex. The mechanism of this targeting may be due to the autosomal dominant CAG amplification in ATN1 leading to the expression of a toxic, misfolded protein and neuronal death. Efficacy has been shown with 70% KD of ATN1. Regarding safety, ATN1 KO mice have been 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.

[0504] AR targeting spinal and medullary muscle atrophy

[0505] Androgen receptor mutations cause spinal and bulbar muscular atrophy (SBMA, Kennedy disease), a progressive muscular dystrophy, among other conditions. This disease is debilitating and ultimately fatal, with no disease-modifying therapies. The prevalence of SBMA is approximately 2 cases per 100,000 men; women have a milder phenotype. Targeting the androgen receptor (AR) via human molecular genetics can be excellent, for example, in familial SBMA, through amplification of the CAG-coding repeat in the AR found in tissues such as the spinal cord or brainstem. The mechanism of this targeting may be due to X-linked CAG-coding amplification in the AR leading to toxic gain-or-function and motor neuron lethality. Efficacy has been shown through 70% KD in the AR. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeats.

[0506] FXN targeting Friedrich Ataxia

[0507] Recessive loss-of-function GAA amplification of FXN leads to Friedrich's ataxia (FA), a progressive degenerative ataxia. This disease is debilitating and ultimately fatal, with no disease-modifying therapies. The prevalence of FA is approximately 2 per 100,000 people worldwide. Targeting FXN via human molecular genetics can be excellent, for example, in familial FA, where intronic GAA repeat amplification in FXN is found in tissues such as the spinal cord, cerebellum, or possibly the retina and heart. The mechanism of this targeting may be due to the death expression of FXN (an important mitochondrial protein) caused by autosomal recessive non-coding FAA amplification of FXN. 70% KD via FXN intronic GAA amplification has shown efficacy. Regarding safety, KD of intronic GAA is safe and effective in mice. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0508] FMR1 targeting FXTAS

[0509] Fragile X-related tremor / ataxia syndrome (FXTAS) is a progressive ataxia and cognitive impairment in adults caused by FMR1 overexpression. This is a debilitating condition with no disease-modifying treatment. The prevalence of FMR1 premutations is approximately 1 in 500 men. Targeting FMR1 via human molecular genetics can be excellent, for example, in FMR1, where premutations encoding CCG repeats are found in tissues such as the spinal cord, cerebellum, or cortex. The mechanism of this targeting may be due to X-linked CCG amplification of FMR1 leading to toxic mRNA. Efficacy has been shown with 70% KD of toxic mRNA. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeats.

[0510] upstream of FMR1 targeting Fragile X syndrome

[0511] Fragile X syndrome (FRAXA), a progressive intellectual disability, can be treated by targeting upstream mRNA of FMR1. This is a debilitating condition with no disease-modifying therapies. The prevalence of FRAXA is approximately 1 in 4,000 men and 1 in 8,000 women. Targeting FMR1 via human molecular genetics can be highly effective, for example, in FRAXA, where amplification of the CCG repeat encoding FMR1 is found in tissues such as the CNS. The mechanism of this targeting may be due to X-linked CCG amplification of FMR1 leading to LOF; and normal FMR1 is used for transporting specific mRNAs from the nucleus. Efficacy has been shown with 70% KD of 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.

[0512] Dominantly inherited amyotrophic lateral sclerosis (ALS) is a devastating disorder with no disease-modifying therapies. Exemplary targets include C9orf72, ATXN2 (which also causes SCA2), and MAPT.

[0513] C9orf72 targeting ALS

[0514] C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These diseases are lethal motor neuron disorders with no disease-modifying therapies. The prevalence of ALS is 2–5 per 100,000 people (10% familial); C9orf72 accounts for 39% of familial ALS and 7% of sporadic ALS in the United States and Europe. Targeting C9orf72 via human molecular genetics can be excellent, for example, in familial and sporadic ALS, in tissues such as upper and lower motor neurons (for ALS); or the cortex (for FTD). The mechanism of this targeting may be due to autosomal dominant hexanucleotide amplification leading to repeat-related non-AUG-dependent translation of toxic dipeptide repeats and neuronal lethality. Efficacy has been shown with 70% KD of C9orf72. Regarding safety, heterozygous LOF mutations in 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.

[0515] TARDBP targeting ALS

[0516] TARDBP mutations cause ALS and frontotemporal dementia (FTD). These diseases are lethal motor neuron disorders with no disease-modifying therapies. The prevalence of ALS is 2–5 per 100,000 people (10% familial); TARDBP causes 5% of familial ALS and 1.5% of sporadic ALS. Targeting TARDBP via human molecular genetics can be excellent, for example, in familial and sporadic ALS, in tissues such as upper and lower motor neurons (for ALS); or the cortex (for FTD). The mechanism of this targeting may be due to autosomal dominant TRDBP mutations leading to toxic TRDBP protein and neuronal lethality. Efficacy has been shown in 70% KD via TARDBP mutant alleles. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF protein.

[0517] FUS targeting ALS

[0518] FUS mutations cause ALS and FTD. These diseases are lethal motor neuron disorders with no disease-modifying therapies. The prevalence of ALS is 2–5 per 100,000 people (10% familial); FUS causes 5% of familial ALS; FUS inclusions are common in sporadic ALS. Targeting FUS via human molecular genetics can be excellent, for example, in familial ALS, where mutations are found in tissues such as those of upper and lower motor neurons associated with ALS. The mechanism of this targeting may be due to autosomal dominant FUS mutations leading to abnormal protein folding and neuronal lethality. Efficacy has been shown through 70% KD targeting of FUS mutant alleles. Regarding safety, KO mice struggled but survived and exhibited an ADHD phenotype. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF protein.

[0519] SOD1 targeting ALS

[0520] Dominant and recessive mutations in SOD1 cause ALS. This disease is a fatal disorder of motor neurons, and there is no disease-modifying therapy. The prevalence of ALS is 2–5 per 100,000 people (10% familial); SOD1 causes 5%–20% of familial ALS. Targeting SOD1 via human molecular genetics can be excellent, for example, in tissues such as the upper and lower motor neurons that contribute to ALS, many SOD1 mutations are associated with familial AD and AR ALS. The efficacy of this targeting may require mutation-specific KD. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers can be mutation-specific.

[0521] Dominantly inherited frontotemporal dementia and progressive supranuclear palsy. Targets include MAPT (because it can be important for AD) or C9orf72.

[0522] Tau, a microtubule-associated protein targeting FTD-17 and PSP

[0523] Familial frontotemporal dementia 17 (FTD-17), a familial form of FTD located on chromosome 17, and familial progressive supranuclear palsy, may be caused by MAPT mutations, which can also cause rare forms of progressive supranuclear palsy, corticobasal degeneration, Tau proteinosis with respiratory failure, and dementia with epilepsy. These diseases are fatal neurodegenerative disorders with no disease-modifying therapies. The prevalence of FTD is 15-22 per 100,000 people; in the Netherlands, the prevalence of FTD-17 is 1 per 1,000,000 people. Targeting MAPT via human molecular genetics can be excellent, for example, in familial and sporadic FTD, with GOF point mutations and splice site mutations of MAPT found in tissues such as the frontal or temporal cortex. The mechanism of this targeting may be due to the toxic Tau peptide and neuronal death caused by autosomal dominant GOF mutations in MAPT. Efficacy has been shown through 70% KD of MAPT. Regarding safety, MAPT KO mice have been reported to be healthy. Possible diagnoses include family history; genetic testing; early symptoms. Biomarkers that can be used include, for example, CSF Tau mRNA and protein.

[0524] Sequestosome targeting FTD and ALS

[0525] Sporadic FTD / ALS is associated with a dominant SQSTM1 mutation. This disease is a fatal neurodegenerative disorder with no disease-modifying therapies. It is a very rare disease. In sporadic cases, targeting skeletal fragment 1 via human molecular genetic associations is plausible in tissues such as the frontal and temporal cortex, or the cerebellum and spinal cord. Possible diagnoses include genetic testing; early symptoms.

