Extrahepatic delivery

Abstract

One aspect of the 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 optionally conjugated to one or more positions on the at least one chain via a linker or carrier. Another aspect of the invention relates to a method of gene silencing comprising administering to a cell or a subject in need thereof a therapeutically effective amount of a lipophilic monomer conjugated compound.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of November 6, 2020, application number 202080093231.5, and invention name “Extrahepatic Delivery”.

[0002] Sequence Listing

[0003] This application includes a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 6, 2022, is named 29520_1503_SL.txt, and is 52,281 bytes in size. Background Art

[0004] Effective delivery of iRNA agents to cells in vivo requires specific targeting and substantial protection from the extracellular environment (particularly serum proteins). RNAi-based therapies show promising clinical data for the treatment of liver-related disorders. However, there are still obstacles to delivering siRNA to extrahepatic tissues, which limits the use of siRNA-based therapies.

[0005] One of the limiting factors for in vivo experiments and therapeutic applications of iRNA agents is the ability to effectively deliver intact siRNA. Particular difficulties have been associated with non-viral gene transfer to the retina in vivo. One of the challenges is overcoming the inner limiting membrane, which hinders retinal transfection. Additionally, negatively charged sugars in the vitreous have been shown to interact with positive DNA-transfection reagent complexes, promoting their aggregation, which hinders diffusion and cellular uptake.

[0006] Because free oligonucleotides cannot pass through the blood-brain barrier (BBB), oligonucleotides cause specific problems to the delivery of central nervous system (CNS). A mode of oligonucleotide delivery in CNS is by intrathecal delivery. However, oligonucleotides also need to be effectively internalized in the target cells of CNS to achieve the therapeutic effect of expectation. Previous research usually uses the delivery reagents such as liposomes, cationic lipids and nanoparticles to form complexes, to help oligonucleotides to be internalized in the cells of neuronal origin.

[0007] Therefore, there is a continuing need for new and improved methods for delivering siRNA molecules in vivo without the use of tissue delivery agents to realize and enhance the therapeutic potential of iRNA agents. Summary of the Invention

[0008] One aspect of the present invention provides a compound (e.g., an oligonucleotide that can be single-stranded or double-stranded) comprising one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand of the oligonucleotide, optionally via a linker or carrier. For example, some embodiments of the present 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 the antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand, optionally via a linker or carrier.

[0009] In some embodiments, the octanol-water partition coefficient log K ow The lipophilicity of the measured lipophilic fraction exceeds 0. The logK of the lipophilic fraction ow It may be more than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5 or more than 10.

[0010] In some embodiments, the hydrophobicity of the compound as measured by the unbound fraction in a plasma protein binding assay of the compound is greater than 0.2. In one embodiment, the plasma protein binding assay measured is an electrophoretic mobility shift assay (EMSA) using human serum albumin. The hydrophobicity of the compound as measured by the fraction of unbound siRNA in the binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 to enhance in vivo delivery of siRNA.

[0011] In some embodiments, the lipophilic moiety is aliphatic, cyclic such as alicyclic, or polycyclic, such as a polyalicyclic compound, such as a steroid (e.g., sterol) or a straight or branched aliphatic hydrocarbon. Exemplary lipophilic moieties are lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexanol, hexadecyl glycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) cholenic acid, ibuprofen, naproxen, dimethoxytrityl or phenoxazine.

[0012] Suitable lipophilic moieties also include saturated or unsaturated C4-C 30 Hydrocarbon chains (e.g. C4-C 30 alkyl or alkenyl) and an optional functional group 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 the iRNA agent. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C 18Hydrocarbon chain (e.g. straight chain C6-C 18 In one embodiment, the lipophilic portion contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g. straight chain C 16 In some embodiments, the lipophilic moiety contains two or more carbon-carbon double bonds.

[0013] In some embodiments, the lipophilic moiety is a C6-C8 olefinic acid having a free terminal carboxylic acid functional group (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid), 30 part.

[0014] In some embodiments, the lipophilic moiety 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 alcohol, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenol, retinol, vitamin E, cholesterol, etc.).

[0015] The lipophilic monomer can include a lipophilic portion conjugated to any portion of the iRNA agent (e.g., a core base, a sugar moiety, or a nucleoside bond). When the lipophilic portion is conjugated to the iRNA agent via a core base, a ribose, or a nucleoside bond directly attached to the iRNA agent, the lipophilic monomer includes a core base, a ribose, or a nucleoside bond and a lipophilic portion. Alternatively, the lipophilic monomer can include a lipophilic portion conjugated to a non-ribose replacement unit, such as a linker or a carrier. When the lipophilic portion is conjugated to the iRNA agent via a non-ribose replacement unit (e.g., a linker or a carrier), the lipophilic monomer includes a non-ribose replacement unit (e.g., a linker or a carrier) and a lipophilic portion.

[0016] In certain embodiments, the lipophilic monomer does not contain a nucleobase.

[0017] In certain embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via one or more linkers (tethers).

[0018] 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, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0019] In some embodiments, at least one linker (tether) is a redox-cleavable linker (such as a reduction-cleavable linker, e.g., a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate ester), or a peptidase-cleavable linker (e.g., a peptide bond).

[0020] In other embodiments, at least one linker (tether) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0021] In certain embodiments, the lipophilic monomer comprises a lipophilic portion conjugated to the compound via a non-ribose replacement unit (i.e., a carrier) replacing one or more nucleotides. The carrier can be a cyclic group or a non-cyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene. In one embodiment, the non-cyclic group is a portion based on a serinol backbone, a glycerol backbone or a diethanolamine backbone.

[0022] In certain embodiments, the carrier replaces one or more nucleotides in a double-stranded iRNA agent. In certain embodiments, the carrier replaces one or more nucleotides in an internal position of a double-stranded iRNA agent. In other embodiments, the carrier replaces the nucleotides at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotides on the 3' end of the sense strand, thereby serving as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group with an amine, for example, the carrier can be a pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl or decahydronaphthyl.

[0023] In some embodiments, the lipophilic monomer can be represented by one of the following formulae:

[0024]

[0025] in:

[0026] J1 and J2 are each independently O, S, NR N , optionally substituted alkyl, OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R P )2)O、or OP(N(R P )2);

[0027] is a cyclic group or an acyclic group;

[0028] R N is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroaryl, or an amino protecting group;

[0029] R P is independently at each occurrence H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, or optionally substituted heteroaryl;

[0030] L 10 is a substituted or unsubstituted, saturated or unsaturated C3-C8 hydrocarbon (e.g., a C3-C8 alkyl, alkenyl or alkynyl group, or a C3-C8 hydrocarbon containing two or more double bonds); substituted groups include those described herein for “substituted” hydrocarbons, alkyls, alkenyls or alkynyls;

[0031] L 11 is a substituted or unsubstituted, saturated or unsaturated C6-C26 hydrocarbon (e.g., a C6-C26 alkyl, alkenyl, or alkynyl group, or a C3-C8 hydrocarbon containing two or more double bonds); substituted groups include those described herein for "substituted" hydrocarbons, alkyls, alkenyls, or alkynyls; and

[0032] When there is no nucleobase on the vector, Q is absent, or Q is a nucleobase that will be converted to L in vivo. 10 From L 11 For example, Q can be a cleavable group that can be cleaved in vivo to release L 11About 10% to 70%, about 15% to 50%, about 20% to 40%, or about 20% to 30% of the cleavable groups are cleaved from the lipophilic monomer. Exemplary cleavable groups include -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R 5 )=N-、-N=C(R 5 )-、-C(R 5 )=NO-、-ON=C(R 5 )-、-C(O)N(R 5 )-、-N(R 5 )C(O)-、-C(S)N(R 5 )-、-N(R 5 )C(S)-、-N(R 5 )C(O)N(R 5 )-、-N(R 5 )C(O)C(R 3 )(R 4 )OC(O)-、-C(O)OC(R 3 )(R 4 )C(O)N(R 5 )-、-OC(O)O-、-OSi(R 5 )2O-、-C(O)(CR 3 R 4 )C(O)O-、-OC(O)(CR 3 R 4 )C(O)-、 or a combination thereof, R 11 is C2-C8 alkyl or alkenyl. 3 、R 4 and R 5 Each is independently H or C1-C4 alkyl.

[0033] In one embodiment, the cleavage of Q is determined by the stability of the ligand in cerebrospinal fluid (CSF), the stability of the ligand in plasma, the stability of the ligand in brain homogenate or tissue homogenate (liver, eye, etc.), or the stability of the ligand in vitreous humor.

[0034] Cyclic and acyclic groups include those already described herein.

[0035] In one embodiment, the acyclic group is a serinol, glycerol, or diethanolamine backbone.

[0036] In one embodiment, the cyclic group is selected from pyrrolidinyl, hydroxyprolinyl, cyclopentyl, cyclohexyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl and decahydronaphthyl.

[0037] In one embodiment, the cyclic group is ribose or a ribose analog. Examples of ribose analogs include arabinose, 4'-thioribose, 2'-O-methylribose, GNA, UNA and LNA analogs.

[0038] In some embodiments, the lipophilic monomer conjugated to one or more positions of the chain of the compound has the following structure:

[0039]

[0040]

[0041]

[0042] In the above structures of the 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 diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.

[0043] In the structure of the above-mentioned lipophilic monomer, the alkylene chain may contain one or more unsaturated bonds.

[0044] Integer m is 0-8. Integer n is 1-21. R2' can be any functional group (which is an acceptable 2' modification for ribose), such as 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropyl) modification, 2'-O-allyl modification, 2'-C-allyl modification, 2'-fluoro modification, 2'-O-N-methylacetamido (2'-O-NMA) modification, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, 2'-O-aminopropyl (2'-O-AP) modification, or 2'-ara-F modification. For example, R2' can be H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-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).

[0045] In some embodiments, the lipophilic monomer conjugated to one or more positions of the chain of the compound has the following structure:

[0046] (R2' is 2'-F, 2'-OMe, 2'-NMA, 2'-deoxy, or 2'-OH);

[0047]

[0048] In these structures, B is a modified or unmodified nucleobase.

[0049] Specific examples of lipophilic monomers include:

[0050]

[0051]

[0052] In these structures, B is a modified or unmodified nucleobase; and R and R' are each independently H, methyl, ethyl, isopropyl, or tert-butyl.

[0053] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to a strand of the compound (a single strand of a single-stranded oligonucleotide; or the sense and / or antisense strand of a double-stranded oligonucleotide) via the following carriers:

[0054] 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.

[0055] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to an internal position of a strand of the compound (a single strand of a single-stranded oligonucleotide; or the sense and / or antisense strand of a double-stranded oligonucleotide) via the following carriers:

[0056] In these embodiments, R is a lipophilic moiety as defined herein. n is an integer from 1 to 21. R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl. B is a modified or unmodified nucleobase.

[0057] Additional examples of lipophilic monomers can be found in the Examples.

[0058] In some embodiments, the sense and antisense strands of the compound are each 15 to 30 nucleotides in length.

[0059] In one embodiment, the sense and antisense strands of the compound are each 19 to 25 nucleotides in length.

[0060] In one embodiment, the sense and antisense strands of the compound are each 21 to 23 nucleotides in length.

[0061] In some embodiments, the compound comprises a single-stranded overhang on at least one of the multiple ends, such as a 3' and / or 5' overhang of 1-10 nucleotides in length, such as an overhang of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, the two chains 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 a blunt end, located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. In one embodiment, the compound comprises a 3' overhang at the 3' end of the antisense strand and optionally comprises a blunt end at the 5' end of the antisense strand. In one embodiment, the compound comprises a 5' overhang at the 5' end of the sense strand and optionally comprises 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 duplex.

[0062] In one embodiment, the sense strand of the compound 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 (with a 2 nucleotide long single-stranded overhang at the 3' end).

[0063] In some embodiments, the sense strand further comprises at least one phosphorothioate bond at the 3' end. In some embodiments, the sense strand further comprises at least two phosphorothioate bonds at the 3' end. In some embodiments, one or more lipophilic monomers are located at the 3' end of the sense strand. In one embodiment, one of these phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide at the 3' end of the sense strand.

[0064] In some embodiments, the sense strand further comprises at least one phosphorothioate bond at the 5' end. In some embodiments, the sense strand further comprises at least two phosphorothioate bonds at the 5' end. In some embodiments, one or more lipophilic monomers are located at the 5' end of the sense strand. In one embodiment, one of these phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide at the 5' end of the sense strand.

[0065] In some embodiments, the antisense strand further comprises at least one phosphorothioate bond at the 3' end. In some embodiments, the antisense strand further comprises at least two phosphorothioate bonds at the 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 phosphorothioate bonds is located between the lipophilic monomer and the first nucleotide at the 3' end of the antisense strand.

[0066] In some embodiments, the compound further comprises a phosphate or a phosphate mimetic at the 5' end of the antisense strand. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0067] In some embodiments, the 5' end of the antisense strand of the compound does not contain 5'-vinylphosphonate (VP).

[0068] In some embodiments, the compound further comprises at least one terminal chiral phosphorus atom.

[0069] Site-specific chiral modification of the internucleotide bond can occur at the 5' end, 3' end, or both the 5' end and the 3' end of the chain. It is referred to herein as a "terminal" chiral modification. The terminal modification can occur at the 3' or 5' terminal position of the terminal region, for example, at the terminal nucleotide of the chain or at a position within the last 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. The chiral modification can occur in the sense strand, the antisense strand, or both the sense strand and the antisense strand. Each chirally pure phosphorus atom can be in the Rp configuration or the Sp configuration and combinations thereof. More details about chiral modifications and chirally modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled "Chirally-Modified Double-Stranded RNA Agents," which is incorporated herein by reference in its entirety.

[0070] In some embodiments, the compound further comprises a terminal chiral modification of the phosphorus atom of the bond with an Sp configuration at the first internucleotide bond at the 3' end of the antisense strand; a terminal chiral modification of the phosphorus atom of the bond with an Rp configuration at the first internucleotide bond at the 5' end of the antisense strand; and a terminal chiral modification of the phosphorus atom of the bond with either an Rp configuration or an Sp configuration at the first internucleotide bond at the 5' end of the sense strand.

[0071] In one embodiment, the compound further comprises a terminal chiral modification of the phosphorus atom of the bond with an Sp configuration at the first internucleotide bond and the second internucleotide bond at the 3' end of the antisense strand; a terminal chiral modification of the phosphorus atom of the bond with an Rp configuration at the first internucleotide bond at the 5' end of the antisense strand; and a terminal chiral modification of the phosphorus atom of the bond with either an Rp or Sp configuration at the first internucleotide bond at the 5' end of the sense strand.

[0072] In one embodiment, the compound further comprises a terminal chiral modification of the phosphorus atom of the bond with an Sp configuration at the first internucleotide bond, the second internucleotide bond, and the third internucleotide bond at the 3' end of the antisense strand; a terminal chiral modification of the phosphorus atom of the bond with an Rp configuration at the first internucleotide bond at the 5' end of the antisense strand; and a terminal chiral modification of the phosphorus atom of the bond with either an Rp or Sp configuration at the first internucleotide bond at the 5' end of the sense strand.

[0073] In one embodiment, the compound further comprises a terminal chiral modification of the phosphorus atom of the bond with an Sp configuration at the first internucleotide bond and the second internucleotide bond at the 3' end of the antisense strand; a terminal chiral modification of the phosphorus atom of the bond with an Rp configuration at the third internucleotide bond at the 3' end of the antisense strand; a terminal chiral modification of the phosphorus atom of the bond with an Rp configuration at the first internucleotide bond at the 5' end of the antisense strand; and a terminal chiral modification of the phosphorus atom of the bond with either an Rp or Sp configuration at the first internucleotide bond at the 5' end of the sense strand.

[0074] In one embodiment, the compound further comprises a terminal chiral modification of the phosphorus atom of the bond with an Sp configuration at the first and second internucleotide bonds at the 3' end of the antisense strand; a terminal chiral modification of the phosphorus atom of the bond with an Rp configuration at the first and second internucleotide bonds at the 5' end of the antisense strand; and a terminal chiral modification of the phosphorus atom of the bond with either an Rp or Sp configuration at the first internucleotide bond at the 5' end of the sense strand.

[0075] In some embodiments, the compound has at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end).

[0076] In some embodiments, the antisense strand comprises two blocks with one, two, or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0077] In some embodiments, the compound further comprises a targeting ligand that targets a receptor that mediates delivery to a specific CNS tissue. In one embodiment, the targeting ligand is selected from the group consisting of: Angiopep-2, a lipoprotein receptor-related protein (LRP) ligand, a bEnd.3 cell-binding ligand, a transferrin receptor (TfR) ligand, a mannose receptor ligand, a glucose transporter, and a LDL receptor ligand.