[0526] Dominantly inherited Parkinson's disease is a devastating disorder with no disease-modifying therapies. Targets include SNCA.

[0527] SNCA targeting Parkinson's disease

[0528] Mutations in alpha synuclein lead to familial Parkinson's disease (PD) and Lewy body dementia. These are fatal neurodegenerative disorders with no disease-modifying therapies. The global prevalence of PD is 4 million; one-third of PD cases are familial; and 1% of fPD is caused by SNCA. Targeting SNCA via human molecular genetics can be excellent, for example, in tissues such as the medulla oblongata or the substantia nigra of the midbrain, where point mutations and duplications of SNCA cause familial PD. The mechanism of this targeting may be due to overexpression of abnormal SNCA proteins or expression leading to toxic peptides and neuronal death. Efficacy has been demonstrated in 70% KD mice using 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.

[0529] LRRK2 targeting Parkinson's disease

[0530] Mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). This is a fatal neurodegenerative disorder with no disease-modifying therapies. The global prevalence of PD is 4 million; one-third of PD cases are familial; and 3%–7% of fPD are caused by LRRK2. Targeting LRRK2 via human molecular genetics can be excellent, for example, in tissues such as the medulla oblongata or the substantia nigra of the midbrain, where point mutations in LRRK2 cause familial PD. Possible diagnoses include family history; genetic testing; and early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0531] GARS targeting spinal muscular atrophy V

[0532] Autosomal dominant glycyl-tRNA synthetase mutations cause spinal muscular atrophy V (SMAV) or remotely inherited motor neuropathy Va. These are neurodegenerative disorders with no disease-modifying therapies. These are very rare diseases. Targeting GARs via human molecular genetics can be beneficial; for example, point mutations in GARs in tissues such as the spinal cord cause familial SMA. Possible diagnoses include family history; genetic testing; and early symptoms.

[0533] Seipin targeting spinal muscular atrophy

[0534] Autosomal dominant seipin mutations cause spinal muscular atrophy (SMA) or remotely inherited motor neuropathy. These are neurodegenerative disorders with no disease-modifying therapies. They are very rare diseases. Targeting seipin via human molecular genetics can be beneficial; for example, point mutations in seipin in tissues such as the spinal cord cause familial SMA. The mechanism of this targeting may be GOF and toxic peptides. Efficacy has been shown with 50% KD. Regarding safety, recessive LOF mutations cause progressive encephalopathy with or without lipodystrophy. Possible diagnoses include family history; genetic testing; or early symptoms.

[0535] Dominant Alzheimer's disease is a devastating disorder with no disease-modifying therapies. Targets include APP (Advanced Perceptual Angiopathy) due to its central mechanistic role in familial disease and its potential role in common AD cases.

[0536] Apps targeting Alzheimer's disease

[0537] Mutations in amyloid precursor proteins lead to early-onset familial Alzheimer's disease (EOFAD); AD in Down syndrome; or AD. These diseases are fatal neurodegenerative disorders with no disease-modifying therapies. The prevalence of EOFAD-APP is 1% in AD; the prevalence of trisomy 21 is 1% in AD; and in the United States, the prevalence of AD is approximately 2.5–5 million. Targeting APP via human molecular genetics can be excellent, for example, in tissues such as the cerebral cortex or hippocampus, where APP duplication and point mutations lead to EOFAD. The mechanism of this targeting may be due to progressive neuronal death caused by APP overexpression or the expression of toxic metabolites. Efficacy has been shown in 70% KD mice via APP. Regarding safety, healthy KD mice with some behavioral abnormalities have been reported; healthy KD mice with some spatial memory impairment have been reported. Possible diagnoses include family history; genetic testing; early symptoms; or MRI. Biomarkers that can be used include, for example, CSF APP mRNA and peptides.

[0538] PSEN1 targeting Alzheimer's disease

[0539] Presenilin 1 mutations cause early-onset familial Alzheimer's disease (EOFAD); or AD. These diseases are fatal neurodegenerative disorders with no disease-modifying therapies. Targeting PSEN1 via human molecular genetics can be excellent, for example, in tissues such as the cerebral cortex or hippocampus, where point mutations in PSEN1 lead to EOFAD. The mechanism of this targeting may be that autosomal dominant mutations in PSEN1 cause abnormal APP metabolism and toxic peptides that induce progressive neuronal death. Efficacy has been demonstrated through APPKD, which eliminates the need for PSEN1-specific therapies. Possible diagnoses include family history; genetic testing; early symptoms; or MRI. Biomarkers that can be used include, for example, CSF, PSEN1, and APP peptides.

[0540] PSEN2 targeting Alzheimer's disease

[0541] Presenilin 2 mutations cause early-onset familial Alzheimer's disease (EOFAD); or AD. These diseases are fatal neurodegenerative disorders with no disease-modifying therapies. Targeting PSEN2 via human molecular genetics can be excellent, for example, in tissues such as the cerebral cortex or hippocampus, where point mutations in PSEN2 lead to EOFAD. The mechanism of this targeting may be that autosomal dominant mutations in PSEN2 cause abnormal APP metabolism and toxic peptides that induce 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.

[0542] Apo E targeting Alzheimer's disease

[0543] Apolipoprotein E4 is associated with sporadic Alzheimer's disease (AD) in older adults. This disease is a fatal neurodegenerative disorder with no disease-modifying therapies. The prevalence of AD in the United States is 2.5–5 million. Targeting Apo E may be effective because the genomic evidence supporting the association between ApoE4 and AD is strong in many populations. Target tissue could be the cerebral cortex. Despite the strong association in many populations, it is unclear whether Apo E4 contributes to the pathogenesis of AD. To date, data suggest that homozygosity of Apo E4 indicates an increased risk of AD in older adults, but not enough to cause AD even in older adults. Regarding safety, KD with ApoE in the CNS may be safe because human LOF mutations in Apo E are not associated with significant neurological deficits, although systemic exposure may cause type III hyperlipoproteinemia. Possible diagnoses include a clinical diagnosis of AD; exclusion of EOFAD mutations; and genetic testing for the Apo E4 genotype. Biomarkers that can be used include, for example, CSF APP, Tau mRNA, and peptides.

[0544] CNS gene duplication disorders. Consistent halving of KD can improve these disorders. Targets include MeCP2.

[0545] MeCP2 targeting X-linked intellectual disability

[0546] MeCP2 gene duplication causes X-linked intellectual disability (XLMR). This is a fatal cognitive impairment with no disease-modifying therapies. 1%–15% of X-linked MR is caused by MeCP2 duplication; 2%–3% of the population has MR. Targeting MeCP2 via human molecular genetics can be advantageous, for example, in tissues such as the cerebral cortex, where MeCP2 duplication leads to XLMR. The mechanism of this targeting may be due to MeCP2 overexpression leading to dysregulation of other genes and neurodegeneration. Efficacy has been shown with 50% KD of MeCP2; and the ASO KD phenotype has been reversed in mouse models. Regarding safety, MeCP2 LOF mutations may 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.

[0547] Dominantly inherited cerebral amyloid angiopathy is a devastating disorder with no disease-modifying therapies. Targets include TTR.

[0548] TTR targeting hATTR CAA

[0549] This targeting could be a low-risk introduction of CNS siRNA. Cerebral amyloid angiopathy (CAA) and meningeal amyloid are lethal disorders with no disease-modifying therapies. Targeting TTR via human genetics and pharmacology could be excellent. Target tissues could be the CNS vascular system or the CNS itself. The mechanism of this targeting could be due to the accumulation of mutant proteins in the adventitia of blood vessels, causing CNS hemorrhage. Efficacy has been shown through 70% KD of TTR. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0550] ITM2B targeting CAA

[0551] Mutations in integrated membrane protein 2B (ITM2B) cause cerebral amyloid angiopathy (CAA), British type, or familial British dementia (FBD). Specific mutations can also cause dominant retinal degeneration. This disease is fatal, and there are no disease-modifying therapies. It is a rare disease. Targeting ITM2B via human molecular genetics can be excellent. Target tissues can be the CNS vascular system or the CNS itself. The mechanism of this targeting may involve GOF mutations. Efficacy has been shown in 70% KD using the ITM2B mutant allele. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and possibly proteins.

[0552] CST3 targeting CAA

[0553] Serum cystatin C mutations cause Icelandic-type familial cerebral amyloid angiopathy. This disease is fatal and there are no disease-modifying therapies. It is a rare disease outside of Iceland and Denmark. Targeting CST3 via human genetics can be excellent. The target tissue can be the CNS vascular system. The mechanism of this targeting may be due to the accumulation of the mutant protein in the adventitia of blood vessels, causing CNS hemorrhage. Efficacy has been shown through a possible 70% KD of the mutant allele. Regarding safety, CST3 KO mice may be at risk of developing arthritis. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and possibly the protein.

[0554] SPAST targeting spastic paraplegia

[0555] SPASTIN mutations cause spastic paraplegia (SP)4 with cognitive loss. This disease is a lower motor neurodegenerative disorder with no disease-modifying treatment. The prevalence of SP is 5 per 100,000 people; SP4 is dominant in 45% of SP cases. Targeting SPAST via human molecular genetics can be excellent, for example, in tissues such as the spinal cord or CNS, where SPAST trinucleotide mutations cause familial SP. The mechanism of this targeting may be due to nonsense and possibly dominant-negative mutations causing 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 possibly proteins.

[0556] KIF5A targeting spastic paraplegia

[0557] Mutations in kinin family member 5A result in spastic paraplegia (SP)10, accompanied by peripheral neuropathy and other impairments. This disease is a lower motor neurodegenerative disorder with no disease-modifying therapies. The prevalence of SP is 5 per 100,000 people; SP10 is 1 per 1,000,000 people. Targeting KIF5A via human molecular genetics can be advantageous; for example, N-terminal missense mutations in KIF5A result in SP10, and KIF5A is expressed in the CNS and encodes microtubule motility proteins. Target tissue could be the spinal cord. The mechanism of this targeting could be due to autosomal dominant missense mutations leading to SP10, which may affect microtubules binding to motor nerves. Efficacy may be provided by possible KD mutations in the mutant allele. Regarding safety, frameshift mutations in KIF5A result in neonatal refractory myoclonus, and splice site mutations may be associated with familial ALS via the LOF mechanism. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and possibly proteins.

[0558] ATL1 targeting spastic paraplegia

[0559] 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 therapy. The prevalence of SP is 5 per 100,000 people; SP3A is a rare dominant form. Targeting ATL1 via human molecular genetics can be excellent, for example, ATL1 point mutations cause familial SP. The target tissue can be the spinal cord. The mechanism of this targeting can be due to autosomal dominant expression of dominant-negative ATL1 protein leading to SP3A; however, LOF mutations lead to sensory neuropathy 1D. Efficacy has been shown through 70% KD of specific ATL1 alleles. Regarding safety, heterozygous LOF mutations of ATL1 lead to HSN1D. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF ATL1 mRNA and protein.

[0560] NIPA1 targeting spastic paraplegia

[0561] LOF NIPA1 mutations cause spastic paraplegia 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 people; SP6 is a rare dominant form. Targeting NIPA1 via human molecular genetics can be excellent, for example, NIPA1 point mutations cause familial SP. Target tissues can be the spinal cord; or the CNS. The mechanism of this targeting can be due to autosomal dominant expression of a defective membrane protein leading to 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 possibly proteins.

[0562] Dominantly inherited tonic dystrophy is a CNS, skeletal muscle, and cardiac disorder requiring both CNS and systemic therapy. Targets include MPK of DM1.

[0563] DMPK targeting muscle tone dystrophy 1

[0564] Effective therapies targeting myotonic dystrophy protein kinases require both CNS and systemic treatment. Myotonic dystrophy 1 (DM1) is a degenerative disorder of muscles and the CNS. It is a fatal disorder with no disease-modifying therapies. The prevalence of DM1 is approximately 1 in 8,000 people worldwide. Targeting DMPK via human molecular genetics can be excellent, for example, DMPK CTG repeat amplification leads to familial DM1. Target tissues can be skeletal muscle, cardiac muscle, or the CNS. The mechanism of this targeting may be due to abnormal RNA processing and dominant-negative effects caused by autosomal dominant noncoding CTG repeats; extreme amplification is expected to lead to early-onset disease. Efficacy has been shown with 70% of DMPKs; and ASO efficacy has been demonstrated in mice. Safety has been demonstrated in KO and ASO KD mice. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, blood and CSF mRNA and proteins.

[0565] ZNF9 targeting muscle tone dystrophy 2

[0566] Mutations in zinc finger protein 9 (ZNF9) cause toneless muscular dystrophy 2 (DM2), a degenerative disorder of skeletal muscle. This is a severe disorder with no disease-modifying therapies. The prevalence of DM2 is 1 in 8,000 people worldwide; it is the most common muscular dystrophy in adults. Targeting ZNF9 via human molecular genetics can be excellent, for example, ZNF9 CTTG repeat amplification in intron 1 leads to familial DM2. Target tissues can be skeletal muscle or cardiac muscle. The mechanism of this targeting may be due to abnormal RNA metabolism and dominant-negative effects caused by autosomal dominant CTTG repeat amplification in intron 1. Efficacy has been shown with 70% ZNF9. Safety has been demonstrated in mice. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, blood mRNA and proteins.

[0567] Dominant hereditary prion diseases are hereditary, sporadic, and infectious PRNP disorders. Targets include PRNP.

[0568] PRNP targeting myotropic prion disease

[0569] Tonic-clonic prion diseases are dominant prion disorders, including PRNP-associated cerebral amyloid angiopathy, Gerstmann-Straussler disease (GSD), Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Huntington's disease 1 (HDL1), and kuru susceptibility. These diseases are fatal neurodegenerative disorders with no disease-modifying therapies. The prevalence of these diseases is approximately 1 in 1,000,000 people. Targeting PRNP via human molecular genetics can be excellent, for example, PRNP mutations cause familial and sporadic prion diseases. Target tissues can be the CNS. The mechanism of this targeting may be due to neurotoxicity caused by mid-folding in autosomal dominant proteins. Efficacy has been shown in 70% of PRNP KD; and PRNP polymorphisms appear to have a protective effect against kuru disease. Regarding safety, PRNP KO mice have been 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.

[0570] Targeting glycogen synthase in lafora's myositis epilepsy

[0571] Mutations in the Laforin (EPM2A) gene cause AR myositis epilepsy, a hereditary progressive seizure disorder. This disease is a fatal disorder characterized by seizures and cognitive decline, and there is no disease-modifying therapy. The prevalence of this disease is 4 per 1,000,000 people. Targeting glycogen synthase via human molecular genetics can be excellent, for example, mutations causing Laforin's AR familial myositis epilepsy. The target tissue can be the CNS. The mechanism of this targeting may be due to autosomal recessive dysfunction of Laforin leading to glycogen misfolding and seizure foci. Efficacy has been shown with 70% KD of the glycogen synthase GYS1. Regarding safety, GYS1 deficiency leads to glycogen deficiency in skeletal and cardiac muscle; surviving GYS1 mice exhibit muscle defects. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0572] In some embodiments, the present invention provides compounds that target disease genes, including but not limited to age-related macular degeneration (AMD) (dry and wet), shotgun chorioretinopathy, retinitis pigmentosa, Fuch's dystrophy, hATTR amyloidosis, hereditary and incidental glaucoma, and Stargardt's disease.

[0573] In some embodiments, the present invention provides compounds of VEGF that target wet (or exudative) AMD.

[0574] In some embodiments, the present invention provides compounds that target C3 of dry (or non-exudative) AMD.

[0575] In some embodiments, the present invention provides compounds that target CFB in dry (or non-exudative) AMD.