[0078] In some embodiments, the compound further comprises a targeting ligand that targets a receptor that mediates delivery to ocular tissue. In one embodiment, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligand. In one embodiment, the targeting ligand is an RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 1) or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

[0079] 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.

[0080] 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 a modification as described herein.

[0081] 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 nearly 100% 2'-O-methyl modified nucleotides.

[0082] 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'-fluoro.

[0083] In one embodiment, the oligonucleotide is antisense and at least 50% of the nucleotides of the antisense are independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0084] In some embodiments, the sense and antisense strands of the compound comprise fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modified nucleotides. In some embodiments, the compound has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications on the sense strand. In some embodiments, the compound has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications on the antisense strand.

[0085] In some embodiments, the compound has one or more 2'-F modifications at any position on the sense or antisense strand.

[0086] In some embodiments, the compound has 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'-O-N-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 abasic sugars, abasic cyclic, and open-chain alkyl groups.

[0087] In some embodiments, the compound has greater than 80%, greater than 85%, greater than 90%, greater than 95%, or nearly 100% natural nucleotides. For the purposes of these embodiments, natural nucleotides can include natural nucleotides with 2'-OH, 2'-deoxy, and 2'-OMe.

[0088] In some embodiments, the antisense strand (e.g., in the seed region of the antisense strand) contains at least one unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA) modification. In one embodiment, the seed region is at positions 2-8 (or positions 5-7) at the 5' end of the antisense strand.

[0089] 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 phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the compound has less than 20%, less than 15%, less than 10%, less than 5% non-natural nucleotides, or is substantially free of non-natural nucleotides.

[0090] 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 phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the compound has greater than 80%, greater than 85%, greater than 95% or nearly 100% natural nucleotides, such as natural nucleotides having 2'-OH, 2'-deoxy or 2'-OMe.

[0091] 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 phosphorothioate internucleotide linkages between the first five nucleotides (counted from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxy modifications on the sense strand and / or the antisense strand; wherein the compound has a double-stranded (duplex) region of between 19 and 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand does not comprise a glycol nucleic acid (GNA).

[0092] It will be understood that the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference. In other words, the compound is capable of inhibiting the expression of the target gene.

[0093] In one embodiment, the compound comprises at least three 2'-deoxy modifications at positions 2 and 14 of the antisense strand (counted from the 5' end of the antisense strand) and position 11 of the sense strand (counted from the 5' end of the sense strand).

[0094] In one embodiment, the compound comprises at least five 2'-deoxy modifications at positions 2, 12, and 14 of the antisense strand (counted from the 5' end of the antisense strand) and positions 9 and 11 of the sense strand (counted from the 5' end of the sense strand).

[0095] In one embodiment, the compound comprises at least seven 2'-deoxy modifications at positions 2, 5, 7, 12, and 14 of the antisense strand (counted from the 5' end of the antisense strand) and positions 9 and 11 of the sense strand (counted from the 5' end of the sense strand).

[0096] In one embodiment, the antisense strand comprises at least five 2'-deoxy modifications located at positions 2, 5, 7, 12, and 14 (counted from the 5' end of the antisense strand).The antisense strand has a length of 18-25 nucleotides or a length of 18-23 nucleotides.

[0097] In one embodiment, the compound may include one or more non-natural nucleotides. For example, the compound may include less than 20%, such as less than 15%, less than 10% or less than 5% non-natural nucleotides, or the compound may not include non-natural nucleotides. For example, the compound includes all natural nucleotides. Some exemplary non-natural nucleotides include, but are not limited to, non-cyclic nucleotides, locked nucleic acids (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-O-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0098] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides (counting from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; wherein the sense strand does not comprise a glycol nucleic acid (GNA); and wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides.

[0099] In one embodiment, at least one of the sense strand and the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more, 2'-deoxy modifications in the central region of the sense strand or antisense strand. Thus, in one embodiment, the compound comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide bonds between the first five nucleotides (counted from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxy nucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand and / or the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more 2'-deoxy modifications in the central region of the sense strand and / or the antisense strand.

[0100] In some embodiments, the sense strand has a length of 18 to 30 nucleotides and comprises 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 comprises at least two 2'-deoxy modifications at positions 7, 8, 9, 10, 11, 12, and 13 (counting from the 5' end of the sense strand).

[0101] In one embodiment, the antisense strand has a length of 18 to 30 nucleotides and comprises 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 comprises at least two 2'-deoxy modifications in positions 10, 11, 12, 13, 14, 15, and 16 (counted from the 5' end of the antisense strand).

[0102] 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 comprises at least one 2'-deoxy modification in the central region of the sense strand; and wherein the antisense strand independently has a length of 17-30 nucleotides and comprises at least two 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; wherein the sense strand has a length of 17-30 nucleotides and comprises 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 comprises at least one 2'-deoxy modification in the central region of the 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 phosphorothioate internucleotide linkages between the first five nucleotides (counted from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the sense strand.

[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 phosphorothioate internucleotide linkages between the first five nucleotides (counted from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the antisense strand.

[0106] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides (counting from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the sense strand and / or the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense strand and / or the antisense strand.

[0107] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides (counting from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the sense strand.

[0108] In one embodiment, the compound comprises a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides (counted from the 5' end of the antisense strand); at least three, four, five, or six 2'-deoxynucleotides on the sense strand and / or the antisense strand; and wherein the compound has a duplex region of between 19 and 25 base pairs; wherein the compound comprises a ligand; wherein the compound comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5%, of non-natural nucleotides, or the compound comprises all natural nucleotides; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the antisense strand.

[0109] In one embodiment, when the compound comprises fewer than 8 non-2'OMe nucleotides, the antisense strand comprises at least one DNA. For example, in any embodiment of the invention, when the compound comprises fewer than 8 non-2'OMe nucleotides, the antisense strand comprises at least one DNA.

[0110] In one embodiment, when the antisense comprises two deoxynucleotides and the nucleotides are located at positions 2 and 14 (counting from the 5' end of the antisense strand), the compound comprises 8 or fewer (e.g., 8, 7, 6, 5, 4, 3, 2, 1 or 0) non-2'OMe nucleotides. For example, in any embodiment of the invention, when the antisense comprises two deoxynucleotides and the nucleotides are located at positions 2 and 14 (counting from the 5' end of the antisense strand), the compound comprises 0, 1, 2, 3, 4, 5, 6, 7 or 8 non-2'OMe nucleotides.

[0111] In another aspect, the present invention further provides a method for delivering a compound of the present invention to a specific target in a subject by subcutaneous or intravenous administration. The present invention further provides a method for using a compound of the present invention to deliver the agent to a specific target in a subject by subcutaneous or intravenous administration.

[0112] Another aspect of the present invention relates to a method for reducing target gene expression 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 containing one or more lipophilic moieties conjugated to one or more positions on at least one strand, optionally via a linker or carrier.

[0113] In the first aspect of the invention relating to compounds, all of the above embodiments relating to lipophilic monomers, lipophilic moieties and conjugation thereof to compounds are suitable for this aspect of the invention relating to a method of reducing target gene expression in a cell.

[0114] In one embodiment, the cell is an extrahepatic cell.

[0115] In one embodiment, the cell is not a hepatocyte.

[0116] Another aspect of the invention relates to a method of reducing target gene expression in a subject, comprising administering to the subject a compound, comprising contacting the cell with a compound comprising an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0117] In the first aspect of the invention relating to compounds, all of the above embodiments relating to lipophilic monomers, lipophilic moieties and conjugation thereof to compounds are applicable to this aspect of the invention relating to a method of reducing target gene expression in a subject.

[0118] In some embodiments, the compound is administered extrahepatically.

[0119] In one embodiment, the compound is administered intrathecally or intraventricularly. By administering the compound intrathecally or intraventricularly, the method can reduce target gene expression in brain or spinal tissue (e.g., cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine).

[0120] 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 administered directly (e.g., intravitreally) to one or more eyes. By administering the compound intravitreally, this method can reduce the expression of the target gene in ocular tissue.

[0121] Another aspect of the present invention relates to a method for treating a subject with a CNS disorder, the method comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, thereby treating the subject. The double-stranded RNAi agent comprises an antisense strand complementary to a target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0122] In the first aspect of the invention directed to compounds, all of the above embodiments directed to lipophilic monomers, lipophilic moieties, and conjugation thereof to compounds are suitable for this aspect of the invention relating to methods of treating a subject suffering from a CNS disorder. 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 diseases, prion diseases, and Lafora's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 is a schematic diagram showing the general structure of ceramide.

[0124] Figure 2 is a graph depicting the stability of siRNA conjugates in rat CSF after incubation of siRNA duplexes with rat CSF for 24 hours.

[0125] Figure 3 is a graph depicting the stability of siRNA conjugates in rabbit and cynomolgus monkey vitreous humor (NHP) over 24 hours. The remaining amount of ligand-conjugated duplex is plotted.

[0126] Figure 4 is a graph depicting the stability of siRNA conjugates in rabbit and cynomolgus monkey vitreous humor (NHP) over 24 hours. The remaining amount of ligand-conjugated duplex is plotted.

[0127] Figure 5A and 5B is a graph depicting the stability of siRNA conjugates in rat brain homogenate for 4 hours. Figure 5AThe remaining amount of ligand-conjugated duplex is plotted in , and Figure 5B The stability of the PS bond is plotted in .

[0128] Figure 6 is a graph depicting the stability of siRNA conjugates with esterase-cleavable conjugates in rabbit and cynomolgus monkey vitreous humor (NHP) for 24 hours. The percentage of ligand-conjugated duplexes that were hydrolyzed is plotted.

[0129] Figure 7 is a graph depicting the stability of siRNA conjugates with esterase-cleavable conjugates in rat plasma, CSF, and brain homogenate for 24 hours. The percentage of ligand-conjugated duplexes that were hydrolyzed is plotted.

[0130] Figure 8 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 the human serum albumin concentration.

[0131] Figure 9 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.

[0132] Figure 10 is a graph depicting inhibition of ocular TTR expression (by qPCR) in mouse eyes following intravitreal administration of a single dose of 7.5 μg of siRNA duplexes compared to PBS controls.

[0133] Figure 11 is a graph depicting inhibition of ocular TTR expression (by qPCR) in rat eyes following intravitreal administration of a single dose of 1 μg of siRNA duplexes compared to PBS control.

[0134] Figure 12 is a graph depicting inhibition of ocular TTR expression (by qPCR) in mouse eyes following intravitreal administration of a single dose of 7.5 μg of siRNA duplexes compared to PBS controls.

[0135] Figure 13 is a graph depicting inhibition of ocular TTR expression (by qPCR) in rat eyes following intravitreal administration of a single dose of 1 μg of siRNA duplexes compared to PBS control.

[0136] Figure 14 is a graph depicting inhibition of ocular TTR expression (by qPCR) in mouse eyes following intravitreal administration of a single dose of 7.5 μg of siRNA duplexes compared to PBS controls.

[0137] Figure 15is a graph depicting inhibition of ocular TTR expression (by qPCR) in rat eyes following intravitreal administration of a single dose of 1 μg of siRNA duplexes compared to PBS control.

[0138] Figure 16 Graph depicting inhibition of TTR gene expression in primary mouse hepatocytes 24 hours after transfection of cells with Q367-modified siRNA duplexes at three different concentrations compared to the control duplex AD-900954. Each nucleotide is modified with Q367 across the sense strand.

[0139] Figure 17 Graph depicting inhibition of SOD1 gene expression in primary mouse hepatocytes 24 hours after transfection of cells with Q367-modified siRNA duplexes at three different concentrations compared to the control duplex AD-900954. Each nucleotide is modified with Q367 across the sense strand.

[0140] Figures 18A-18D Figure depicts the changes in the spinal cord ( Figure 18A ), cerebellum( Figure 18B ), frontal cortex ( Figure 18C ) and heart ( Figure 18D ) in the figure showing the inhibition of SOD1 expression (by qPCR).

[0141] Figures 19A-19E Figure depicts the changes in the spinal cord ( Figure 19A ), brainstem( Figure 19B ), cerebellum( Figure 19C ), frontal cortex ( Figure 19D ) and heart ( Figure 19E ) in the figure showing the inhibition of SOD1 expression (by qPCR).

[0142] Figure 20 Graph depicting inhibition of SOD1 expression (by qPCR) in rat brain (cerebellum and frontal cortex) and spinal cord (thoracic spinal cord) after 14 days following IT administration of a single 0.9 mg siRNA duplex / rat compared to an artificial CSF dosing control group.

[0143] Figure 21A and 21B It depicts 14 days ( Figure 21A ) and 7 days ( Figure 21B ) after ICV administration of a single 50 μg ( Figure 21A ) and 110 μg ( Figure 21BFigure 2 shows the inhibition of SOD1 expression (by qPCR) in the mouse brain (right hemisphere) and heart after administration of siRNA duplexes of α, β, and β-actin / mouse. DETAILED DESCRIPTION

[0144] In particular, the present inventors have discovered that conjugating a lipophilic monomer containing a lipophilic moiety to one or more positions on at least one chain of the compound provides surprisingly good results for in vivo ocular delivery (e.g., intravitreal delivery) and intrathecal or intracerebroventricular delivery of double-stranded iRNA, resulting in efficient entry into CNS and ocular tissues and efficient internalization into cells of the CNS and ocular systems.

[0145] One aspect of the present invention provides a compound comprising:

[0146] 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.

[0147] The term "lipophile" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient log K ow , where K ow is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase when a two-phase system is in equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the structural components of the chemical substance, which are 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 the tendency of a substance to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilicity / lipophilicity balance). In principle, when logK ow When it exceeds 0, the chemical is lipophilic. Typically, the logK of the lipophilic part is ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10. For example, the logK of 6-aminohexanol is predicted to be ow Using the same method, the logK of cholesteryl N-(hexan-6-ol)carbamate was predicted. ow It is 10.7.

[0148] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K) of the lipophilic moiety. ow )value.

[0149] 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 can be positively correlated with the silencing activity of the double-stranded iRNA agent.

[0150] In one embodiment, the plasma protein binding assay measured is an electrophoretic mobility shift assay (EMSA) using human serum albumin. The hydrophobicity of the double-stranded iRNA agent, as measured by the fraction of unbound siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 to enhance in vivo delivery of the siRNA.

[0151] Therefore, conjugating a lipophilic monomer (containing a lipophilic moiety) to the compound provides optimal hydrophobicity for enhancing the in vivo delivery of siRNA.

[0152] In certain embodiments, the lipophilic moiety is aliphatic, cyclic such as alicyclic, or polycyclic such as a polyalicyclic compound, such as a steroid (e.g., sterol) or a straight or branched aliphatic hydrocarbon. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may comprise various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic moieties 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. monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes) and other polyalicyclic hydrocarbons. For example, the lipophilic portion may contain C4-C 30 Hydrocarbon chains (e.g. C4-C 30 In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C 18 Hydrocarbon chain (e.g. straight chain C6-C 18 In one embodiment, the lipophilic portion contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g. straight chain C16 alkyl or alkenyl).

[0153] The lipophilic monomer containing a lipophilic moiety can be attached to the iRNA agent by any method known in the art, including through a functional group already present in the lipophilic monomer or introduced into the iRNA agent, such as a hydroxyl group (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, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0154] Conjugation of the iRNA agent to the lipophilic monomer can occur, for example, by forming an ether or carboxyl or carbamoyl ester bond between a hydroxyl group and an alkyl R-, alkanoyl RCO-, or substituted carbamoyl RNHCO-. The alkyl R group can be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). The alkyl R group can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl, among others.

[0155] In some embodiments, a lipophilic monomer comprising a lipophilic moiety is conjugated to the compound via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0156] In another embodiment, the lipophilic moiety is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentaphenanthrene 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 derivatives" refer to compounds derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0157] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" refers to both monoaromatic and polyaromatic hydrocarbons. Aryl groups include, but are not limited to, C6-C8 aromatic hydrocarbons containing one to three aromatic rings. 14 Aryl moieties, which may be optionally substituted; "aralkyl" or "arylalkyl" comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; 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 about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O) and sulfur (S) in addition to carbon atoms.

[0158] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group is a group having one to about four, preferably one to about three, and more preferably one or two non-hydrogen substituents. Suitable substituents include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, amido, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxyl, hydroxyalkyl, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, arylsulfonamido, alkylcarbonyl, acyloxy, cyano, and urea.