[0576] In some embodiments, the present invention provides compounds that target MYOC in glaucoma.

[0577] In some embodiments, the present invention provides compounds that target ROCK2 in glaucoma.

[0578] In some embodiments, the present invention provides compounds that target ADRB2 in glaucoma.

[0579] In some embodiments, the present invention provides compounds that target CA2 in glaucoma.

[0580] In some embodiments, the present invention provides compounds of CRYGC that target cataracts.

[0581] In some embodiments, the present invention provides compounds of PPP3CB that target dry eye syndrome.

[0582] ligand

[0583] In some embodiments, the compounds of the present invention are further modified by covalently attaching one or more conjugation groups. Generally, the conjugation groups modulate one or more properties of the attached compounds of the present invention, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugation groups are commonly used in chemical techniques and are directly or via optional linking moieties or linking groups to parent compounds, such as oligomeric compounds. A preferred list of conjugation groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinones, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0584] In some embodiments, the compound further comprises a targeting ligand that targets and mediates delivery to a receptor in a specific CNS tissue. These targeting ligands may be conjugated with a lipophilic moiety to achieve specific intrathecal and systemic delivery.

[0585] Exemplary targeting ligands for receptors that are targeted to CNS tissues include peptide ligands such as angiotensin-2, lipoprotein receptor-associated protein (LRP) ligands, bEnd.3 cell-binding ligands; transferrin receptor (TfR) ligands (which utilize the iron transport system in the brain and load transport to the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells and glial cells), glucose transporters, and LDL receptor ligands.

[0586] In some embodiments, the compound further comprises a targeting ligand that targets and mediates delivery to receptors in specific ocular tissues. These targeting ligands may be conjugated with a lipophilic moiety to achieve specific ocular delivery (e.g., intravitreal delivery) and systemic delivery. Exemplary targeting ligands that target and mediate delivery to receptors in ocular tissues are lipophilic ligands such as all-trans retinol (which targets retinal pigment epithelial cells); RGD peptides (which target retinal pigment epithelial cells), such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO:1) or Cyclo(-Arg-Gly-Asp-D-Phe-Cys; LDL receptor ligands); and carbohydrate-based ligands (which target endothelial cells in the posterior eye).

[0587] Preferred conjugation groups suitable for use in this invention include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); and thiocholesterols (Oberhauser et al., Nucl. Acids). Res. [Nucleic Acid Research], 1992, 20, 533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J. [Journal of the European Society for Molecular Biology], 1991, 10, 111; Kabanov et al., FEBS Lett. [Communications of the Federation of European Biochemical Societies], 1990, 259, 327; Svinarchuk et al., Biochimie [Biochemistry], 1993, 75, 49); phospholipids, such as di-hexadecyl-racemic-glycerol or triethylammonium-1,2-di-O-hexadecyl-racemic-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. [Tetrahedron Communications], 1995, 36, 3651; Shea et al., Nucl. Acids Res. [Nucleic Acid Research], 1990, 18, 3777); polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides [Nucleosides and Nucleotides], 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett. [Tetrahedron Communications], 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta [Chinese Journal of Biochemistry and Biophysics], 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-hydroxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. [Journal of Pharmacology and Experimental Therapy], 1996, 277, 923).

[0588] In general, a wide variety of entities (e.g., ligands) can be coupled with the oligomeric compounds described herein. Ligands may 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-glycolic acid) copolymer, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, such as PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazene, polyethyleneimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide mimic polyamine, dendritic polymer polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrin, quaternary salts of polyamines. Thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamate, polyaspartic acid, aptamers, desialylated fetoglobulin, hyaluronic acid, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, glyco-albumin conjugates, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantine, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-o-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-Propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine), peptides (e.g., α-helical peptides, amphiphilic peptides, RGD peptides, cell-penetrating peptides, endosome cleavage / membrane fusion peptides), alkylating agents, phosphate esters, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., naproxen, aspirin). Lin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazolium conjugates, tetraazamacyclohexane Eu3+ complexes), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, such as folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 Activators of MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, 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 permeators (e.g., spiral cell permeators).

[0589] Peptides and peptide mimic ligands include ligands having naturally occurring or modified peptides, such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azide peptides; peptides having one or more amides, i.e., peptides whose bonds are replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides. Peptide mimics (also referred to herein as oligopeptide mimics) are molecules capable of folding into a defined three-dimensional structure similar to that of a natural peptide. Peptides or peptide mimic ligands can be about 5–50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0590] Exemplary amphiphilic 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), magainine, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentini-1, and caerin.

[0591] As used herein, the term "endosome cleavage ligand" refers to a molecule possessing endosome cleavage properties. Endosome cleavage ligands facilitate the dissolution of the compositions or components thereof of the present invention and / or their transport from intracellular cellular compartments, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, perosomes, or other vesicles, to the cytoplasm of the cell. Some exemplary endosome cleavage ligands include, but are not limited to, imidazoles, polyimide or oligoimide, linear or branched polyethyleneimine (PEI), linear and branched polyamines (e.g., spermine, cationic linear and branched polyamines), polycarboxylate esters, polycationic, masked oligomers or polycationic or anionic, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers having masked or unmasked cationic or anionic charges, dendritic polymers having masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimics, pH-sensitive peptides, natural and synthetic membrane-fused lipids, and natural and synthetic cationic lipids.

[0592] Exemplary endosomal lysis / membrane fusion peptides include but are not limited to AALEALAEALEALAEALEALAEAAAAGGC(GALA)(SEQ ID NO:3); AALAEALAEALAEALAEALAEALAAAAGGC(EALA)(SEQ ID NO:4); ALEALAEALEALAEA(SEQ ID NO:5); GLFEAIEGFIENGWEGMIWDYG(INF-7)(SEQ ID NO:6); GLFGAIAGFIENGWEGMIDGWYG(Inf HA-2)(SEQ ID NO:7); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC(diINF-7)(SEQ ID NO:8); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC(diINF-3)(SEQ ID NO:9); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC(GLF)(SEQ ID NO:10); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC(GALA-INF3)(SEQ ID NO:11); GLF EAI EGFI ENGW EGnI DG KGLF EAI EGFI ENGW EGnI DG(INF-5, n is norleucine)(SEQID NO:12); LFEALLELLESLWELLLEA(JTS-1)(SEQ ID NO:13); GLFKALLKLLKSLWKLLLKA(ppTG1)(SEQ ID NO:14); GLFRALLRLLRSLWRLLLRA(ppTG20)(SEQ ID NO:15); WEAKLAKALAKALAKHLAKALAKALKACEA(KALA)(SEQ ID NO:16); GLFFEAIAEFIEGGWEGLIEGC(HA)(SEQ ID NO:17); GIGAVLKVLTTGLPALISWIKRKRQQ(melittin)(SEQ ID NO:18); H5WYG(SEQ IDNO:19); and CHK6HC(SEQ ID NO:20).

[0593] Unwilling to be bound by theory, membrane-fused lipids fuse with membranes and thus destabilize them. Membrane-fused lipids typically have small head groups and unsaturated acyl chains. Exemplary membrane-fused lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptadecano-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadecano-9,12-dienyl)-1,3-dioxolane-4-yl)methylamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadecano-9,12-dienyl)-1,3-dioxolane-4-yl)ethylamine (also referred to herein as XTC).

[0594] Synthetic polymers with endosome cleavage activity suitable for use in this invention are described in U.S. Patent Application Publications Nos. 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the contents of which are incorporated herein by reference in their entirety.

[0595] Exemplary cell permeation peptides include, but are not limited to, RQIKIWFQNRRMKWKK (membrane-penetrating peptide) (SEQ ID NO:21); GRKKRRQRRRPPQC (Tat fragment 48-60) (SEQ ID NO:22); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence-based peptide) (SEQ ID NO:23); LLIILRRRIRKQAHAHSK (PVEC) (SEQ ID NO:24); GWTLNSAGYLLKINLKALAALAKKIL (transport peptide) (SEQ ID NO:25); KLALKLALKALKAALKLA (amphiphilic model peptide) (SEQ ID NO:26); RRRRRRRRR (Arg9) (SEQ ID NO:27); KFFKFFKFFK (bacterial cell wall permeation peptide) (SEQ ID NO:28); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO:28). NO:29); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (bactericidal peptide P1) (SEQ ID NO:30); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO:31); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO:32); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO:33); ILPWKWPWWPWRR-NH2 (peptide antibiotic) (SEQ ID NO:34); AAVALLPAVLLALLAP (RFGF) (SEQ ID NO:35); AALLPVLLAAP (RFGF analog) (SEQ ID NO:36); and RKCRIVVIRVCR (bovine antimicrobial peptide) (SEQ ID NO:37).

[0596] Exemplary cationic groups include, but are not limited to, protonated amino groups derived from, for example, O-amines (amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy groups, such as O(CH2). nAmines (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); amino groups (e.g., NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); and NH (CH2CH2NH). n CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino or diheteroarylamino).

[0597] As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0598] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, polygalactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc such as GalNAc2 and GalNAc3 (GalNAc and polyvalent GalNAc are collectively referred to as GalNAc conjugates herein); D-mannose, polymannose, polylactose, N-acetyl-glucosamine, glucose, polyglucose, polytrehalose, glycosylated polyamino acids, and lectins. The term polyvalent indicates the presence of more than one monosaccharide unit. These monosaccharide subunits can be linked to each other or to the backbone molecule via glycosidic bonds.

[0599] Many folic acid and folic acid analogues suitable as ligands for the present invention are described in U.S. Patent Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the contents of which are incorporated herein by reference in their entirety.

[0600] As used herein, the terms “PK-modifying ligand” and “PK modulator” refer to molecules that 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, phenoxazine, aspirin, naproxen, ibuprofen, sulofen, ketoprofen, (S)-(+)-pranoprofen, carboprofen, PEG, biotin, and thyroxine transporter binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). It is also known that oligomers containing multiple thiophosphate glycosidic bonds bind to serum proteins. Therefore, short oligomers, such as oligonucleotides containing about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and containing multiple thiophosphate bonds in the main chain, are also suitable as ligands (e.g., as PK-regulating ligands) in this invention. PK-regulating oligonucleotides may contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more thiophosphate and / or dithiophosphate bonds. In some embodiments, all nucleotide internucleotide bonds in the PK-regulating oligonucleotide are thiophosphate and / or dithiophosphate bonds. Additionally, aptamers that bind serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in this invention. Binding to serum components (e.g., serum proteins) can be predicted from albumin binding assays, such as those described in Oravcova et al., Journal of Chromatography B (1996), 677:1-27.

[0601] 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 others have different properties. For example, ligands may have targeting properties, endosome cleavage activity, or PK modulation properties. In a preferred embodiment, all ligands have different properties.

[0602] When a monomer is incorporated into a component of a compound of the present invention (e.g., a compound or linker of the present invention), a ligand or chain ligand may be present on the monomer. In some embodiments, after a “precursor” monomer has been incorporated into a component of a compound of the present invention (e.g., a compound or linker of the present invention), a ligand may be incorporated into the “precursor” monomer via coupling. For example, a monomer having, for example, an amino-terminated chain (i.e., without an associated ligand), such as monomer-linker-NH2, may be incorporated into a component of a compound of the present invention (e.g., a compound or linker of the present invention). In a subsequent operation, i.e., after the precursor monomer has been incorporated into a component of a compound of the present invention (e.g., a compound or linker of the present invention), a ligand having an electrophilic group, such as a pentafluorophenyl ester or an aldehyde group, may subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the chain of the precursor monomer.

[0603] In another example, a chain linker / connector with a monomer having a chemical group suitable for participating in click chemistry, such as an azide or alkyne-terminated chain, can be incorporated. In subsequent operations, i.e., after the precursor monomer has been incorporated into the chain, a ligand with a complementary chemical group (e.g., alkyne or azide) can be attached to the precursor monomer by coupling the alkyne and azide together.

[0604] In some embodiments, the ligand may conjugate to a nucleobase, sugar moiety, or nucleoside internucleotide of the compound of the present invention. Conjugation with a purine nucleobase or a derivative thereof may occur at any position, including inner and outer ring atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugation moiety. Conjugation with a pyrimidine nucleobase or a derivative thereof may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase may be substituted by the conjugation moiety. When the ligand conjugates to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., one that does not interfere with the hydrogen bonding interactions required for base pairing.

[0605] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to the conjugated moiety include 2', 3', and 5' carbon atoms. The 1' position can also be attached to the conjugated moiety, such as in a base-free residue. Nucleoside interbonds can also have conjugated moieties. For phosphorus-containing bonds (e.g., phosphate diesters, thiosulfates, dithiophosphates, aminophosphodiesters, etc.), the conjugated moiety can be directly attached to the phosphorus atom or the O, N, or S atom bonded to that phosphorus atom. For nucleoside interbonds containing amines or amides (e.g., PNA), the conjugated moiety can be attached to the nitrogen atom of the amine or amide or an adjacent carbon atom.

[0606] There are many methods for preparing oligonucleotide conjugates. Generally, oligonucleotides are attached to a conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, or similar group) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the others are nucleophilic.

[0607] For example, the electrophilic group can be a carbonyl functional group and the nucleophilic group can be an amine or a thiol. Methods for conjugating nucleic acids and related oligomers with and without linker groups are well described in the literature, such as Manoharan, in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0608] Ligands may be attached to the compounds of the present invention via a linker or a carrier monomer (e.g., a ligand carrier). The carrier includes (i) at least one “main chain attachment point,” preferably two “main chain attachment points,” and (ii) at least one “tether attachment point.” As used herein, a “main chain attachment point” refers to a functional group, such as a hydroxyl group, or generally a bond, that can be used and is suitable for incorporating a carrier monomer into the main chain (e.g., a phosphate ester of an oligonucleotide or a modified phosphate ester, such as a sulfur-containing main chain). A “tether attachment point” (TAP) refers to an atom, such as a carbon atom or a heteroatom (different from the atom providing the main chain attachment point), that connects a selected portion of the carrier monomer. The selected portion may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected portion is connected to the carrier monomer by an inserted tether. Thus, the carrier will generally include a functional group, such as an amino group, or generally provide a bond suitable for incorporating or tethering another chemical entity (e.g., a ligand) to the constituent atoms.

[0609] Representative U.S. patents teaching the preparation of nucleic acid conjugates include, but are not limited to, U.S. patent numbers 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; and 5,486,603. 5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,149,782;

[0610] 5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,37 1; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; 6,559,279; The contents of these references are incorporated herein by reference in their entirety.

[0611] 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.

[0612] Because the ligand can be conjugated to the iRNA agent via a linker or carrier, and because the linker or carrier can contain branching heads, the iRNA agent can subsequently be attached via the same or different backbone sites of the carrier, or via branching heads containing multiple ligands. For example, the branching points of the branching heads can be divalent, trivalent, tetravalent, pentavalent, or hexavalent atoms, or present such multivalent groups. In some embodiments, the branching points are -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; wherein Q is independently H or optionally a substituted alkyl group each time it appears. In other embodiments, the branching point is glycerol or a glycerol derivative.