[0159] In some embodiments, the lipophilic moiety is an aralkyl group, such as a 2-arylpropionyl moiety. The structural features of the aralkyl group are selected such that the lipophilic moiety will bind to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected such that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, immunoglobulins, lipoproteins, alpha-2-macroglobulin, or alpha-1-glycoprotein.

[0160] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Methods for synthesizing naproxen are described in U.S. Pat. No. 3,904,682 and U.S. Pat. No. 4,009,197, which are incorporated herein by reference in their entirety. Naproxen's chemical name is (S)-6-methoxy-α-methyl-2-naphthylacetic acid, and its structure is

[0161] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. Methods for synthesizing ibuprofen are described in U.S. Patent No. 3,228,831, which is incorporated herein by reference in its entirety. The structure of ibuprofen is

[0162] Additional exemplary aralkyl groups are described in US Pat. No. 7,626,014, which is incorporated herein by reference in its entirety.

[0163] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0164] In some embodiments, the lipophilic moiety 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 alcohol, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenol, retinol, vitamin E, cholesterol, etc.).

[0165] In certain embodiments, the lipophilic monomer containing one or more lipophilic moieties can be incorporated into the double-stranded iRNA agent, particularly when the lipophilic moiety has low lipophilicity or hydrophobicity. In one embodiment, the lipophilic monomer containing two or more lipophilic moieties is incorporated into the same chain of the double-stranded iRNA agent. In one embodiment, each chain of the double-stranded iRNA agent has the lipophilic monomer containing one or more lipophilic moieties incorporated therein. In one embodiment, the lipophilic monomer containing two or more lipophilic moieties is incorporated into the same position (i.e., same core base, same sugar moiety or same internucleoside bond) of the double-stranded iRNA agent. This can be achieved by, for example, using a lipophilic monomer containing a carrier and / or a straight linker and / or one or more linkers (two or more lipophilic moieties can be connected).

[0166] When the lipophilic moiety is conjugated to the iRNA agent via a nucleobase, ribose or internucleoside bond directly attached to the iRNA agent, the lipophilic monomer comprises a nucleobase, ribose or internucleoside bond and a lipophilic moiety. Alternatively, the lipophilic monomer can comprise a lipophilic moiety conjugated to a non-ribose replacement unit, such as a linker or carrier. When the lipophilic moiety is conjugated to a double-stranded iRNA agent via a non-ribose replacement unit (such as a linker or carrier), the lipophilic monomer comprises a non-ribose replacement unit (such as a linker or carrier) and a lipophilic moiety.

[0167] In certain embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the iRNA agent via one or more linkers (tethers).

[0168] In one embodiment, the lipophilic monomer comprises a lipophilic moiety conjugated to the compound via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0169] Connectors / tethers

[0170] The linker / tether is connected to the lipophilic moiety at a "tether attachment point (TAP)". The linker / tether may comprise any C1-C 100 Carbon-containing part (e.g. C1-C 75 、C1-C 50 、C1-C 20 、C1-C 10 ; C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 ), and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) on the linker / tether, which may serve as a point of attachment for the lipophilic moiety. Non-limiting examples of linkers / tethers (underlined) include TAP -(CH2) n NH- ;TAP- C(O)(CH2) n NH- ;TAP- NR””(CH2) n NH- ;TAP- C(O)-(CH2) n -C (O)- ;TAP- C(O)-(CH2) n -C(O)O-; TAP- C(O)-O- ;TAP- C(O)-(CH2) n -NH-C(O)- ;TAP- C(O)- (CH2) n - ;TAP- C(O)-NH- ;TAP- C(O)- ;TAP- (CH2) n -C(O)- ;TAP- (CH2) n -C(O)O- ;TAP- (CH2) n - ; or TAP- (CH2) n -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, the nitrogen may form part of a terminal oxyamino group such as -ONH2 or a hydrazine group -NHNH2. The linker / tether may optionally be substituted, for example, with hydroxyl, alkoxy, perhaloalkyl, and / or optionally be interrupted by one or more additional heteroatoms, such as N, O, or S. Preferred tethering 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)(with body) ;TAP- C(O)-(CH2) n (ligand) ;TAP- C(O)-NH(ligand) ;TAP- C(O)(ligand) ;TAP- (CH2) n -C(O)(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 / tether (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH2, ONH2, NH2NH2) can be acylated, for example, with C(O)CF3.

[0171] In some embodiments, the linker / tether can be terminated with a thiol (ie, SH) or an olefin (eg, CH=CH2). For example, the tether can be TAP -(CH2) n -SH 、TAP- C(O)(CH2) n SH 、TAP -(CH2) n -(CH=CH2) or TAP- C(O)(CH2) n (CH= CH2) , wherein n may be as described elsewhere. The tether may be optionally substituted, for example, with hydroxy, alkoxy, perhaloalkyl, and / or optionally interrupted by one or more additional heteroatoms, such as N, O, or S. The double bond may be cis or trans or E or Z.

[0172] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at a terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, aldehydes, alkyl halides, mesylates, tosylates, nitrobenzenesulfonates, or bromobenzenesulfonates, or activated carboxylates, such as NHS esters or pentafluorophenyl esters. Preferred linkers / tethers (underlined) include TAP - (CH2) n GIVE ;TAP- C(O)(CH2) n GIVE ; or TAP- NR””(CH2) n GIVE , wherein n is 1-6 and R"" is C1-C6 alkyl; or TAP -(CH2) n C(O)ONHS ;TAP- C(O)(CH2) n C(O)ONHS ; or TAP- NR””(CH2) n C(O)ONHS , wherein n is 1-6 and R"" is C1-C6 alkyl; TAP- (CH2) n C(O)OC6F5 ;TAP- C(O)(CH2) n C(O)OC6F5 ; or TAP- NR””(CH2) n C(O) OC6F5 , wherein n is 1-11 and R"" is C1-C6 alkyl; or -(CH2) n CH2LG ;TAP- C(O)(CH2) n CH2LG ; or TAP- NR”” (CH2) n CH2LG, wherein n can be as described elsewhere and R"" is C1-C6 alkyl (LG can be a leaving group such as halide, mesylate, tosylate, nitrobenzenesulfonate, bromobenzenesulfonate). Tether attachment can be performed by coupling a nucleophilic group of the ligand (e.g., thiol or amino group) to an electrophilic group on the tether.

[0173] In other embodiments, it may be desirable for the monomer to include a phthalimido group (K) at the terminal position of the linker / tether.

[0174] In other embodiments, other protected amino groups can be present at the terminal position of the linker / tether, such as allyloxycarbonyl, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety can be o-nitrophenyl or o- or p-dinitrophenyl).

[0175] Any linker / tether described herein may further include one or more additional linking groups, such as -O-(CH2) n -、-(CH2) n -SS-, -(CH2) n -or -(CH=CH)-.

[0176] Cleavable linker / tether

[0177] In some embodiments, at least one of the linkers / tethers can be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0178] In one embodiment, at least one of the linkers / tethers may be a reduction-cleavable linker (eg, a disulfide group).

[0179] In one embodiment, at least one of the linkers / tethers can be an acid-cleavable linker (eg, a hydrazone, ester, acetal, or ketal group).

[0180] In one embodiment, at least one of the linkers / tethers may be an esterase cleavable linker (eg, an ester group).

[0181] In one embodiment, at least one of the linkers / tethers can be a phosphatase cleavable linker (eg, phosphate ester).

[0182] In one embodiment, at least one of the linkers / tethers may be a peptidase cleavable linker (eg, a peptide bond).

[0183] Cleavable linking groups are susceptible to the effects of cleavage agents (e.g., pH, redox potential, or the presence of degradation molecules). In general, cleavage agents are more prevalent or found at higher levels or activity in cells than in serum or blood. Examples of such degradants include: redox agents selected for specific substrates or having no substrate specificity, including, for example, oxidative enzymes or reductive enzymes or reducing agents such as thiols present in cells, which can degrade redox-cleavable linking groups by reduction; esterases; endosomes or agents that can produce an acidic environment, such as an endosome or agent that results in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0184] Cleavable linking groups (such as disulfide bonds) can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging between about 7.1-7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0. Some tethers will have linking groups that crack at a preferred pH, thereby releasing the iRNA agent from the intracellular ligand (e.g., targeting or cell permeability ligand, such as cholesterol), or entering the desired compartment of the cell.

[0185] The chemical junction (e.g., a linking group) connecting the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is absorbed into the cell by endocytosis, the acidic environment of the endosome will cause the disulfide bond to cleave, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. [Drug Research] 19: 1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. [Current Views in Chemistry and Biology] 6: 466-471, 2002). The ligand can be a targeting ligand or a second therapeutic agent that can supplement the therapeutic effect of the iRNA agent.

[0186] The tether can include a linking group that is cleavable by a specific enzyme. The type of linking group incorporated into the tether can be determined by the cell targeted by the visual iRNA agent. For example, an iRNA agent targeting an mRNA in a hepatocyte can be conjugated to a tether that includes an ester group. Hepatocytes are rich in esterases, and therefore the tether will be more effectively cracked in hepatocytes than in cell types that are not rich in esterases. The cracking of the tether releases the iRNA agent from the part attached to the distal end of the tether, thereby potentially enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0187] Tethers containing peptide bonds can be conjugated to iRNA agents targeting peptidase-rich cell types (such as hepatocytes and synoviocytes). For example, iRNA agents targeting synoviocytes, such as those used to treat inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to tethers containing peptide bonds.

[0188] In general, the suitability of a candidate cleavable linking group can be assessed by the ability of a test degradation agent (or condition) cracking candidate linking group. It will also be desirable to test the ability of a candidate cleavable linking group in blood or when contacting with other non-target tissues (such as when applied to a subject, iRNA agent will be exposed to tissue therein) to resist cracking. Therefore, the relative susceptibility to cracking between the first and second conditions can be determined, wherein the first condition is selected to indicate the cracking in the target cell, and the second condition is selected to indicate the cracking in other tissues or biofluids (such as blood or serum). Assessment can be carried out in a cell-free system, cell, cell culture, organ or tissue culture or whole animal. Initial assessment is carried out in a cell-free or culture condition and it can be useful to confirm by further assessing in whole animal. In a preferred embodiment, the cracking ratio of useful candidate compounds in cells (or under the in vitro conditions selected for simulating intracellular conditions) is at least 2, 4, 10 or 100 times faster in blood or serum (or under the in vitro conditions selected for simulating extracellular conditions).

[0189] Redox-cleavable linker

[0190] A class of cleavable linking groups is a redox cleavable linking group, which cracks when reduced or oxidized. The example of a reduction cleavable linking group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable linking group is suitable for a "reduction cleavable linking group" or, for example, whether it is suitable for use with a specific iRNA portion and a specific targeting agent, the methods described herein can be considered. For example, a candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, and its simulation will be evaluated by the cleavage rate observed in cells (such as target cells). The candidate can also be evaluated under conditions selected for simulating blood or serum conditions. In a preferred embodiment, the candidate compound cracks at most 10% in blood. In a preferred embodiment, the degradation ratio of useful candidate compounds in cells (or under in vitro conditions selected for simulating intracellular conditions) is at least 2, 4, 10 or 100 times faster in blood (or under in vitro conditions selected for simulating extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetics under conditions selected for simulating intracellular mediators, and compared with the conditions selected for simulating extracellular mediators.

[0191] Phosphate-based cleavable linkers

[0192] Phosphate-based linking groups are cleaved by agents that degrade or hydrolyze the phosphate. Examples of agents that cleave phosphate groups in cells are enzymes in the cell, such as phosphatases. Examples of phosphate-based linking 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 examples 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—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred example is —O—P(O)(OH)—O—. These candidates can be evaluated using methods similar to those described above.

[0193] Acid cleavable linker

[0194] Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In a preferred embodiment, the acid cleavable linking group is cleaved in an acidic environment of about pH 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or by an agent such as an enzyme that can act as a general acid. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid cleavable linking group. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and amino acid esters. The acid cleavable group may have the general formula -C═NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon of the oxygen (alkoxy) attached to the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as a dimethylpentyl group or a tertiary butyl group. These candidates can be evaluated using a method similar to that described above.

[0195] Ester-based linking groups

[0196] Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0197] Peptide-based cleavage groups

[0198] Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene groups. Peptide bonds are special types of amide bonds formed between amino acids to produce peptides and proteins. Peptide-based cleavable groups are generally limited to the peptide bonds (i.e., amide bonds) formed between the amino acids that produce peptides and proteins, and do not include the entire amide functional group. Peptide-cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)-, where R 1 and R 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0199] Biocleavable linkers / tethers

[0200] Linkers can also include biodegradable linkers, which are nucleotide and non-nucleotide linkers or combinations thereof, which connect two parts of a molecule, such as one or two strands of two separate siRNA molecules to produce a bis(siRNA). In some embodiments, only electrostatic or stacking interactions between two separate siRNAs may represent a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides and derivatives thereof, aliphatics, alicyclics, heterocycles and combinations thereof.

[0201] In some embodiments, at least one of the linkers (tethers) is a biocleavable 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.

[0202] In one embodiment, the biodegradable carbohydrate linker can have 1 to 10 sugar units with at least one mutarotational anomeric bond capable of linking two siRNA units. When two or more sugars are present, these units can be linked via 1-3, 1-4, or 1-6 sugar bonds or via an alkyl chain.

[0203] Exemplary biocleavable linkers include:

[0204]

[0205]

[0206]

[0207] Further discussion of biocleavable linkers can be found in PCT application number PCT / US18 / 14213, filed January 18, 2018, entitled “Endosomal Cleavable Linkers,” the contents of which are incorporated herein by reference in their entirety.

[0208] carrier

[0209] In certain embodiments, the lipophilic monomer comprises a lipophilic moiety conjugated to the iRNA agent via a non-ribose replacement unit (ie, a carrier) that replaces one or more nucleotides.

[0210] The carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0211] The carrier may replace one or more nucleotides of the double-stranded iRNA agent.

[0212] In some embodiments, the vector replaces one or more nucleotides in an internal position of the double-stranded iRNA agent.

[0213] In other embodiments, the carrier replaces the nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby acting as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl or decahydronaphthyl.

[0214] A ribonucleotide subunit in which the ribose of the subunit has been so replaced is referred to herein as a ribose replacement modified subunit (RRMS). The carrier can be a cyclic or non-cyclic moiety and comprises two "backbone attachment points" (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). The lipophilic moiety can be attached directly to the carrier or indirectly to the carrier via an intervening linker / tether, as described above.

[0215]

[0216] The ligand-conjugated monomer subunit can be the 5' or 3' terminal subunit of the iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group, and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit can occupy an internal position, and both "W" groups can be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit can be present in an iRNA agent.

[0217] Sugar-based monomers, such as ligand-conjugated monomers (cyclic)

[0218] Sugar-substituted cyclic monomers, such as sugar-substituted ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The carrier may have the general formula (LCM-2) provided below (in this structure, the preferred main chain attachment point 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 (Two positions are chosen to give two main chain attachment points, e.g. 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, which can be incorporated into the chain. Therefore, it is understood that the structure also covers the following situation, where one (in the case of terminal position) or two (in the case of 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 10When, for example, a phosphate or modified phosphate containing a sulfur backbone is connected. For example, one of the R groups can be -CH2-, wherein one bond is connected to the support and one bond is connected to a backbone atom (e.g., to an oxygen or central phosphorus atom).

[0219]

[0220] in:

[0221] X is N(CO)R 7 NR 7 or CH2;

[0222] Y is NR 8 , O, S, CR 9 R 10 ;

[0223] Z is CR 11 R 12 or does not exist;

[0224] R 1 、R 2 、R 3 、R 4 、R 9 and R 10 Each of which is independently H, OR a or (CH2) n OR b , 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 ;

[0225] R 5 、R 6 、R 11 and R 12 Each of the ligands is independently H, optionally 1-3 R 13 Substituted C1-C6 alkyl, or C(O)NHR 7 ; or R 5 With R 11 Together for optionally through R 14 substituted C3-C8 cycloalkyl;

[0226] R 7 It can be a ligand, such as R 7 It can be R d , or R 7This can be, for example, through a tethering moiety (e.g., via NR c R d Substituted C1-C 20 alkyl) ligands tethered to the support indirectly; or via NHC(O)R d Substituted C1-C 20 alkyl;

[0227] R 8 is H or C1-C6 alkyl;

[0228] R 13 is hydroxy, C1-C4 alkoxy or halogen;

[0229] R 14 NR c R 7 ;

[0230] R 15 is a C1-C6 alkyl group optionally substituted with a cyano group, or a C2-C6 alkenyl group;

[0231] R 16 C1-C 10 alkyl;

[0232] R 17 It is a liquid or solid phase support reagent;

[0233] L is -C(O)(CH2) q C(O)- or -C(O)(CH2) q S-;

[0234] R a is a protecting group, such as CAr3; (such as dimethoxytrityl) or Si(X 5 ')(X 5 ”)(X 5 ”'), where (X 5 '),(X 5 ”) and (X 5 ”') as described elsewhere.