[0613] Evaluation of candidate iRNAs

[0614] We can evaluate candidate iRNA agents, such as modified RNA, against selected properties by exposing the agent or modified molecule and control molecule to appropriate conditions and assessing the presence of the selected properties. For example, resistance to degrading agents can be assessed as follows: The candidate modified RNA (and the control molecule, typically in its unmodified form) can be exposed to degrading conditions, such as exposure to an environment containing degrading agents (e.g., nucleases). For example, we can use biological samples, such as those similar to the environment that might be encountered for therapeutic purposes, such as blood or cellular portions, such as cell-free homogenates or lysed cells. The resistance of the candidate and control to degradation can then be assessed using any of a variety of methods. For example, the candidate and control can be labeled with, for example, radioactive or enzyme-labeled or fluorescent labels (such as Cy3 or Cy5) prior to exposure. The control and modified RNA can be incubated with the degrading agent and optional control (e.g., inactivated, heat-inactivated) degrading agent. The physical parameters, such as size, of the modified molecule and control molecule are then determined. It can be determined by physical methods, such as polyacrylamide gel electrophoresis or fractionation columns, to assess whether the molecule retains its original length or its function. Alternatively, RNA blotting can be used to determine the length of unlabeled modified molecules.

[0615] Functional assays can also be used to evaluate candidate agents. Functional assays can be applied initially or after earlier non-functional assays (e.g., assays 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, such as 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, for example, WO 00 / 44914). For example, the ability of a modified dsiRNA homologous to GFP mRNA to inhibit GFP expression can be determined by monitoring a decrease in cellular fluorescence, compared to control cells transfected without the candidate dsiRNA (e.g., a control without the agent and / or a control with unmodified RNA). The efficacy of a candidate agent on gene expression can be assessed by comparing cellular fluorescence in the presence of modified and unmodified dssiRNA compounds.

[0616] In another functional assay, candidate dssiRNA compounds homologous to endogenous mouse genes (e.g., 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, WO 01 / 36646). Oocyte phenotypes (e.g., the ability to maintain arrest in metaphase II) can be monitored as indicators of the agent's inhibitory effect. For example, cleavage of c-mos mRNA by a dssiRNA compound leads to oocyte exit from metaphase arrest and initiation of parthenogenesis (Colledge et al., Nature 370:65-68, 1994; Hashimoto et al., Nature 370:68-71, 1994). The effect of the modified agent on target RNA levels compared to a negative control can be verified by RNA blotting to determine a decrease in target mRNA levels or by protein blotting to determine a decrease in target protein levels. Controls may include cells in which no agent was added and / or cells in which unmodified RNA was added.

[0617] Physiological effects

[0618] The siRNA compounds described herein can be designed to more easily determine therapeutic toxicity through the complementarity of siRNA with human and non-human animal sequences. Through these methods, siRNA can consist of sequences that are fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal (e.g., a non-human mammal, such as a rodent, ruminant, or primate). For example, non-human mammals can be mice, rats, dogs, pigs, goats, sheep, cattle, monkeys, bonobos, chimpanzees, rhesus monkeys, or cynomolgus monkeys. The sequence of the siRNA compound can be complementary to sequences within homologous genes (e.g., oncogenes or tumor suppressor genes) in both non-human mammals and humans. By determining the toxicity of the siRNA compound in non-human mammals, we can infer its toxicity in humans. For more demanding toxicity testing, siRNA can be complementary to humans and more than one (e.g., two or three or more) non-human animals.

[0619] The methods described herein can be used to associate any physiological effects of siRNA compounds on humans, such as any unwanted effects, like toxic effects, or any positive or desired effects.

[0620] Increase cellular uptake of siRNA

[0621] This article describes various siRNA compositions containing covalently attached conjugates that increase siRNA uptake and / or intracellular targeting.

[0622] Additionally, a method of the present invention is provided, comprising administering an siRNA compound and a drug that affects siRNA uptake into cells. The drug may be administered before, after, or simultaneously with the administration of the siRNA compound. The drug may be covalently or non-covalently linked to the siRNA compound. The drug may be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drug may have a transient effect on cells. The drug may increase siRNA compound uptake into cells, for example, by disrupting the cellular cytoskeleton, such as by disrupting microtubules, microfilaments, and / or intermediate filaments. The drug may be, for example, tacrolimus, vincristine, vinblastine, cytochalasin, nocodazole, jepkenlide, lenokulin A, phalloidin, svenholid A, indanoxin, or mesene. The drug may also increase siRNA compound uptake into a given cell or tissue by, for example, activating an inflammatory response. Exemplary drugs with such effects include tumor necrosis factor α (TNFα), interleukin-1β, CpG motifs, gamma interferon, or more generally agents that activate Toll-like receptors.

[0623] siRNA production

[0624] siRNA can be produced in large quantities, for example, through a variety of methods. Exemplary methods include organic synthesis and RNA lysis, such as in vitro lysis.

[0625] Organic synthesis. siRNA can be prepared by synthesizing the individual strands of a single-stranded or double-stranded RNA molecule separately, followed by annealing the constituent strands.

[0626] Large bioreactors, such as the 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 1.5 moles of excess phosphoramidine nucleotides. Ribonucleotide amino acids are used to produce the RNA strands. A standard cycle of monomer addition can be used to synthesize 21 to 23 nucleotide strands of siRNA. Typically, the two complementary strands are prepared separately and subsequently annealed, for example, after release from the solid support and deprotection.

[0627] Organic synthesis can be used to generate discrete siRNA species. The complementarity of the species to a specific target gene can be precisely specified. For example, the species can be complementary to a region that includes polymorphisms (e.g., single nucleotide polymorphisms). Furthermore, the location of the polymorphism can be precisely determined. In some embodiments, the polymorphism is located in an internal region, such as at least 4, 5, 7, or 9 nucleotides from one or both ends.

[0628] dsiRNA cleavage. siRNA can also be prepared by cleaving larger siRNAs. Cleavage can be mediated in vitro or in vivo. For example, to generate iRNA through in vitro cleavage, the following methods can be used:

[0629] In vitro transcription. dsiRNA is produced by transcribing segments of nucleic acid (DNA) in both directions. For example, HiScribe TM The RNAi transcription kit (New England Biolabs) provides a vector and method for generating dsiRNA from a nucleic acid segment cloned into the vector at a position flanked by the T7 promoter on either side. Separate templates are generated for T7 transcription of the two complementary strands of the dsiRNA. The template is transcribed in vitro using T7 RNA polymerase to produce dsiRNA. Similar methods using PCR and / or other RNA polymerases (e.g., T3 or SP6 polymerases) can also be contaminated with toxins that may contaminate the recombinant enzyme preparation.

[0630] In vitro lysis. In one embodiment, the RNA produced by this method is carefully purified to remove terminal siRNA, which is cleaved in vitro into siRNA, for example, using Dicer or similar RNase III-based enzymes. For example, dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, such as purified RNase or RISC complex (RNA-induced silencing complex). See, for example, Ketting et al. Genes Dev [Genes and Development] 15 Oct 2001; 15(20):2654-9; and Hammond Science [Science] 10 Aug 2001; 293(5532):1146-50.

[0631] dsiRNA cleavage typically produces multiple siRNA species, each being a specific 21 to 23 nt fragment of the source dsiRNA molecule. For example, siRNAs may include sequences that overlap with and are complementary to adjacent regions of the source dsiRNA molecule.

[0632] Regardless of the synthesis method, siRNA formulations can be prepared in solutions suitable for formulation (e.g., aqueous and / or organic solutions). For example, siRNA formulations can be precipitated and redissolved in double-distilled water and then lyophilized. The dried siRNA can then be resuspended in a solution suitable for the intended formulation method.

[0633] Manufacturing double-stranded iRNA agents conjugated to the lipophilic portion

[0634] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the compound via a nucleobase, sugar moiety, or nucleoside bond.

[0635] Conjugation with a purine nucleobase or a derivative thereof can occur at any position, including both inner and outer ring atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugation moiety. Conjugation with a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase may be substituted by the conjugation moiety. When the lipophilic moiety is conjugated with 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). In one embodiment, a lipophilic monomer containing a lipophilic moiety can be conjugated with a nucleobase via a linker containing an alkyl, alkenyl, or amide bond.

[0636] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety to which the lipophilic moiety can attach include 2', 3', and 5' carbon atoms. The lipophilic moiety can also attach to the 1' position, such as in a base-free residue. In one embodiment, the lipophilic moiety can be conjugated to the sugar moiety via a 2'-O modification with or without a linker.

[0637] Nucleoside bonds can also have a lipophilic moiety. For phosphorus-containing bonds (such as phosphate diesters, thiophosphate esters, thiophosphate diesters, phosphorylamino acids, etc.), the lipophilic moiety can be directly attached to the phosphorus atom or the O, N, or S atom bonded to the phosphorus atom. For nucleoside bonds containing amines or amides (such as PNA), the lipophilic moiety can be attached to the nitrogen atom of the amine or amide or an adjacent carbon atom.

[0638] There are many methods for preparing oligonucleotide conjugates. Generally, oligonucleotides are attached to a conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, or similar group) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the others are nucleophilic.

[0639] For example, the electrophilic group can be a carbonyl functional group and the nucleophilic group can be an amine or a thiol. Methods for conjugating nucleic acids and related oligomers with and without linker groups are well described in the literature, such as Manoharan, in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0640] In one embodiment, a first (complementary) RNA strand and a second (sense) RNA strand can be synthesized separately, wherein one of the RNA strands contains a lipophilic moiety, and the first and second RNA strands can be mixed to form dsRNA. The step of synthesizing the RNA strand preferably involves solid-phase synthesis, wherein the individual nucleotides are end-to-end joined by forming internucleotide 3'-5' phosphodiester bonds in a series of synthesis cycles.

[0641] In one embodiment, a lipophilic molecule having a phosphoramidite group is coupled to the 3' or 5' end of a first (complementary) or second (sense) RNA strand in the final synthetic cycle. In solid-phase RNA synthesis, the nucleotide is initially in the form of a nucleoside phosphoramidite. In each synthetic cycle, another nucleoside phosphoramidite is linked to the -OH group of the previously incorporated nucleotide. If the lipophilic molecule has a phosphoramidite group, it can be coupled to the free OH end of the RNA previously synthesized in solid-phase synthesis in a manner similar to that of a nucleoside phosphoramidite. Synthesis can be performed in an automated and standardized manner using a conventional RNA synthesizer. The synthesis of a lipophilic molecule having a phosphoramidite group may include phosphorylation of the free hydroxyl group to generate a phosphoramidite group.

[0642] Generally, oligonucleotides can be synthesized using methods known in the art, for example, as described in Caruthers et al., Methods in Enzymology (1992) 211:3-19; 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, each of which is incorporated herein by reference in its entirety. Generally, the synthesis of oligonucleotides involves conventional nucleic acid protection and coupling groups, such as dimethoxytriphenylmethyl at the 5' end and phosphoramidite at the 3' end. In a non-limiting example, nucleoside phosphoramide, marketed by ChemGenes Corporation (Ashland, Massachusetts), was synthesized on a small scale using an Expedite 8909 RNA synthesizer marketed by Applied Biosystems, Inc. (Weiterstadt, Germany). Alternatively, the synthesis can be performed on a 96-well disc synthesizer, such as that manufactured by Protogene (Palo Alto, California), or by methods 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, each of which is incorporated herein by reference in its entirety.

[0643] The nucleic acid molecules of the present invention can be synthesized individually and then concatenated together, for example, by ligation after synthesis and / or deprotection (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. The nucleic acid molecules can be purified by gel electrophoresis using conventional methods, or by high-performance liquid chromatography (HPLC; see Wincott et al., above, all of which are incorporated herein by reference) and resuspended in water.

[0644] Pharmaceutical Composition

[0645] In one aspect, the invention is characterized by a pharmaceutical composition comprising an siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof), the compound comprising a nucleotide sequence complementary (e.g., substantially and / or precisely complementary) to a target RNA. The target RNA may be a transcript of an endogenous human gene. In one embodiment, the siRNA compound (a) is 19-25 nucleotides long, for example 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and optionally, (c) includes at least one 3' overhang of 1-5 nt in length. In one embodiment, the pharmaceutical composition may be an emulsion, microemulsion, cream, gel, or liposome.

[0646] In one example, the pharmaceutical composition comprises an siRNA compound mixed with a local delivery agent. The local delivery agent may be a plurality of microvesicles. The microvesicles may be liposomes. In some embodiments, the liposomes are cationic liposomes.

[0647] In another aspect, the pharmaceutical composition comprises a siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding a siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof), mixed with a local permeation enhancer. In one embodiment, the local permeation enhancer is a fatty acid. The fatty acid may be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, tridecanoate, monooleate, dilaurate, 1-monodecanoate, 1-dodecylazine-2-one, acylcarnitine, acylcholine, or C 1-10 Alkyl esters, monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof.

[0648] In another embodiment, the local permeability enhancer is a bile salt. The bile salt may be cholic acid, dehydrocholic acid, deoxycholic acid, glutamic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium taurine-24,25-dihydro-fusidic acid, sodium diol dihydrofusidic acid, polyoxyethylene-9-lauryl ether, or a pharmaceutically acceptable salt thereof.

[0649] In another embodiment, the penetration enhancer is a chelating agent. The chelating agent may be EDTA, citric acid, salicylate, N-acyl derivatives of collagen, lauryl ether-9, N-aminoacyl derivatives of β-diketone, or mixtures thereof.

[0650] In another embodiment, the penetration enhancer is a surfactant, such as an ionic or nonionic surfactant. The surfactant may be sodium dodecyl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-hexadecyl ether, perfluorinated chemical emulsions, or mixtures thereof.

[0651] In another embodiment, the penetration enhancer may be selected from the group consisting of unsaturated cycloureas, 1-alkyl-alkane ketones, 1-alkenylazine-propane ketones, steroid anti-inflammatory agents, and mixtures thereof. In another embodiment, the penetration enhancer may be a diol, pyrrole, azone, or terpene.

[0652] In one aspect, the invention is characterized by a pharmaceutical composition in a form suitable for oral delivery, comprising a siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding a siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, oral delivery can be used to deliver the siRNA compound composition to cells or regions of the gastrointestinal tract, such as the small intestine, colon (e.g., for treating colon cancer), etc. The oral delivery form may be a tablet, capsule, or gel capsule. In one embodiment, the siRNA compound of the pharmaceutical composition modulates the expression of cell adhesion proteins, regulates cell proliferation rates, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition comprises an enteric material that substantially prevents the tablet, capsule, or gel capsule from dissolving in the mammalian stomach. In some embodiments, the enteric material is a coating. The coating may be phthalate acetate, propylene glycol, sorbitan monooleate, cellulose trimellitate acetate, hydroxypropyl methylcellulose phthalate, or cellulose acetate.

[0653] In another embodiment, the oral dosage form of the pharmaceutical composition includes a permeation enhancer. The permeation enhancer may be a bile salt or a fatty acid. The bile salt may be ursodeoxycholic acid, chenodeoxycholic acid, or their salts. The fatty acid may be decanoic acid, lauric acid, or their salts.

[0654] 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 glyceryl stearate (precirol).

[0655] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer may be diethyl phthalate, glyceryl triacetate, dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0656] In one aspect, the invention is characterized by a pharmaceutical composition comprising an siRNA compound and a delivery medium. In one embodiment, the siRNA compound (a) is 19-25 nucleotides long, for example 21-23 nucleotides long, (b) is complementary to an endogenous target RNA, and optionally, (c) includes at least one 3' overhang of 1-5 nucleotides long.

[0657] In one embodiment, the delivery medium can deliver siRNA compounds, such as double-stranded siRNA compounds or ssiRNA compounds (e.g., precursors, such as larger siRNA compounds that can be processed into ssiRNA compounds, or DNA encoding siRNA compounds (e.g., double-stranded siRNA compounds or ssiRNA compounds or their precursors) to cells via a local administration route. The delivery medium can be microvesicles. In one example, the microvesicles are liposomes. In some embodiments, the liposomes are cationic liposomes. In another example, the microvesicles are microcells. In one aspect, the invention is characterized by pharmaceutical compositions in an injectable dosage form comprising siRNA compounds, such as double-stranded siRNA compounds or ssiRNA compounds (e.g., precursors, such as larger siRNA compounds that can be processed into ssiRNA compounds, or DNA encoding siRNA compounds (e.g., double-stranded siRNA compounds or ssiRNA compounds or their precursors)). In one embodiment, the injectable dosage form of the pharmaceutical composition comprises a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile solution may include a diluent such as water; a saline solution; a non-volatile oil, polyethylene glycol, glycerin, or propylene glycol.