[0235] R b P(O)(O - )H、P(OR 15 )N(R 16 )2 or LR 17 ;

[0236] R c is H or C1-C6 alkyl;

[0237] R d is H or ligand;

[0238] Each Ar is independently a C6-C4 alkoxy group optionally substituted with a C1-C4 alkoxy group. 10 aryl;

[0239] n is 1-4; and q is 0-4.

[0240] Exemplary vectors include those wherein, for example, X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 and Z is absent; 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 indane 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).

[0241] In certain embodiments, the carrier may be based on a pyrroline 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 linked to one of the carbons in the five-membered ring, such as an exocyclic alkylene group, such as a methylene group (-CH2OFG in D) 1 ). OFG 2 Preferably directly attached to one carbon of the five-membered ring (-OFG in D 2). For pyrroline-based supports, -CH2OFG 1 Can be attached to C-2 and OFG 2 Can be attached to C-3; or -CH2OFG 1 Can be attached to C-3 and OFG 2 Can be attached to C-4. In certain embodiments, CH2OFG 1 and OFG 2 Can be substituted orally to one of the carbons mentioned above. For 3-hydroxyproline based carriers, -CH2OFG 1 Can be attached to C-2 and OFG 2 Can be attached to C-4. Pyrroline and 4-hydroxyproline based monomers may therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond, e.g., due to the presence of a ring. Thus, CH2OFG 1 and OFG 2 In any pairing described above, the monomers may be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. 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 monomers (e.g., with CHOFG) are expressly included. 1 and OFG 2 The centers of the two groups may both have an R configuration; or both have an S configuration; or one center may have an R configuration and the other may have an S configuration, or vice versa). The tether attachment point is preferably nitrogen. Preferred examples of carrier D include the following:

[0242]

[0243] In certain embodiments, the support 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 attached to a primary carbon linked to one of the carbons in the six-membered ring, such as an exocyclic alkylene group, such as methylene (n=1) or ethylene (n=2) [-(CH2) in E n OFG 1 ]. OFG 2 Preferably attached directly to one carbon of the six-membered ring (-OFG in E 2 ). -(CH2) n OFG 1 and OFG2 Can be arranged in a geminal manner on the ring, that is, two groups can be attached to the same carbon, such as C-2, C-3 or C-4. Alternatively, -(CH2) n OFG 1 and OFG 2 The groups may be arranged in an ortho position on the ring, ie, the two groups may be attached to adjacent ring carbon atoms, for example -(CH2) n OFG 1 Can be attached to C-2 and OFG 2 Can be attached to C-3; -(CH2) n OFG 1 Can be attached to C-3 and OFG 2 Can be attached to C-2; -(CH2) n OFG 1 Can be attached to C-3 and OFG 2 Can be attached to C-4; or -(CH2) n OFG 1 Can be attached to C-4 and OFG 2 Can be attached to C-3. Piperidine-based monomers may therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond, such as due to the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 In any pairing described above, the monomers may be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. 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 monomers (e.g., with CHOFG) are expressly included. 1 and OFG 2 The centers of can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other can have the S configuration, or vice versa). The tether attachment point is preferably nitrogen.

[0244] In certain embodiments, the support 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 a morpholine ring system (G), such as 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 linked to one of the carbons in the six-membered ring, such as an exocyclic alkylene group, such as a methylene group (-CH2OFG in F or G) 1 ). OFG 2 Preferably directly attached to one carbon of the six-membered ring (-OFG in F or G) 2 ). For both F and G, -CH2OFG 1 Can be attached to C-2 and OFG 2 Can be attached to C-3; or vice versa. In certain embodiments, CH2OFG 1 and OFG 2 can be substituted geminal to one of the carbons mentioned above. Monomers based on piperazine and morpholine may therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond, e.g., due to the presence of a ring. Thus, CH2OFG 1 and OFG 2 In any pairing described above, the monomers may be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. 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 monomers (e.g., with CHOFG) are expressly included. 1 and OFG 2 The centers of the two groups may both have the R configuration; or both have the S configuration; or one center may have the R configuration and the other may have the S configuration, or vice versa). R'' may be, for example, a C1-C6 alkyl group, preferably CH3. The tether attachment point is preferably the nitrogen in F and G.

[0245] In certain embodiments, the support may be based on a decalin 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 C6 cycloalkyl (H, z = 2) or indane 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). OFG 1 Preferably attached to a primary carbon, such as an exocyclic methylene (n=1) or ethylene (n=2) group linked to one of C-2, C-3, C-4, or C-5 [-(CH2) in Hn OFG 1 ]. OFG 2 Preferably directly attached to one of C-2, C-3, C-4 or C-5 (-OFG in H 2 ). -(CH2) n OFG 1 and OFG 2 Can be arranged in a geminal manner on the ring, that is, two groups can be attached to the same carbon, such as C-2, C-3, C-4 or C-5. Alternatively, -(CH2) n OFG 1 and OFG 2 The groups may be arranged in an ortho position on the ring, ie, the two groups may be attached to adjacent ring carbon atoms, for example -(CH2) n OFG 1 Can be attached to C-2 and OFG 2 Can be attached to C-3; -(CH2) n OFG 1 Can be attached to C-3 and OFG 2 Can be attached to C-2; -(CH2) n OFG 1 Can be attached to C-3 and OFG 2 Can be attached to C-4; or -(CH2) n OFG 1 Can be attached to C-4 and OFG 2 Can be attached to C-3; -(CH2) n OFG 1 Can be attached to C-4 and OFG 2 Can be attached to C-5; or -(CH2) n OFG 1 Can be attached to C-5 and OFG 2 May be attached to C-4. Decalin or indane based monomers may therefore contain bonds (e.g. carbon-carbon bonds) where bond rotation is restricted around that particular bond, e.g. due to the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 In any pairing described above, the monomers may be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. 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 monomers (e.g., with CHOFG) are expressly included. 1 and OFG 2In some embodiments, the substituents at C-1 and C-6 are trans relative to each other. The point of tether attachment is preferably C-6 or C-7.

[0246] Other vectors may include 3-hydroxyproline based vectors (J). Therefore, -(CH2) n OFG 1 and OFG 2 The monomers may be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. 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 monomers (e.g., with CH2OFG 1 and OFG 2 The centers of can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other can have the S configuration, or vice versa). The tether attachment point is preferably nitrogen.

[0247] Details regarding more representative sugar-substituted based circular vectors can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0248] Sugar-substituted monomers (acyclic)

[0249] Sugar-substituted acyclic monomers, such as sugar-substituted ligand-conjugated monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers may have the formula LCM-3 or LCM-4:

[0250]

[0251] In some embodiments, each of x, y, and z can be independently 0, 1, 2, or 3. In formula LCM-3, when y and z are different, the tertiary carbon can have an R or S configuration. In a preferred embodiment, x in formula LCM-3 is zero and y and z are each 1 (e.g., based on serinol), and y and z in formula LCM-3 are each 1. Each of the following formulas LCM-3 or LCM-4 can be optionally substituted, for example, with a hydroxyl group, an alkoxy group, or a perhaloalkyl group.

[0252] Details regarding more representative sugar-substituted based acyclic vectors can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0253] In some embodiments, the compound comprises one or more lipophilic monomers comprising a lipophilic moiety conjugated to the 5' end of the sense strand or the 5' end of the antisense strand.

[0254] In certain embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the 5' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to the 5' end of the chain via a carrier having the formula: wherein R is a ligand, such as a lipophilic moiety.

[0255] In some embodiments, the compound comprises one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to the 3' end of the sense strand or the 3' end of the antisense strand.

[0256] In certain embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the 3' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to the 3' end of the chain via a carrier having the formula: wherein R is a ligand, such as a lipophilic moiety.

[0257] In certain embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to an interior position of the chain via a carrier and / or a linker. In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to an interior position of the chain via a carrier having the formula: wherein R is a ligand, such as a lipophilic moiety.

[0258] In some embodiments, the compound comprises one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to both ends of the sense strand.

[0259] In some embodiments, the compound comprises one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to both ends of the antisense strand.

[0260] In certain embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to an internal position of a sense strand or an antisense strand. In certain embodiments, one or more lipophilic moieties are conjugated to ribose, a core base and / or a nucleotide bond. In certain embodiments, one or more lipophilic moieties are conjugated to ribose at the 2' position, 3' position, 4' position and / or 5' position of ribose. In certain embodiments, one or more lipophilic moieties are conjugated to a natural core base (such as A, T, G, C or U) or a modified core base as defined herein. In certain embodiments, one or more lipophilic moieties are conjugated to a phosphate group or a modified phosphate group as defined herein.

[0261] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to the 5' end or 3' end of the sense strand, and one or more lipophilic monomers containing one or more lipophilic moieties conjugated to the 5' end or 3' end of the antisense strand.

[0262] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the terminus of the chain via one or more linkers (tethers) and / or carriers.

[0263] In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to the terminus of the chain via one or more linkers (tethers).

[0264] In one embodiment, the lipophilic monomer contains a lipophilic moiety conjugated to the 5' end of the sense or antisense strand via a circular carrier, optionally via one or more intervening linkers (tethers).

[0265] In some embodiments, at least one lipophilic monomer is located at one or more terminal positions of the sense strand or the antisense strand. In one embodiment, at least one lipophilic monomer is located at the 3' end or the 5' end of the sense strand. In one embodiment, at least one lipophilic monomer is located at the 3' end or the 5' end of the antisense strand.

[0266] 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 of a chain refers to a nucleotide at any position of the chain, excluding the terminal positions at the 3' and 5' ends of the chain (e.g., excluding two positions: position 1 counting from the 3' end and position 1 counting from the 5' end).

[0267] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, including all positions except the terminal two positions at each end of the chain (e.g., excluding 4 positions: positions 1 and 2 counted from the 3' end and positions 1 and 2 counted from the 5' end). In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, including all positions except the terminal three positions at each end of the chain (e.g., excluding 6 positions: positions 1, 2, and 3 counted from the 3' end and positions 1, 2, and 3 counted from the 5' end).

[0268] In one embodiment, at least one lipophilic monomer is located at one or more positions at at least one end of the duplex region, including all positions within the duplex region excluding the overhang region or the carrier that replaces the terminal nucleotide at the 3' end of the sense strand.

[0269] In one embodiment, at least one lipophilic monomer is located on the sense strand within the first five, four, three, two, or one base pairs of the 5' end of the antisense strand of the duplex region.

[0270] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one strand, excluding the cleavage site region of the sense strand, e.g., the lipophilic monomer is not located at positions 9-12, counted from the 5' end of the sense strand, e.g., the lipophilic monomer is not located at positions 9-11, counted from the 5' end of the sense strand. Alternatively, the internal positions do not include positions 11-13, counted from the 3' end of the sense strand.

[0271] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one chain, which do not include the cleavage site region of the antisense strand. For example, the internal positions do not include positions 12-14 counted from the 5' end of the sense strand.

[0272] In one embodiment, at least one lipophilic monomer is located at one or more internal positions on at least one strand, excluding positions 11-13 counting from the 3' end on the sense strand and positions 12-14 counting from the 5' end on the antisense strand.

[0273] In one embodiment, the one or more lipophilic monomers are located at one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0274] In one embodiment, the one or more lipophilic monomers are located at one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0275] definition

[0276] Unless specific definitions are provided, the nomenclature used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and the methods and techniques of these disciplines, are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses and chemical analyses. Certain such techniques and methods can be found, for example, in "Carbohydrate Modifications in Antisense Research," ed. 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," ed. Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press, 1989, which are incorporated by reference for any purpose. Where permitted, all patents, applications, published applications, and other publications and other data cited throughout this disclosure are incorporated by reference in their entirety.

[0277] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target protein (including but not limited to mRNA precursors and mRNA or portions thereof), as well as cDNA derived from such RNA, and miRNA. For example, the 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, the target nucleic acid can be a nucleic acid molecule from an infectious agent.

[0278] As used herein, the term "iRNA" refers to an agent that mediates the targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein 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 microRNA and microRNA precursors. In addition, as used herein, the "compound (compound or compounds)" of the present invention also refers to an iRNA agent and can be used interchangeably with an iRNA agent.

[0279] iRNA agent should include a region with sufficient homology to the target gene and with sufficient length in terms of nucleotides so that the iRNA agent or its fragment can mediate the downregulation of the target gene. (For ease of explanation, the term nucleotide or ribonucleotide is sometimes used in this article to refer to one or more monomer subunits of an iRNA agent. It should be understood in this article that the term "ribonucleotide" or "nucleotide" can be used in the context of modified RNA or nucleotide substitutes, and also refers to a modified nucleotide or alternative replacement portion at one or more positions.) Therefore, an iRNA agent is or includes a region that is at least partially, and in some embodiments, completely complementary to the target RNA. It is not necessary to have perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent or its cleavage product to guide sequence-specific silencing, such as by RNAi cleavage target RNA, such as mRNA. The degree of complementarity or homology with the target chain is the most critical in the antisense chain. Although perfect complementarity is generally required, particularly in the antisense chain, some embodiments can include, particularly in the antisense chain, one or more or, for example, 6, 5, 4, 3, 2 or less mismatches (relative to the target RNA). The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded character of the molecule.

[0280] iRNA agents include: molecules that are long enough to trigger an interferon response (these molecules can be cleaved by Dicer (Bernstein et al. 2001. Nature [Nature], 409: 363-366) and enter RISC (RNAi-induced silencing complex)); and molecules that are short enough that these molecules do not trigger an interferon response (these molecules can also be cleaved by Dicer and / or enter RISC), for example, molecules with a size that allows entry into RISC, such as molecules that resemble Dicer cleavage products. Molecules that are short enough not to trigger an interferon response are referred to herein as siRNA agents or shorter iRNA agents. As used herein, "siRNA agents or shorter iRNA agents" refer to iRNA agents that are short enough that they do not induce a harmful interferon response in human cells, such as double-stranded RNA agents or single-stranded agents, for example, duplex regions having less 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 target RNA, wherein the target can comprise endogenous or pathogen target RNA.

[0281] As used herein, "single-stranded iRNA agent" is an iRNA agent consisting of a single molecule. It may include a duplex region formed by intrachain pairing, for example, it may be or include a hairpin or pan-handle structure. Relative to the target molecule, the single-stranded iRNA agent may be antisense. The single-stranded iRNA agent may be long enough to enter RISC and participate in the cleavage of the target mRNA mediated by RISC. 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 certain embodiments, its length is less than 200, 100 or 60 nucleotides.

[0282] A loop is a region where one portion of an iRNA strand is unpaired with an opposing nucleotide in the duplex when one portion of the iRNA strand forms a base pair with the other strand or with another portion of the same strand.

[0283] The hairpin iRNA agent will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the duplex region may be equal to or less than 200, 100, or 50. In certain embodiments, the length of the duplex 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 terminal unpaired region, in some embodiments at the 3', and in certain embodiments at the antisense side of the hairpin. In some embodiments, the overhang is 2-3 nucleotides in length.

[0284] As used herein, a "double-stranded (ds) iRNA agent" is an iRNA agent that includes more than one strand, and in some cases two strands, wherein hybridization between the strands can form a region of duplex structure.

[0285] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0286] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in the level of mRNA in a cell of a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to and including 100%, and any integers therebetween, of the level of mRNA found in the cell in the absence of the miRNA or RNA interference molecule. 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 integers therebetween.

[0287] As used herein, the term "modulate 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 so that the expression, level, or activity is greater or less than that observed in the absence of the modulator. For example, the term "modulate" may mean "inhibit," but the use of the word "modulate" is not limited to this definition.

[0288] As used herein, modulation of gene expression 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, is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more that observed in the absence of an siRNA (e.g., RNAi agent). The % and / or fold difference can be calculated relative to a control or non-control, for example,

[0289]

[0290] or

[0291]

[0292] As used herein, the terms "inhibit," "downregulate," or "reduce" in relation to gene expression mean that gene expression, or the level of RNA molecules encoding one or more proteins or protein subunits, or equivalent RNA molecules, or the activity of one or more proteins or protein subunits is reduced to below that observed in the absence of a modulator. Gene expression is downregulated when gene expression, or the level of RNA molecules encoding one or more proteins or protein subunits, or equivalent RNA molecules, or the activity of one or more proteins or protein subunits is reduced by at least 10%, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or optimally 100% (i.e., no gene expression) relative to a corresponding non-modulated control.

[0293] As used herein, the term "increase" or "up-regulate" in relation to 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, or the activity of one or more proteins or protein subunits is increased to a level higher than that observed in the absence of a modulator. Gene expression is upregulated 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, or the activity of one or more proteins or protein subunits is increased by at least 10% relative to a corresponding non-modulated control, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or more.

[0294] As used herein, the term "increased" or "increase" generally means an increase by a statically significant amount; for the avoidance of any doubt, "increase" 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% compared to a reference level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or greater compared to a reference level.

[0295] As used herein, the term "reduce" generally means a statistically significant amount of reduction. However, for the avoidance of doubt, "reduce" means a reduction of at least 10% compared to a reference level, such as a reduction 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% (i.e., no level compared to a reference sample), or any reduction between 10%-100%, compared to a reference level.

[0296] Double-stranded iRNA comprises two oligonucleotide chains, which are fully complementary to form a duplex structure with hybridization. Typically, the length of the duplex structure is 15 to 30, more typically 18 to 25, also more typically 19 to 24 and most typically 19 to 21 base pairs. In certain embodiments, a longer double-stranded iRNA of 25 to 30 base pairs in length is preferred. In certain embodiments, a shorter double-stranded iRNA of 10 to 15 base pairs in length is preferred. In another embodiment, the double-stranded iRNA is at least 21 nucleotides long.

[0297] 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 duplex region is 14-30 nucleotides in length. Similarly, the complementary region of the target sequence is 14 to 30, more typically 18 to 25, still more typically 19 to 24, and most typically 19 to 21 nucleotides in length.

[0298] As used herein, the term "compound" refers to an oligomeric compound, which can be an oligonucleotide, an antisense or an iRNA agent, such as siRNA.

[0299] As used herein, the phrase "antisense strand" refers to an oligomeric compound that is substantially or 100% complementary to a target sequence of interest. The phrase "antisense strand" includes the antisense regions of both oligomeric compounds formed from two separate strands, as well as single-molecule oligomeric compounds capable of forming a hairpin or dumbbell-shaped structure. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0300] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence identical to all or part of a target sequence, such as a messenger RNA or DNA sequence. The terms "sense strand" and "follower strand" are used interchangeably herein.

[0301] By "specifically hybridizable" and "complementary" is meant that a nucleic acid can form hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types. With respect to the core molecules of the present invention, the binding free energy of a nucleic acid molecule to 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 Symposium on Quantitative Biology] 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. [American Chemical Society] 109:3783-3785). Percent complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "Perfect complementarity" or 100% complementarity means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Less than perfect complementarity refers to a situation where some, but not all, nucleoside units in the two chains can hydrogen bond with each other. "Significant complementarity" refers to a polynucleotide chain exhibiting 90% or greater 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 oligomeric compound to a non-target sequence under conditions where specific binding is required, i.e., under physiological conditions in the case of an in vivo assay or therapeutic treatment, or under conditions where the assay is performed in the case of an in vitro assay. Non-target sequences typically differ by at least 5 nucleotides.

[0302] In some embodiments, 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 in length.

[0303] In some embodiments, the antisense strand 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 in length.

[0304] In some embodiments, the sense strand 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 in length.

[0305] In one embodiment, the sense and antisense strands of the compound are each 15 to 30 nucleotides in length.

[0306] In one embodiment, the sense and antisense strands of the compound are each 19 to 25 nucleotides in length.

[0307] In one embodiment, the sense and antisense strands of the compound are each 21 to 23 nucleotides in length.

[0308] In some embodiments, a chain has at least one section of 1-5 single-stranded nucleotides in the double-stranded region. "Single-stranded nucleotide section in the double-stranded region" means that there is at least one nucleotide base pair at both ends of the single block. In some embodiments, the two chains have at least one section of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. When the two chains have a section of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides can be opposite to each other (e.g., a mismatch) or it can be positioned so that the second chain does not have a single-stranded nucleotide opposite to the single-stranded iRNA of the first chain, and vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides at either end, such as 8, 7, 6, 5, 4, 3, or 2 nucleotides at the 5' or 3' end of the complementary region between the two chains.

[0309] In one embodiment, the compound comprises a single-stranded overhang on at least one of the plurality of ends. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0310] In one embodiment, the sense strand of the iRNA agent 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 (with a 2 nucleotide long single-stranded overhang at the 3' end).

[0311] In some embodiments, each strand of the double-stranded iRNA has a ZXY structure, such as described in PCT Publication No. 2004080406, which is herein incorporated by reference in its entirety.

[0312] In certain embodiments, the two chains of the double-stranded oligomeric compound can be linked together. The two chains can be linked to each other at both ends, or only at one end. Linked 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 ; wherein 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: GNR A, (G) 4, (U) 4, and (dT) 4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some nucleotides in the linker may involve base pair interactions with other nucleotides in the linker. The two chains may also be linked together by a non-nucleoside linker, such as a linker described herein. It will be appreciated by those skilled in the art that any oligonucleotide chemical modification or variation described herein may be used in oligonucleotide linkers.

[0313] Hairpin and dumbbell oligomeric compounds have a duplex region of equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the duplex region may be equal to or less than 200, 100, or 50 nucleotide pairs. In some embodiments, the length of the duplex region ranges from 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0314] The hairpin oligomeric compound can have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3', and in some embodiments at the antisense side of the hairpin. In some embodiments, the overhang is 1-4, more typically 2-3 nucleotides in length. Hairpin oligomeric compounds that can induce RNA interference are also referred to herein as "shRNA."

[0315] In certain embodiments, the two oligomeric chains specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0316] As used herein, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which an antisense compound will hybridize to its target sequence but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and the "stringent conditions" under which an antisense compound hybridizes to a target sequence are determined by the nature and composition of the antisense compound and the assay being studied.

[0317] It should be understood in the art that, compared with unmodified compounds, incorporation of nucleotide affinity modification can allow a greater number of mispairings. Similarly, some oligonucleotide sequences can be more tolerant to mispairings than other oligonucleotide sequences. Those of ordinary skill in the art can determine the appropriate number of mispairings between oligonucleotides or between oligonucleotides and target nucleic acids, such as by measuring melting temperature (Tm). Tm or ΔTm can be calculated by the technology familiar to those of ordinary skill in the art. For example, the technology described in Freier et al. (Nucleic Acids Research [nucleic acid research], 1997, 25, 22: 4429-4443) allows those of ordinary skill in the art to assess the ability of nucleotide modification to increase the melting temperature of RNA:DNA duplexes.

[0318] siRNA design

[0319] In one embodiment, the iRNA agent is a double-ended blunt-ended product having a length of 19 nt, wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0320] In one embodiment, the iRNA agent is a double-ended blunt-ended RNA of 20 nt in length, wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0321] In one embodiment, the iRNA agent is a double-ended blunt-ended RNA of 21 nt in length, wherein the sense strand contains at least one motif having 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 having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0322] 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 having 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 having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 at the 5' end, wherein one end of the iRNA is blunt and the other end comprises 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).

[0323] 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 and comprises 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 comprises at least 8 ribonucleotides starting from the 3'-terminal nucleotide at positions that pair with positions 1-23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5'-terminal end of the antisense strand is unpaired with the sense strand, and The invention relates to a nucleic acid comprising 10-30 consecutive nucleotides that are unpaired with the sense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5'-terminal and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand, thereby forming a substantially duplex region between the sense strand and the antisense strand; and when the double-stranded nucleic acid is introduced into a mammalian cell, the antisense strand is sufficiently complementary to the target RNA along a length of at least 19 ribonucleotides of the antisense strand to reduce target gene expression; and wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site. The antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0324] 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, there is at least one motif of three 2'-O-methyl modifications 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 duplex region is at least 25 nucleotides in length, and when the iRNA agent is introduced into a mammalian cell, the second strand is sufficiently complementary to the target mRNA along a length of at least 19 nt of the second strand to reduce target gene expression, and wherein dicer cleavage of the iRNA preferably produces an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0325] In one embodiment, the sense strand of the iRNA agent contains at least one motif having three identical modifications on three consecutive nucleotides, wherein one motif occurs at the cleavage site of the sense strand. For example, the sense strand may contain at least one motif having three 2'-F modifications on three consecutive nucleotides within 7-15 positions of the 5' end.

[0326] In one embodiment, the antisense strand of the iRNA agent may also contain at least one motif having three identical modifications on three consecutive nucleotides, wherein one motif occurs at or near the cleavage site of the antisense strand. For example, the antisense strand may contain at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides within 9-15 positions of the 5' end.

[0327] For iRNA agents with duplex regions of 17-23 nt in length, the cleavage sites of the antisense strand are typically near positions 10, 11, and 12 of the 5' end. Thus, a motif having three identical modifications can appear at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide of the 5' end of the antisense strand, or from the first paired nucleotide within the duplex region of the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the iRNA from the 5' end.

[0328] 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 occurs at or near the cleavage site within the strand, and at least one motif occurs in another portion of the strand (the motif is 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 occurs at or near the cleavage site within the strand. The modifications in the motifs occurring at or near the cleavage site in the sense strand are different from the modifications in the motifs occurring at or near the cleavage site in the antisense strand.

[0329] 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 having three 2'-F modifications on three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site in the strand. In one embodiment, the antisense strand also contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0330] 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 having three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 of the 5' terminus, and wherein the antisense strand contains at least one motif having three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 of the 5' terminus.

[0331] In one embodiment, the iRNA agent comprises one or more mismatches with the target within the duplex, or a combination thereof. Mismatches can be present in the protruding region or duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melt (e.g., based on the free energy of association or dissociation of a specific pairing, the simplest method is to check pairing on the basis of a single pair, but the next adjacent or similar analysis can also be used). In terms of promoting dissociation: A: U is better than G: C; G: U is better than G: C; and I: C is better than G: C (I = inosine). Mismatches (e.g., atypical or not typical pairing (as described elsewhere herein)) are better than typical (A: T, A: U, G: C) pairings; and pairings including universal bases are better than typical pairings.

[0332] In one embodiment, the iRNA agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region at the 5' end of the antisense strand, which can be independently selected from the group consisting of A:U, G:U, I:C, and mismatched pairings, e.g., atypical pairings including universal bases or in addition to canonical pairings, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0333] In one embodiment, the nucleotide at position 1 in the 5' duplex 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' duplex region of the antisense strand is an AU base pair. For example, the first base pair in the 5' duplex region of the antisense strand is an AU base pair.

[0334] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the dsRNA agents are modified. For example, when 50% of the dsRNA agents are modified, 50% of all nucleotides present in the dsRNA agent contain modifications as described herein.

[0335] In one embodiment, each of the sense and antisense strands is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0336] In one embodiment, each of the sense and antisense strands of the dsRNA agent contains at least two different modifications.

[0337] In one embodiment, the dsRNA agent does not contain any 2'-F modifications.

[0338] In one embodiment, the sense strand and / or antisense strand of the dsRNA agent comprises one or more blocks having a bond between phosphorothioate or methylphosphonate nucleotides. In one example, the sense strand comprises a block having a bond between two phosphorothioate or methylphosphonate nucleotides. In one example, the antisense strand comprises two blocks having a bond between two phosphorothioate or methylphosphonate nucleotides. For example, two blocks having a bond between phosphorothioate or methylphosphonate nucleotides are separated by a bond between 16-18 phosphate nucleotides.

[0339] 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.

[0340] 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.

[0341] In one embodiment, the antisense strand of the dsRNA agent is 100% complementary to the target RNA to hybridize therewith 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.

[0342] In one aspect, the present invention relates to a dsRNA agent as defined herein that is capable of inhibiting target gene expression. 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 thermally destabilized nucleotide, wherein at least one of the thermally destabilized nucleotides occurs at or near a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5' end of the antisense strand).

[0343] When the sense strand is 21 nucleotides long, the thermally destabilized nucleotide can be present, for example, between positions 14-17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification. Preferably, the two modified nucleic acids that are smaller than the sterically demanding 2'-OMe modification are separated by 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0344] In one embodiment, the dsRNA agent comprises:

[0345] (a) a sense strand having:

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

[0347] (ii) three consecutive 2'-F modifications at positions 7-15; and

[0348] (b) an antisense strand having:

[0349] (i) 18-23 nucleotides in length;

[0350] (ii) at least a 2'-F modification anywhere on the strand; and

[0351] (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5' end);

[0352] 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 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 duplex are blunt ended.

[0353] In one embodiment, the dsRNA agent comprises:

[0354] (a) a sense strand having:

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

[0356] (ii) fewer than four 2′-F modifications;

[0357] (b) an antisense strand having:

[0358] (i) 18-23 nucleotides in length;

[0359] (ii) fewer than twelve 2'-F modifications; and

[0360] (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5' end);

[0361] 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 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 duplex are blunt ended.

[0362] In one embodiment, the dsRNA agent comprises:

[0363] (a) a sense strand having:

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

[0365] (ii) fewer than four 2′-F modifications;

[0366] (b) an antisense strand having:

[0367] (i) 19-35 nucleotides in length;

[0368] (ii) fewer than twelve 2'-F modifications; and

[0369] (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5' end);

[0370] wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); and 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 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 duplex are blunt.

[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 phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex 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 has less than 20%, less than 15%, and less than 10% non-natural nucleotides.

[0372] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP) or 2'-ara-F, etc.

[0373] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex 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 has greater than 80%, greater than 85%, and greater than 90% natural nucleotides, such as 2'-OH, 2'-deoxy, and 2'-OMe as natural nucleotides.

[0374] In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex 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 has 100% natural nucleotides, such as 2'-OH, 2'-deoxy, and 2'-OMe as natural nucleotides.

[0375] 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):

[0376] 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0377] (I)

[0378] in:

[0379] i and j are each independently 0 or 1;

[0380] p and q are each independently 0-6;

[0381] Each N a independently represent an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0382] Each N b independently represents an oligonucleotide sequence comprising 1, 2, 3, 4, 5 or 6 modified nucleotides;

[0383] Each n p and n q Overhang nucleotides are represented independently;

[0384] where N b and Y does not have the same modification;

[0385] wherein XXX, YYY and ZZZ each independently represent a motif having three identical modifications on three consecutive nucleotides;

[0386] 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

[0387] wherein the antisense strand of the dsRNA comprises two blocks having one, two or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 phosphate internucleotide linkages.

[0388] Various disclosures describe multimeric siRNAs, and all can be used with the iRNA of the present invention. Such disclosures include WO 2007 / 091269, U.S. Patent No. 7858769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, which are incorporated herein by reference in their entirety.

[0389] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agents of the invention are modified with 2'-OMe.

[0390] In some embodiments, each of the sense and antisense strands of the iRNA agent is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0391] In some embodiments, each of the sense and antisense strands of the iRNA agent contains at least two different modifications.

[0392] In some embodiments, the compounds of the invention do not contain any 2'-F modifications.

[0393] In some embodiments, the compounds of the 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 invention contain nine or ten 2'-F modifications.

[0394] The iRNA agent of the present invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may occur on any nucleotide of the sense strand or the antisense strand or both at any position in the strand. For example, the internucleotide bond modification may occur on each nucleotide on the sense strand or the antisense strand; each internucleotide bond modification may occur on the sense strand or the antisense strand in an alternating pattern; or the sense strand or the antisense strand may contain two internucleotide bond modifications in an alternating pattern. The alternating pattern of internucleotide bond modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide bond modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide bond modifications on the antisense strand.

[0395] In one embodiment, iRNA comprises phosphorothioate or methylphosphonate internucleotide key modification in the overhang region.For example, the overhang region can contain two nucleotides, with a phosphorothioate or methylphosphonate internucleotide key between the two nucleotides.Also can carry out internucleotide key modification to connect the overhang nucleotide with the terminal pairing nucleotide in the duplex region.For example, at least 2,3,4 or all overhang nucleotides can be connected by a key between phosphorothioate or methylphosphonate nucleotides, and optionally, there can be an extra phosphorothioate or methylphosphonate internucleotide key that the overhang nucleotide is connected to the pairing nucleotide next to the overhang nucleotide.For example, between the terminal three nucleotides, there can be at least two phosphorothioate internucleotide connections, wherein two of these three nucleotides are overhanging nucleotides, and the third is the nucleotide next to the pairing of the overhanging nucleotide.Preferably, these terminal three nucleotides can be at the 3 ' end of the antisense strand.