[0658] In one aspect, the invention is characterized by an oral dosage form of a pharmaceutical composition comprising a siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding a siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules, and gel capsules. In another embodiment, the pharmaceutical composition comprises an enteric coating material that substantially prevents the tablet, capsule, or gel capsule from dissolving in the mammalian stomach. In some embodiments, the enteric coating material is a coating. The coating may be phthalate acetate, propylene glycol, sorbitan monooleate, cellulose trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate. In one embodiment, the oral dosage form of the pharmaceutical composition comprises a penetration enhancer, such as those described herein.

[0659] 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 glyceryl stearate (precirol).

[0660] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer may be diethyl phthalate, glyceryl triacetate, dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0661] In one aspect, the invention is characterized by a pharmaceutical composition in the form of a rectal suppository comprising a siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding a siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

[0662] In one aspect, the invention is characterized by a pharmaceutical composition in the form of a vaginal suppository comprising a siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding a siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

[0663] In one aspect, the invention is characterized by a pharmaceutical composition in a pulmonary or nasal dosage form comprising an siRNA compound, such as a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed into an ssiRNA compound, or DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, the siRNA compound is incorporated into particles, such as large particles, such as microspheres. The particles can be produced by spray drying, lyophilization, evaporation, fluidized bed drying, vacuum drying, or a combination thereof. The microspheres can be formulated as a suspension, powder, or implantable solid.

[0664] Treatment methods and delivery routes

[0665] Another aspect of the invention relates to a method for reducing the expression of target genes in cells, the method comprising contacting the cells with a compound of the invention. In one embodiment, the cells are extrahepatic cells.

[0666] Another aspect of the present invention relates to a method for reducing the expression of target genes in a subject, the method comprising administering the compound of the present invention to the subject.

[0667] Another aspect of the invention relates to a method for treating a subject suffering from a CNS disorder, the method comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the invention, thereby treating the subject. Exemplary CNS disorders treatable by the method of the invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prions, and Lafra disease.

[0668] The compounds of the present invention can be delivered to subjects via a variety of routes, depending on the type of gene targeted and the type of disorder to be treated. In some embodiments, the compounds are administered extrahepatically, such as via ocular administration (e.g., intravitreal administration), or via intrathecal or intraventricular administration.

[0669] In one embodiment, the compound is administered intrathecally or intraventricularly. This method reduces the expression of target genes in brain or spinal tissues (e.g., cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae) by intrathecal or intraventricular administration of the double-stranded iRNA agent.

[0670] 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, the compound can be applied directly (e.g., intravitreally) to one or more eyes. This method reduces the expression of target genes in ocular tissue by intravitreal administration of a double-stranded iRNA agent.

[0671] For ease of explanation, the formulations, compositions, and methods described in this section are primarily directed toward modified siRNA compounds. However, it is understood that these formulations, compositions, and methods can be practiced with other siRNA compounds (e.g., unmodified siRNA compounds), and such practices are within the scope of this invention. Compositions comprising iRNA can be delivered to the subject via a variety of routes. Exemplary routes include intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular delivery.

[0672] The iRNA molecules of this invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise one or more iRNA species 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 agents, and absorption delay agents and the like, compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Additional active compounds may also be incorporated into the composition.

[0673] The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be local (including ocular, vaginal, rectal, intranasal, and percutaneous), oral, or parenteral. Non-enteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal, intracardiac, or intraventricular administration.

[0674] The route and site of administration can be chosen to enhance targeting. For example, intramuscular injection into the muscle of interest would be a logical choice to target muscle cells. Lung cells can be targeted by administering iRNA in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with iRNA and mechanically introducing DNA.

[0675] Formulations for topical application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powdered, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

[0676] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or sugar tablets. In the case of tablets, carriers that may 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 an aqueous suspension is required for oral use, nucleic acid compositions may be combined with emuls...

Claims

1. A compound comprising: The antisense strand is complementary to the target gene; a sense strand that is complementary to the antisense strand; and One or more lipophilic monomers, wherein the lipophilic monomer is selected from the group consisting of: in: m is an integer from 0 to 8; n is an integer from 1 to 21; B is a modified or unmodified nucleobase; and R" are each independently H or C1-C4 alkyl; and At least one lipophilic monomer is conjugated to one or more internal positions on at least one chain.

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

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

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

5. The compound of claim 4, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having 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 the plurality of ends.

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 1-7, wherein the sense strand and antisense strand comprise less than ten 2'-fluoro-modified nucleotides.

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

10. The compound of claim 1, wherein the lipophilic monomer is selected from the group consisting of:

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

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

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

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

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

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

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

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

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

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

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

22. The compound of claim 21, wherein the targeting ligand is an RGD peptide and the RGD peptide is H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 1) or Cyclo (-Arg-Gly-Asp-D-Phe-Cys).

23. The compound of claim 20, wherein the targeting ligand is a carbohydrate-based ligand.

24. The compound of claim 1, wherein the internal position includes all positions except the terminal positions at each of the 3' and 5' ends of the chain, or except the terminal two positions at each of the 3' and 5' ends of the chain, or except the terminal three positions at each of the 3' and 5' ends of the chain.

25. The compound of claim 1, wherein the internal position does not include the cleavage site region of the sense strand.

26. The compound of claim 25, wherein the internal positions do not include positions 9-12 counted from the 5' end of the sense strand, or positions 11-13 counted from the 3' end of the sense strand.

27. The compound of claim 1, wherein the internal position does not include the cleavage site region of the antisense strand.

28. The compound of claim 27, wherein the internal positions do not include positions 12-14 counted from the 5' end of the antisense strand.

29. The compound of claim 1, wherein the internal positions do not include positions 11-13 counted from the 3' end on the sense strand and positions 12-14 counted from the 5' end on the antisense strand.

30. The compound of any one of claims 1-17, wherein at least one lipophilic monomer is located on the sense strand within the first five base pairs of the 5' end of the antisense strand of the duplex region.

31. The compound of any one of claims 1-17, wherein 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.

32. The compound of any one of claims 1-17, wherein 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.

33. The compound of claim 9, wherein the sense strand and the antisense strand comprise at least 60% 2'-OMe modified nucleotides.

34. The compound of claim 9, wherein the sense strand and the antisense strand comprise at least 70% 2'-OMe modified nucleotides.

35. The compound of any one of claims 1-17, wherein at least one lipophilic monomer is located on the sense strand within the first four base pairs of the 5' end of the antisense strand of the duplex region.

36. The compound of any one of claims 1-17, wherein at least one lipophilic monomer is located on the sense strand within the first three base pairs of the 5' end of the antisense strand of the duplex region.

37. The compound of any one of claims 1-17, wherein at least one lipophilic monomer is located on the sense strand within the first two base pairs of the 5' end of the antisense strand of the duplex region.

38. The compound of any one of claims 1-17, wherein at least one lipophilic monomer is located on the sense strand within one base pair before the 5' end of the antisense strand of the duplex region.

39. The compound of any one of claims 25-32, wherein the compound contains only one lipophilic monomer.

40. The compound of any one of claims 1-17 and 39, wherein one lipophilic monomer is located at position 5 on the sense strand, counting from the 5' end of the strand.

41. The compound of any one of claims 1-17 and 39, wherein one lipophilic monomer is located at position 6 on the sense strand, counting from the 5' end of the strand.

42. The compound of any one of claims 1-17 and 39, wherein one lipophilic monomer is located at position 7 on the sense strand, counting from the 5' end of the strand.

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