[0396] In some embodiments, the sense strand and / or antisense strand of the iRNA agent comprises one or more blocks having a phosphorothioate or methylphosphonate internucleotide bond. In one example, the sense strand comprises a block having two phosphorothioate or methylphosphonate internucleotide bonds. In one example, the antisense strand comprises two blocks having two phosphorothioate or methylphosphonate internucleotide bonds. For example, two blocks having a phosphorothioate or methylphosphonate internucleotide bond are separated by 16-18 phosphate internucleotide bonds.

[0397] In some embodiments, the antisense strand of an iRNA agent is 100% complementary to the target RNA to hybridize therewith and inhibit its expression by RNA interference. In another embodiment, the antisense strand of an iRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0398] Nucleic acid modification

[0399] 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 a modified internucleoside bond, a modified nucleobase, a modified sugar, and any combination thereof. Without limitation, such a modification may be present at any position in the compound. For example, the modification may be present in one of the RNA molecules.

[0400] Nucleic acid modifications (nucleobases)

[0401] The naturally occurring base moiety of a nucleoside is typically a heterocyclic base. The two most common categories of such heterocyclic bases are purines and pyrimidines. For those nucleosides comprising furanose sugars, the phosphate group can be attached to the 2', 3' or 5' hydroxyl moiety of the sugar. When forming an oligonucleotide, those phosphates covalently link adjacent nucleosides to form a linear polymeric compound. In an oligonucleotide, phosphate is commonly referred to as the internucleoside backbone that forms an oligonucleotide. The naturally occurring bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.

[0402] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimetics known to those of skill in the art are suitable for use in the compounds described herein. Unmodified or natural nucleobases can be modified or replaced to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or using any of the synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be employed. When a natural base is replaced by a non-natural and / or universal base, the nucleotide is referred to as comprising a modified nucleobase and / or nucleobase modification herein. Modified nucleobases and / or nucleobase modifications also include natural, non-natural, and universal bases, which comprise a conjugated moiety, such as a ligand as described herein. Preferred conjugated moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobase via a suitable alkyl, alkenyl, or linker having an amide bond.

[0403] The oligomeric compounds described herein may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, muscimol, isoguanosine, tuberculin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(Isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxy)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6 -(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-( uracil, 5-(aminoalkyl)uracil, 5-(guanidinyl)uracil, 5-(1,3-oxadiazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio) ) pseudouracil, 1-(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)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl 1-yl, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, muscimol, tuberculin, isoguanosine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isoquinolone, 5-(methyl)isoquinolone, 3-(methyl)-7-(propynyl)isoquinolone, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyrazinyl, isoquinolone keto, 7-(propynyl)isoquinolone, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrene, stilbene, tetracene, pentacene, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N, 2 -substituted purine, N 6 -substituted purine, O 6 -substituted purine, substituted 1,2,4-triazole, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, p-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, o-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, di-o-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, p-(aminoalkylhydroxy)-6-phenyl In some embodiments, the present invention provides a pyrimidin-2-one-3-yl group, a pyrimidin-2-one-3-yl group, a pyrimidin-3-yl group, a pyrimidin-2-one-3-yl group, a pyrimidin-3-yl group, a pyrimidin-2-one-3-yl group, a pyrimidin-3-yl group, a pyrimidin-2-one-3-yl group, a pyrimidin-3-yl group, a pyrimidin-7-amino-pyrimidin-3-yl group, a pyrimidin-2-one ...4-yl group, a pyrimidin-4-yl group, a pyrimidin-5-yl group, a pyrimidin-6-yl group, a

[0404] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, intracellular enzymatic recognition, or activity of the iRNA duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisoquinolonyl, 5-methylisoquinolonyl, 3-methyl-7-propynylisoquinolonyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyrazinyl, isoquinolonyl, 7-propynylisoquinolonyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenyl, tetracenyl, pentacene, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Nucleic Acids Research, 29, 2437-2447).

[0405] Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808; International Application No. PCT / US09 / 038425, filed March 26, 2009; Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990; English et al., Angewandte Chemie, International Edition, 1991, 30, 613; Modified Nucleosides in Biochemistry, Biotechnology and Nucleobases disclosed in Modified Nucleosides in Biochemistry, Biotechnology, and Medicine, Herdewijin, P., ed., Wiley-VCH, 2008; and Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., eds., CRC Press, 1993. All of the foregoing are incorporated herein by reference.

[0406] In certain embodiments, the modified nucleobase is a nucleobase that is structurally very similar to the parent nucleobase, such as 7-deazapurine, 5-methylcytosine, or a G-clamp. In certain embodiments, the nucleobase mimetic comprises a more complex structure, such as a tricyclic phenoxazine nucleobase mimetic. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.

[0407] Nucleic acid modification (sugar)

[0408] The compounds of the invention provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers, including nucleosides or nucleotides having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in a variety of ways, including but not limited to adding substituents, bridging two non-geminal ring atoms to form locked nucleic acids or bicyclic nucleic acids. In certain embodiments, the oligomeric compound comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNAs.

[0409] In some embodiments of locked nucleic acids, the 2' position of the furanosyl 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)-;

[0410] in:

[0411] x is 0, 1, or 2;

[0412] n is 1, 2, 3, or 4;

[0413] each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfo (S(═O)—J1); and

[0414] 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, heterocyclyl, substituted heterocyclyl, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0415] 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.

[0416] Certain LNAs have been prepared and disclosed in the patent literature and in the scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Patent Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807).

[0417] Also provided herein are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') linkage to form a bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patent Nos. 6,268,490 and 6,670,461). The bond can be a methylene group (-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 group at this position, the term ethyleneoxy (4'-CH2CH2-O-2') LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy (4'-CH2-O-2') LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability (Tm = +3 to +10°C) with complementary DNA and RNA, stability against 3'-exonucleolytic degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0418] An isomer of methyleneoxy (4'-CH2-O-2') LNA that has also been discussed is α-L-methyleneoxy (4'-CH2-O-2') LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy (4'-CH2-O-2') LNA has been incorporated into antisense gapmers and chimeras that exhibit potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0419] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0420] Analogs of methyleneoxy (4'-CH2-O-2') LNA, phosphorothioate-methyleneoxy (4'-CH2-O-2') LNA and 2'-thio-LNA, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs comprising oligodeoxynucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). In addition, the synthesis of 2'-amino-LNA, a novel, conformationally constrained, high-affinity oligonucleotide analog, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-LNAs have been prepared and their thermal stability in duplexes with complementary RNA and DNA strands has been previously reported.

[0421] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars, including methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2' bridge) ENA; substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; and 4'-sulfanyl modified sugars. Sugars can also be substituted with sugar-mimicking groups and other substitutions. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; and WO 2005 / 121371.

[0422] Examples of "oxy"-2' hydroxy modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O); n CH2CH2OR, n=1-50; "locked" nucleic acids (LNA), in which the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic system; O-amine or O-(CH2) n Amine (n = 1-10, amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0423] "Deoxy" modifications include hydrogen (i.e., deoxyribose, which is particularly relevant to single-stranded overhangs); halo (e.g., fluorine); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) nCH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; sulfhydryl; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which may be optionally substituted, for example, with an amino functional group.

[0424] Other suitable 2'-modifications (eg, modified MOE) are described in US Patent Application Publication No. 20130130378, the contents of which are herein incorporated by reference in their entirety.

[0425] The modification at the 2' position may be present in the arabinose configuration. The term "arabinose configuration" refers to the position of the substituent on C2' of the ribose sugar having the same configuration as the 2'-OH in arabinose.

[0426] Sugars can contain two different modifications at the same carbon in the sugar, such as a geminal modification. The glycosyl group can also contain one or more carbons that have a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, an oligomeric compound can include one or more monomers containing, for example, arabinose as a sugar. The monomer can have an α bond at the 1' position of the sugar, such as an α-nucleoside. The monomer can also have an opposite configuration at the 4'-position, such as C5' and H4' or the substituents replacing them interchangeably. When C5' and H4' or the substituents replacing them interchangeably, the sugar is said to be modified at the 4' position.

[0427] 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 in place of a nucleobase at C1'. See, for example, U.S. Patent No. 5,998,203, the contents of which are incorporated herein in their entirety. These abasic sugars may also further contain modifications at one or more of the constituent sugar atoms. The compounds of the present invention may also contain one or more sugars in the form of L isomers, such as L-nucleosides. Modifications to the sugar group may also include replacement of 4'-O with sulfur, optionally substituted nitrogen, or a CH2 group. In some embodiments, the bond between C1' and the nucleobase is in an α configuration.

[0428] Sugar modifications may also include "acyclic nucleotides," which refer to any nucleotide having a non-cyclic ribose sugar, e.g., wherein a C-C bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, the acyclic nucleotide is wherein 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 a sugar.

[0429] 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 geminal 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0430] It is 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., a nucleotide linked via its 2'-position). The modification at the 3' position can be present in a xylose configuration. The term "xylose configuration" refers to the position of the substituent on C3' of ribose having the same configuration as the 3'-OH in xylose.

[0431] The hydrogen attached to C4' and / or C1' may be replaced by a linear or branched optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, wherein the backbone of the alkyl, alkenyl, and alkynyl groups 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'), a phosphorus-containing bond, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycle, or an optionally substituted cycloalkyl group, wherein R' is hydrogen, acyl, or an optionally substituted aliphatic, 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 41 R 51、N(R 21 )C(=NR 31 )NR 21 R 31 、N(R 21 )C(O)NR 21 R 31 、N(R 21 )C(S)NR 21 R 31 、OC(O)NR 21 R 31 、SC(O)NR 21 R 31 、N(R 21 )C(S)OR 11 、N(R 21 )C(O)OR 11 、N(R 21 )C(O)SR 11 、N(R 21 )N=CR 41 R 51 、ON=CR 41 R 51 、SO2R 11 、SOR 11 SR 11 and a substituted or unsubstituted heterocycle; R 21 and R 31 is independently at each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclyl, OR 11 、COR 11 、CO2R 11 or NR 11 R 11 '; or R 21 and R 31 Together with the atoms to which they are attached, they form a heterocyclic ring; R 41 and R 51 is independently at each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclyl, OR 11 、COR 11 or CO2R 11 or NR 11 R 11 '; and R 11 and R 11 ' is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclyl. In some embodiments, the hydrogen of C4' attached to the 5' terminal nucleotide is replaced.

[0432] In some embodiments, C4' and C5' together form an optionally substituted heterocyclyl, preferably comprising 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, at each occurrence, an alkali metal or transition metal with a total charge of +1; and Y is O, S, or NR', wherein R' is hydrogen, an optionally substituted aliphatic group. Preferably, this modification is at the 5' end of the iRNA.

[0433] In certain embodiments, the compounds of the present invention comprise at least two regions comprising at least two consecutive monomers of the formula above. In certain embodiments, the compounds of the present invention comprise a gapped motif. In certain embodiments, the compounds of the present invention comprise at least one region comprising from about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, the compounds of the present invention comprise at least one region comprising from about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0434] In certain embodiments, compounds of the invention comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) (S)-cEt monomer having the formula:

[0435]

[0436] Wherein Bx is a heterocyclic base moiety.

[0437] In certain embodiments, monomers include sugar mimetics. In certain such embodiments, mimetics are used to replace sugar or sugar-nucleoside bond combinations, and the core base is kept to hybridize with the selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or N-morpholinyl. Representative examples of mimetics of sugar-nucleoside bond combinations include, but are not limited to, peptide nucleic acids (PNAs) and N-morpholinyl connected by uncharged achiral bonds. In some cases, mimetics are used to replace core bases. Representative core base mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. [Nucleic Acids Research] 2000, 28: 2911-14, which are incorporated herein by reference). The synthetic methods of sugar, nucleoside and core base mimetics are well known to those skilled in the art.

[0438] Nucleic acid modifications (intersugar bonds)

[0439] Described herein are linking groups that link monomers (including but not limited to modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, such as oligonucleotides. Such linking groups are also referred to as intersugar bonds. The 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), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P═S). Representative phosphorus-free linking groups include but are not limited to methylenemethylimino (—CH2—N(CH3)—O—CH2-), thiodiester (—OC(O)—S-), thiocarbamate (—OC(O)(NH)—S-); siloxane (—O—Si(H)2-O-); and N,N'-dimethylhydrazine (—CH2—N(CH3)—N(CH3)-). Modified bonds can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester bonds. In certain embodiments, bonds with chiral atoms can be prepared as racemic mixtures, prepared as individual enantiomers. Representative chiral bonds include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known to those skilled in the art.

[0440] The phosphate in the linking group can be modified by replacing one oxygen with a different substituent. One result of this modification can be to increase the resistance of the oligonucleotide to nucleolytic decomposition. Examples of modified phosphates include phosphorothioates, phosphoroselenoates, boranophosphates, boranophosphates, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. 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 the unmodified phosphate is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups of atoms makes the phosphorus atom chiral; in other words, the phosphorus atom in the phosphate modified in this way is a stereosymmetric center. The stereosymmetric phosphorus atom can have an "R" configuration (herein, Rp) or an "S" configuration (herein, Sp).

[0441] Phosphorodithioate has two non-bridging oxygens replaced by sulfur.The phosphorus center in phosphorodithioate is achiral, and it stops the formation of oligonucleotide diastereomers.Therefore, although do not wish to be bound by theory, it can be desirable to eliminate chiral center (such as phosphorodithioate forms) to the modification of two non-bridging oxygens, because it cannot produce diastereoisomer mixtures.Therefore, non-bridging oxygen can independently be any one in O, S, Se, B, C, H, N or OR (R is alkyl or aryl).

[0442] Phosphate linkers can also be modified by replacing the bridging oxygen (i.e., the oxygen connecting the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylenephosphonates). Replacement can occur at either or both oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, replacement with nitrogen is preferred.

[0443] A modified phosphate linkage, in which at least one of the oxygens attached to the phosphate has been replaced or the phosphate has been replaced by a non-phosphorus group, is also referred to as a "non-phosphodiester intersugar linkage" or a "non-phosphodiester linker."

[0444] In certain embodiments, phosphates can be replaced by phosphorus-free linkers, such as dephospholinkers. Dephospholinkers are also referred to herein as non-phosphodiester linkers. Although not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center for nucleolytic degradation, its replacement with a neutral structural mimetic should confer enhanced nuclease stability. Similarly, although not wishing to be bound by theory, in some embodiments, it may be necessary to introduce a change in which the charged phosphate is replaced by a neutral moiety.

[0445] Examples of moieties that can replace the phosphate group 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, siloxane (dialkylsiloxane), formamide, carbonate, carboxymethyl, carbamate, carboxylate, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate, thiomethyl acetal (3'-S-CH2-O-5'), methylal (3'-O-CH2-O-5'), oxime, methyleneimino, methylenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazine, methylenedimethylhydrazine, 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, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and P.D. Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred examples include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0446] Those skilled in the art are well aware that, in certain circumstances, replacement of a non-bridging oxygen can result in enhanced cleavage of an inter-sugar bond by the adjacent 2'-OH group, and therefore, in many cases, modification of the non-bridging oxygen group may require modification of the 2'-OH group, such as modifications that do not participate in the cleavage of an adjacent inter-sugar bond, such as arabinose, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0447] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates having an enantiomeric excess of the Sp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorothioates having an enantiomeric excess of the Rp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphoroates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranephosphonates.

[0448] In some embodiments, the compounds of the 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 up to all) modified or non-phosphodiester bonds. In some embodiments, the compounds of the 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 up to all) phosphorothioate bonds.

[0449] Compounds of the present invention can also be constructed in which phosphate linkers and sugars are replaced with nuclease-resistant nucleosides or nucleotide substitutes. Although not wishing to be bound by theory, it is believed that the absence of a repeated charged main chain reduces binding to proteins (e.g., nucleases) that recognize polyanions. Similarly, although not wishing to be bound by theory, in some embodiments, it may be necessary to introduce changes in which bases are bound by neutral alternative main chains. Examples include N-morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and main chain-extended pyrrolidine PNA (bepPNA) nucleoside substitutes. Preferred substitutes are PNA substitutes.

[0450] The compounds of the invention described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. The compounds of the invention provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0451] Nucleic acid modification (terminal modification)

[0452] In some embodiments, the compound further comprises a phosphate or a phosphate mimetic at the 5' end of the antisense strand. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0453] In some embodiments, the 5' end of the antisense strand of the compound does not contain 5'-vinylphosphonate (VP).

[0454] The end of the iRNA agent of the present invention can be modified.Such modification can be at one end or both ends.For example, the 3' and / or 5' ends of iRNA can be conjugated with other functional molecular entities, such as labeling moieties, such as fluorophores (for example, pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based on, for example, sulfur, silicon, boron or esters). Functional molecular entities can be attached to sugars by phosphates and / or joints. The terminal atom of the joint can be connected to or replace the connecting atom of the C-3' or C-5' O, N, S or C groups of phosphates or sugars. Alternatively, the joint can be connected to or replace the terminal atom of a nucleotide substitute (such as PNA).

[0455] When the linker / phosphate-functional molecular entity-linker / phosphate array is inserted between the two strands of a double-stranded oligomeric compound, the array can replace the hairpin loop in the hairpin-type oligomeric compound.

[0456] Terminal modifications that can be used to modulate activity include modifying the 5' end of the iRNA with a phosphate or phosphate analog. In certain embodiments, the 5' end of the iRNA is phosphorylated or includes a phosphoryl analog. Exemplary 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Modifications at the 5' end can also be used to stimulate or suppress the subject's immune system. In some embodiments, the 5' end of the oligomeric compound comprises a modification wherein W, X and Y are each independently selected from the group consisting of: O, OR (R is hydrogen, alkyl, aryl), S, Se, BR3 (R is hydrogen, alkyl, aryl), 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 at each occurrence, wherein the backbone of the alkylene may contain one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and / or terminally; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen attached to the 5' carbon of the sugar. When n is 0, W and Y together with the P to which they are attached may form an optionally substituted 5-8 membered heterocyclic ring, wherein W and Y are each independently O, S, NR' or alkylene. Preferably, the heterocyclic ring is substituted with aryl or heteroaryl. In some embodiments, one or two hydrogens on C5' of the 5' terminal nucleotide are replaced with halogen (e.g., F).

[0457] 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'-monothioate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithioate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-thioate ((HO)2(O)PS-5'); 5'-α-thiotriphosphate; 5'-β-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkylphosphonates (R(OH)(O)PO-5', R = alkyl, such as methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyletherphosphonates (R(OH)(O)PO-5', R = alkyl ether, such as methoxymethyl (CH2OMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is optionally substituted with an alkyl radical 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 terminal phosphates and phosphate mimetics: HO[-(CH2) a -OP(X)(OH)-O] b -5'、H2N[-(CH2) a -OP(X)(OH)-O] b -5'、H[-(CH2) a -OP(X)(OH)-O] b -5'、Me2N[-(CH2) a -OP(X)(OH)-O] b -5'、HO[-(CH2) a -P(X)(OH)-O] b -5'、H2N[-(CH2) a -P(X)(OH)-O] b -5'、H[-(CH2) a -P(X)(OH)-O] b-5'、Me2N[-(CH2) a -P(X)(OH)-O] b -5', wherein a and b are each independently 1-10. Other embodiments include using BH3, BH3 - and / or Se replace oxygen and / or sulfur.

[0458] 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 Alexa 488. Terminal modifications can also be used to enhance uptake, and useful modifications for this include targeting ligands. Terminal modifications can also be used to crosslink the oligonucleotide to another moiety; useful modifications for this include mitomycin C, psoralen, and its derivatives.

[0459] Thermal destabilization modification

[0460] Compounds of the invention (such as iRNA or dsRNA agents) can be optimized for RNA interference by introducing a thermal destabilizing modification in the sense strand at a site opposite the seed region of the antisense strand (i.e., at positions 2-8 of the 5' end of the antisense strand) that increases the propensity of the iRNA duplex to dissociate or melt (reduces the free energy of duplex association). This modification can increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand.

[0461] Thermally destabilizing modifications can include abasic modifications; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications, such as 2'-deoxy modifications or non-cyclic nucleotides, eg, unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA).

[0462] Exemplary abasic modifications are:

[0463]

[0464] Exemplary sugar modifications are:

[0465]

[0466] The term "UNA" refers to an unlocked non-cyclic nucleic acid in which any bond to the sugar has been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) is removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entirety. Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' bonds.

[0467] The term 'GNA' refers to diol nucleic acids, which are polymers similar to DNA or RNA but differ in their "backbone" composition, consisting of repeating glycerol units linked by phosphodiester bonds:

[0468]

[0469] Thermal destabilization modification can be a mismatch (i.e., non-complementary base pair) between a thermal destabilization nucleotide within a dsRNA duplex and a relative nucleotide in the relative chain. Exemplary mismatch base pairings 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 a combination thereof. Other mismatch base pairings known in the art are also applicable to the present invention. Mismatches can occur between nucleotides (naturally occurring nucleotides or modified nucleotides), that is, mismatch base pairings can occur between the core bases of the corresponding nucleotides, regardless of the modification on the ribose of the nucleotide. In certain embodiments, the compounds of the present invention (such as siRNA or iRNA agents) contain at least one core base, i.e., a 2'-deoxynucleobase, in the mismatch pairing; for example, a 2'-deoxynucleobase is in the sense strand.

[0470] Further examples of abasic nucleotides, non-cyclic 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.

[0471] Thermally destabilizing modifications may also include universal bases that have reduced or eliminated ability to form hydrogen bonds with opposing bases, as well as phosphate modifications.

[0472] Nucleobase modifications with impaired or completely abolished ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of dsRNA duplexes as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

[0473]

[0474] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester bonds are:

[0475]

[0476] In some embodiments, the compounds of the present invention may comprise a 2'-5' bond (having 2'-H, 2'-OH, and 2'-OMe and having P=O or P=S). For example, the 2'-5' bond modification may be used to promote nuclease resistance or inhibit the binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0477] In another embodiment, the compounds of the present invention may include an L sugar (e.g., L-ribose, L-arabinose with 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 the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0478] In one embodiment, the iRNA agent of the present invention is conjugated to the ligand via a carrier, wherein the carrier can be a cyclic group or a non-cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene; preferably, the non-cyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0479] In some embodiments, at least one strand of an iRNA agent disclosed herein is 5' phosphorylated or includes a phosphoryl analog at the 5' terminus. 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothioate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithioate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothioate ((HO)2(O)PS-5'); any combination of monophosphates, diphosphates and triphosphates with additional oxygen / sulfur substitutions (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., such as RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., such as RP(OH)(O)-O-5'-).

[0480] target gene

[0481] Without limitation, target genes for siRNA include, but are not limited to, genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, genes that express kinases, attachment protein genes, genes encoding G protein superfamily molecules, genes encoding transcription factors, genes that mediate angiogenesis, viral genes, genes required for viral replication, cellular genes that mediate viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes that mediate unwanted immune responses, genes that mediate pain processing, genes that mediate neurological diseases, alleles found in cells characterized by loss of heterozygosity, or one allele of a polymorphic gene.

[0482] 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 gene; Erb-B gene, Src gene; CRK gene; GRB2 gene; RAS gene; MEKK gene; JNK gene; RAF gene; Erk1 / 2 gene; PCNA (p21) gene; MYB gene; c-MYC gene; JUN gene; FOS gene; BCL- 2 gene; cyclin D gene; VEGF gene; EGFR gene; cyclin A gene; cyclin E gene; WNT-1 gene; β-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; topoisomerase I gene; topoisomerase IIα gene; p73 gene; p21 (WAF1 / CIP1) gene, p27 (KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor gene; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, 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; tubulin gene; human papillomavirus gene, gene required for human papillomavirus replication, human immunodeficiency virus gene, gene required for human immunodeficiency virus replication, hepatitis A virus gene, hepatitis A Genes required for viral replication, hepatitis B virus gene, gene required for hepatitis B virus replication, hepatitis C virus gene, gene required for hepatitis C virus replication, hepatitis D virus gene, gene required for hepatitis D virus replication, hepatitis E virus gene, gene required for hepatitis E virus replication, hepatitis F virus gene, gene required for hepatitis F virus replication, hepatitis G virus gene, gene required for hepatitis G virus replication, hepatitis H virus gene, gene required for hepatitis H virus replication, respiratory syncytial virus gene, gene required for respiratory syncytial virus replication, herpes simplex virus gene, gene required for herpes simplex virus replication, herpes cytomegalovirus gene, gene required for herpes cytomegalovirus replication, herpes Epstein-Barr Virus gene, gene required for herpes Epstein-Barr Virus replication, Kaposi's sarcomaSarcoma-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, Forest Encephalitis virus genes, genes required for Forest Encephalitis virus replication, Murray Valley Encephalitisencephalitis virus genes, genes required for replication of Murray Valley encephalitis virus, dengue virus genes, genes required for replication of dengue virus genes, simian virus 40 genes, genes required for replication of simian virus 40, human T-cell lymphotropic virus genes, genes required for replication of human T-cell lymphotropic virus, Moloney-Murine leukemia virus genes, genes required for replication of Moloney murine leukemia virus, encephalomyocarditis virus genes, genes required for replication of encephalomyocarditis virus, measles virus genes, genes required for replication of measles virus, varicella-zoster Virus genes, genes required for varicella-zoster virus replication, adenovirus genes, genes required for adenovirus replication, yellow fever virus genes, genes required for yellow fever virus replication, poliovirus genes, genes required for poliovirus replication, poxvirus genes, genes required for poxvirus replication, Plasmodium genes, genes required for Plasmodium genes 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 genes, genes required for Streptococcus pneumoniae replication, Streptococcus pyogenes genes, genes required for Streptococcus pyogenes replication, Chlamydia pneumoniae genes, genes required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, 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 for components of ion channels, neurotransmitter receptor genes, neurotransmitter ligand genes, amyloid family genes, presenilin genes, 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 a polymorphic gene, and combinations thereof.

[0483] Loss of heterozygosity (LOH) can result in hemizygosity of sequences (e.g., genes) in the LOH region. This can result in significant genetic differences between normal cells and diseased cells (e.g., cancer cells), and provide useful differences between normal cells and diseased cells (e.g., cancer cells). This difference can be caused by genes or other sequences that are heterozygous in diploid cells but hemizygous in cells with LOH. LOH regions typically include genes whose deletion causes unwanted proliferation, such as tumor suppressor genes, and other sequences, including, for example, other genes, which in some cases are necessary for normal function (e.g., growth). The methods of the present invention rely in part on the specific regulation of one allele of an essential gene with the compositions of the present invention.

[0484] In certain embodiments, the present invention provides compounds of the present invention that modulate microRNA.

[0485] Targeting the CNS

[0486] In some embodiments, the present invention provides a compound that targets APP for early-onset familial Alzheimer's disease, ATXN2 for spinocerebellar ataxia 2 and ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia.

[0487] In some embodiments, the present invention provides a compound that targets TARDBP in ALS, MAPT (Tau) in frontotemporal dementia, and HTT in Huntington's disease.

[0488] 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, the genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, prion diseases, recessive CNS disorders: PRNP for Lafora disease, DMPK for DM1 (CNS and skeletal muscle), and TTR for hATTR (CNS, eye, and systemic).

[0489] Spinocerebellar ataxia is an inherited brain disorder. Dominantly inherited forms of spinocerebellar ataxia (such as SCA1-8) are devastating disorders with no disease-modifying therapies. Exemplary targets include SCA2, SCA3, and SCA1.

[0490] Targeting ATXN2 in SCA2

[0491] Spinocerebellar ataxia 2 (SCA2), a progressive ataxia, is the second most common SCA. Another disease associated with this target is amyotrophic lateral sclerosis (ALS). These diseases are debilitating and ultimately fatal, with no disease-modifying therapies. The prevalence of SCA is 2-6 per 100,000 people; ATXN2 causes 15% of the SCA population worldwide and is much higher in some countries, especially Cuba (40 per 100,000 people). Targeting ATXN2 via human molecular genetics can be excellent, for example, in familial and sporadic SCA and ALS, the coding CAG repeat expansion in ATXN2 is found in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion of ATXN2 leads to the expression of toxic, misfolded proteins and the death of Purkinje cells and neurons. Efficacy has been demonstrated by 70% knockdown (KD) of ATXN2 mRNA; and mATXN2 mouse KD POC 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 repeat protein

[0492] Targeting ATXN3 in SCA3

[0493] Spinocerebellar ataxia 3 (SCA3), a progressive ataxia, is the most common SCA worldwide. The disease is debilitating and ultimately fatal, with no disease-modifying therapies. It is the most common cause of SCA, with a prevalence of 2-6 per 100,000 people; ATXN3 causes 21% of SCA cases in the United States and significantly more in Europe, particularly Portugal. Targeting ATXN3 via human molecular genetics may be advantageous. For example, in familial and sporadic SCA, the coding CAG repeat expansion in ATXN3 is found in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in ATXN3 leads to the expression of toxic, misfolded proteins, Purkinje cell death, and neuronal death. Efficacy has been demonstrated by 70% knockout of ATXN3 mRNA, and mATXN3 knockout mice have been demonstrated for POC. Regarding safety, mATXN3 knockout 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.

[0494] Targeting ATXN1 in SCA1

[0495] Spinocerebellar ataxia 1 (SCA1), a progressive ataxia, was the first SCA gene discovered in 1993. The disease is debilitating and ultimately fatal, with no disease-modifying therapies. The prevalence of SCA is 2-6 per 100,000 people; ATXN1 causes 6% of SCA cases in the United States and globally, and significantly more in some countries (25% in Japan), particularly Poland (64%) and Siberia (100%). Targeting ATXN1 via human molecular genetics may be advantageous. For example, in familial and sporadic SCA, coding CAG repeat expansions in ATXN1 are found in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in ATXN1 leads to the expression of toxic, misfolded proteins, Purkinje cell death, and neuronal death. Efficacy has been demonstrated with a 70% knockout (KD) of ATXN1 mRNA, and mATXN1 mouse POC has been demonstrated. Regarding safety, mATXN1 KO mice have been reported to be healthy. Possible diagnostic options include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0496] Targeting ATXN7 in SCA7

[0497] Spinocerebellar ataxia 7 (SCA7) causes progressive ataxia and retinal degeneration. This disease is a debilitating and ultimately fatal disorder of the retina and cerebellum, with no disease-modifying therapy. The prevalence of SCA is 2-6 per 100,000 people; ATXN7 causes 5% of the SCA population worldwide and significantly more in some countries, particularly South Africa. Targeting ATXN7 via human molecular genetics may be advantageous. For example, in familial and sporadic SCA, the coding CAG repeat expansion in ATXN7 is found in tissues such as the spinal cord, brainstem, cerebellum, or retina. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion of ATXN1 leads to the expression of a toxic, misfolded protein, triggering cone and rod degeneration, Purkinje cell and neuronal lethality. Efficacy has been demonstrated with a 70% knockout (KD) of ATXN1 mRNA via intrathecal (IT) and intravitreal (IVT) administration. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0498] ATXN8 targeting SCA8

[0499] Spinocerebellar ataxia 8 (SCA8), a progressive neurodegenerative ataxia, is caused by a CTG repeat expansion in ATXN8. The disease is debilitating and ultimately fatal, with no disease-modifying treatments. Prevalence: SCA affects 2-6 people per 100,000; ATXN8 causes 3% of the SCA population worldwide, and significantly more in some countries (particularly Finland). Targeting ATXN8 via human molecular genetics may be advantageous. For example, in familial and sporadic SCA, the coding CTG repeat expansion in ATXN8 is found in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be that the autosomal dominant coding CTG expansion in ATXN8 leads to the expression of a toxic, misfolded protein, triggering Purkinje cell and neuronal lethality. Efficacy has been demonstrated with a 70% knockdown 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 repeat proteins.

[0500] Targeting CACNA1A in SCA6

[0501] Spinocerebellar ataxia 6 (SCA6) is a progressive ataxia. It is a debilitating and ultimately fatal disease with no disease-modifying therapies. The prevalence of SCA is 2-6 per 100,000 people; and CACNA1A causes 15% of the SCA population worldwide. Targeting CACNA1A via human molecular genetics may be advantageous. For example, in familial and sporadic SCA, the coding CAG repeat expansion in CACNA1A is found in tissues such as the spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in CACNA1A leads to the expression of toxic, misfolded proteins and the death of Purkinje cells and neurons. Efficacy has been demonstrated with a 70% KD for the CACNA1A CAG expansion. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0502] Exemplary targets for inherited polyglutamine disorders include Huntington's disease (HD).

[0503] Targeting HTT in Huntington's disease

[0504] Huntington's mutation causes HD, a progressive degenerative disease of the CNS. The disease is debilitating and ultimately fatal, with no disease-modifying therapy. The global prevalence of HD is 5-10 per 100,000 people, and is more common in certain countries, particularly Venezuela. Targeting HTT via human molecular genetics may be advantageous, for example, in familial and sporadic HD, the coding CAG repeat expansion in HTT found in tissues such as the striatum or cortex. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion of HTT leads to the expression of toxic, misfolded proteins and neuronal death. Efficacy has been shown by a 70% KD of only the HTT CAG expansion; and mouse POC has been confirmed. Regarding safety, KO of HTT in mice can be lethal; KD has been confirmed in humans. Possible diagnoses include family history; genetic testing; early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat protein.

[0505] ATN1 targeting DRPLA

[0506] Atrophin 1 mutations cause dentatorubral pallidum Lewy body atrophy (DRPLA), a progressive spinocerebellar disorder similar to HD. The disease is debilitating and ultimately fatal, with no disease-modifying therapy. In Japan, the prevalence of DRPLA is 2-7 per 1,000,000 people. Targeting ATN1 via human molecular genetics may be advantageous, for example, in familial and sporadic SCA, coding CAG repeat expansions in ATN1 are found in tissues such as the spinal cord, brainstem, cerebellum, or cortex. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion of ATN1 leads to the expression of toxic, misfolded proteins and neuronal death. Efficacy has been shown by a 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 protein.

[0507] Targeting AR in spinal bulbar muscular atrophy

[0508] Androgen receptor mutations lead to spinal and bulbar muscular atrophy (SBMA, Kennedy disease), a progressive muscle wasting disease, among other diseases. This disease is debilitating and ultimately fatal, and there is no disease-modifying therapy. The prevalence of SBMA is 2 cases per 100,000 males; women have a mild phenotype. Targeting the AR via human molecular genetics can be advantageous, for example, in familial SBMA, the coding CAG repeat in the AR found in tissues such as the spinal cord or brainstem is expanded. The mechanism of this targeting may be that the X-linked coding CAG expansion of the AR leads to toxic gain-or-function and motor neuron lethality. Efficacy has been shown by a 70% KD of 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 repeat proteins.

[0509] Targeting FXN for Friedrich Ataxia

[0510] A recessive loss-of-function GAA expansion in FXN causes Friedreich's ataxia (FA), a progressive, degenerative ataxia. This disease is debilitating and ultimately fatal, with no disease-modifying therapy. The prevalence of FA is 2 per 100,000 people worldwide. Targeting FXN via human molecular genetics may be advantageous. For example, in familial FA, intronic GAA repeat expansions in FXN are found in tissues such as the spinal cord, cerebellum, and possibly the retina and heart. The mechanism of this targeting may be that autosomal recessive noncoding FAA expansions in FXN lead to lethal expression of FXN, an important mitochondrial protein. Efficacy has been shown by a 70% KD of the FXN intronic GAA expansion. Regarding safety, KD of the intronic GAA is safe and effective in mice. Possible diagnostics include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat protein.

[0511] Targeting FMR1 with FXTAS

[0512] Fragile X-associated tremor / ataxia syndrome (FXTAS), a progressive ataxia and cognitive loss disorder in adults, is caused by overexpression of FMR1. The disease is debilitating and there is no disease-modifying therapy. The prevalence of FMR1 premutation is 1 in 500 males. Targeting FMR1 via human molecular genetics can be advantageous, for example, in FXTAS, a coding CCG repeat expansion premutation in FMR1 is found in tissues such as the spinal cord, cerebellum, or cortex. The mechanism of this targeting may be that the X-linked coding CCG expansion of FMR1 leads to toxic mRNA. Efficacy has been shown by 70% KD of the toxic mRNA. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0513] Targeting upstream of FMR1 in fragile X syndrome

[0514] Fragile X syndrome (FRAXA), a progressive mental retardation disorder, can be treated by targeting the upstream mRNA of FMR1. This disease is debilitating and there is no disease-modifying therapy. The prevalence of FRAXA is 1 in 4,000 males and 1 in 8,000 females. Targeting FMR1 via human molecular genetics can be advantageous, for example, in FRAXA, coding CCG repeat expansions in FMR1 are found in tissues such as the CNS. The mechanism of this targeting may be because the X-linked coding CCG expansion of FMR1 leads to LOF; and normal FMR1 is used to transport specific mRNA from the cell nucleus. Efficacy has been shown by 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.

[0515] Dominantly inherited amyotrophic lateral sclerosis is a devastating disorder with no disease-modifying therapies. Exemplary targets include C9orf72, ATXN2 (also responsible for SCA2), and MAPT.

[0516] Targeting C9orf72 in ALS

[0517] C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These diseases are fatal disorders of motor neurons with no disease-modifying therapies. The prevalence of ALS is 2-5 per 100,000 people (10% familial); C9orf72 is responsible for 39% of familial ALS and 7% of sporadic ALS in the United States and Europe. Targeting C9orf72 via human molecular genetics may be advantageous, for example, in familial and sporadic ALS, hexanucleotide expansions are found in tissues such as upper and lower motor neurons (for ALS) or the cortex (for FTD). The mechanism of this targeting may be that the autosomal dominant hexanucleotide expansion leads to repeat-associated AUG-independent translation of a toxic dipeptide repeat protein and neuronal lethality. Efficacy has been demonstrated with a 70% KD of C9orf72. Regarding safety, heterozygous LOF mutations in C9orf72 appear 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.

[0518] Targeting TARDBP in ALS

[0519] TARDBP mutations cause ALS and frontotemporal dementia (FTD). These diseases are fatal disorders of motor neurons and there are no disease-modifying therapies. The prevalence of ALS is 2-5 per 100,000 people (10% is familial); TARDBP causes 5% of familial ALS and 1.5% of sporadic ALS. Targeting TARDBP via human molecular genetics can be advantageous, for example, in familial and sporadic ALS, mutations are found in tissues such as upper and lower motor neurons (for ALS); or cortex (for FTD). The mechanism of this targeting may be because autosomal dominant TRDBP mutations lead to toxic TRDBP protein and neuronal lethality. Efficacy has been shown by 70% KD of TARDBP mutant alleles. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF protein.

[0520] Targeting FUS in ALS

[0521] FUS mutations cause ALS and FTD. These diseases are fatal disorders of motor neurons and there are no disease-modifying therapies. The prevalence of ALS is 2-5 per 100,000 people (10% are familial); FUS causes 5% of familial ALS; FUS inclusions are common in sporadic ALS. Targeting FUS via human molecular genetics may be advantageous, for example, in familial ALS, mutations are found in tissues such as upper and lower motor neurons for ALS. The mechanism of this targeting may be because autosomal dominant FUS mutations lead to abnormal protein folding and neuronal lethality. Efficacy has been shown by 70% KD of FUS mutant alleles. Regarding safety, KO mice struggle but survive and have an ADHD phenotype. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF protein.

[0522] Targeting SOD1 in ALS

[0523] Dominant and recessive mutations in SOD1 cause ALS. This disease is a fatal disorder of motor neurons with 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 advantageous, for example, in tissues such as upper and lower motor neurons for ALS, where many SOD1 mutations are associated with AD and AR ALS in families. 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.

[0524] Dominant frontotemporal dementia and progressive supranuclear palsy. Targets include MAPT (as it may be important for AD) or C9orf72.

[0525] Targeting the microtubule-associated protein Tau, which is involved in FTD-17 and PSP

[0526] Familial frontotemporal dementia 17 (FTD-17), a familial form of FTD located on chromosome 17, and familial progressive supranuclear palsy may be caused by mutations in MAPT, which can also cause rare forms of progressive supranuclear palsy, corticobasal degeneration, tauopathies 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; the prevalence of FTD-17 in the Netherlands is 1 in 1,000,000 people. Targeting MAPT via human molecular genetics may be advantageous, for example, in familial and sporadic FTD, GOF point mutations and splice site mutations of MAPT are found in tissues such as the frontal or temporal cortex. The mechanism of this targeting may be that autosomal dominant GOF mutations in MAPT lead to toxic tau peptides and neuronal death. Efficacy has been demonstrated with a 70% KD of MAPT. Regarding safety, MAPT KO mice have been reported to be healthy. Possible diagnostic options include family history, genetic testing, and early symptoms. Biomarkers that can be used include, for example, CSF tau mRNA and protein.

[0527] Targeting Sequestosome 1 in FTD and ALS

[0528] Sporadic FTD / ALS is associated with dominant SQSTM1 mutations. This disease is a fatal neurodegenerative disorder with no disease-modifying treatments. It is a very rare disorder. In sporadic cases, targeting sequestrum 1 via human molecular genetic targeting in tissues such as the frontal and temporal cortices, or the cerebellum and spinal cord is reasonable. Possible diagnostic options include genetic testing; early symptoms are indicated.

[0529] Dominantly inherited Parkinson's disease is a devastating disorder with no disease-modifying therapies. Targets include SNCA.

[0530] Targeting SNCA in Parkinson's disease

[0531] Mutations in alpha-synuclein cause familial Parkinson's disease (PD) and Lewy body dementia. These diseases are fatal neurodegenerative disorders with no disease-modifying therapies. The prevalence of PD is 4 million worldwide; one-third of PD is familial; and 1% of fPD is caused by SNCA. Targeting SNCA via human molecular genetics may be advantageous, for example, in tissues such as the medulla oblongata or the substantia nigra of the midbrain, where SNCA point mutations and duplications cause familial PD. The mechanism of this targeting may be due to overexpression or expression of abnormal SNCA protein leading to toxic peptides and neuronal death. Efficacy has been demonstrated with a 70% KD of SNCA. Regarding safety, SNCA KO mice are healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF SNCA mRNA and protein.

[0532] Targeting LRRK2 in Parkinson's disease

[0533] Mutations in leucine-rich repeat kinase 2 cause familial Parkinson's disease. This disease is a fatal neurodegenerative disorder with no disease-modifying therapies. The prevalence of PD is 4 million worldwide; one-third of PD cases are familial; and 3%-7% of fPD cases are caused by LRRK2. Targeting LRRK2 via human molecular genetics may be advantageous; for example, point mutations in LRRK2 in tissues such as the medulla oblongata or the substantia nigra of the midbrain cause familial PD. Possible diagnostics include family history; genetic testing; and early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0534] Targeting GARS in spinal muscular atrophy V

[0535] Autosomal dominant glycyl-tRNA synthetase mutations cause spinal muscular atrophy V (SMAV) or telencephalic motor neuropathy Va. These diseases are neurodegenerative disorders with no disease-modifying therapies. They are very rare. Targeting GARs via human molecular genetics may be beneficial; for example, GARS point mutations in tissues such as the spinal cord cause familial SMA. Possible diagnoses include family history, genetic testing, and early symptoms.

[0536] Seipin targeting spinal muscular atrophy

[0537] Autosomal dominant Seipin mutations cause spinal muscular atrophy (SMA) or telencephalic motor neuropathy. These diseases are neurodegenerative disorders with no disease-modifying therapies. These are very rare diseases. Targeting Seipin via human molecular genetics may be beneficial, for example, in tissues such as the spinal cord, Seipin point mutations cause familial SMA. The mechanism of this targeting may be GOF and toxic peptides. Efficacy has been shown with a 50% KD. Regarding safety, recessive LOF mutations cause progressive encephalopathy with or without lipodystrophy. Possible diagnoses include family history; genetic testing; or early symptoms.

[0538] Dominantly inherited Alzheimer's disease is a devastating disorder with no disease-modifying therapies. Targets include APP due to its central mechanistic role in familial disease and its possible role in common AD.

[0539] APP targeting Alzheimer's disease

[0540] Mutations in the amyloid precursor protein cause 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% AD; the prevalence of trisomy 21 is 1% AD; and in the United States, the prevalence of AD is approximately 2.5-5 million. Targeting APP via human molecular genetics can be advantageous, for example, in tissues such as the cerebral cortex or hippocampus, APP duplications and point mutations cause EOFAD. The mechanism of this targeting may be because APP overexpression or expression of toxic metabolites causes progressive neuronal death. Efficacy has been shown by 70% KD of APP. Regarding safety, KD mice have been reported to be healthy with some behavioral abnormalities; KD mice have been reported to be healthy with some spatial memory effects. Possible diagnostics include family history; genetic testing; early symptoms; or MRI. Biomarkers that can be used include, for example, CSF APP mRNA and peptides.

[0541] Targeting PSEN1 in Alzheimer's disease

[0542] 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 may be advantageous, for example, in tissues such as the cerebral cortex or hippocampus, PSEN1 point mutations cause EOFAD. The mechanism of this targeting may be because autosomal dominant mutations in PSEN1 cause abnormal APP metabolism and toxic peptides cause progressive neuronal death. Efficacy has been shown to obviate the need for PSEN1-specific therapies for APPKD. Possible diagnoses include family history; genetic testing; early symptoms; or MRI. Biomarkers that may be used include, for example, CSF PSEN1 and APP peptides.

[0543] Targeting PSEN2 for Alzheimer's disease

[0544] 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 may be advantageous, for example, in tissues such as the cerebral cortex or hippocampus, PSEN2 point mutations cause EOFAD. The mechanism of this targeting may be that autosomal dominant mutations in PSEN2 cause abnormal APP metabolism and toxic peptides cause progressive neuronal death. Possible diagnoses include family history; genetic testing; early symptoms; or MRI. Biomarkers that may be used include, for example, CSF PSEN2 and APP peptides.

[0545] Targeting Apo E in Alzheimer's disease

[0546] Apolipoprotein E4 is associated with sporadic AD in the elderly. This disease is a fatal neurodegenerative disorder with no disease-modifying therapy. In the United States, the prevalence of AD is 2.5-5 million. Targeting Apo E may be effective because genomic evidence supporting an association between ApoE4 and AD is strong in many populations. The target tissue may be the cerebral cortex. Despite the strong association in many populations, it is unclear whether Apo E4 contributes to the pathogenesis of AD. To date, data suggest that Apo E4 homozygosity indicates an increased risk of AD in the elderly, but is insufficient to cause AD even in the elderly. Regarding safety, KD of ApoE in the CNS may be safe because the human LOF mutation in Apo E is not associated with overt neurological deficits, although systemic exposure may cause type III hyperlipoproteinemia. Possible diagnoses include clinical diagnosis of AD; exclusion of the EOFAD mutation; and genetic testing for the Apo E4 genotype. Biomarkers that can be used include, for example, CSF APP and Tau mRNA and peptide.

[0547] CNS gene duplication disorders. Consistent KD halving improves these disorders. Targets include MeCP2.

[0548] Targeting MeCP2 in X-linked mental retardation

[0549] Duplication of the methyl CpG binding protein 2 gene causes X-linked mental retardation (XLMR). This disease is a fatal cognitive disorder with no disease-modifying therapy. 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 causes XLMR. The mechanism of this targeting may be that overexpression of MeCP2 leads to dysregulation of other genes and neurodegeneration. Efficacy has been shown by 50% KD of MeCP2; and ASO KD in mouse models reverses the phenotype. 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 peptide.

[0550] Dominantly inherited cerebral amyloid angiopathy is a devastating disorder with no disease-modifying therapies. Targets include TTR.

[0551] TTR targeting hATTR CAA

[0552] This targeting may be a low-risk introduction of CNS siRNA. Cerebral amyloid angiopathy (CAA) and meningeal amyloid are fatal disorders with no disease-modifying therapies. Targeting TTR via human genetics and pharmacology may be superior. The target tissue may be the CNS vasculature or the CNS. The mechanism of this targeting may be that the mutant protein accumulates in the adventitia, causing CNS bleeding. Efficacy has been shown with a 70% KD of TTR. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that may be used include, for example, CSF mRNA and protein.

[0553] Targeting ITM2B in CAA

[0554] Mutations in the integral membrane protein 2B cause cerebral amyloid angiopathy (CAA), British type, or familial British dementia (FBD). Specific mutations can also cause dominant retinal degeneration. This disease is a fatal disorder with no disease-modifying therapies. This is a rare disease. Targeting ITM2B via human molecular genetics may be advantageous. The target tissue may be the CNS vasculature or the CNS. The mechanism of this targeting may involve GOF mutations. Efficacy has been shown with a 70% KD of the ITM2B mutant allele. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that may be used include, for example, CSF mRNA and possibly protein.

[0555] Targeting CST3 in CAA

[0556] Mutations in serum cystatin C cause familial cerebral amyloid angiopathy, Icelandic type. This disorder is fatal and there are no disease-modifying therapies. It is a rare disease outside Iceland and Denmark. Targeting CST3 via human genetics may be advantageous. The target tissue may be the CNS vasculature. The mechanism of this targeting may be that the mutant protein accumulates in the adventitia of blood vessels, causing CNS bleeding. Efficacy has been shown with a possible 70% KD of the mutant allele. Regarding safety, CST3 KO mice may be at risk for arthritis. Possible diagnostics include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and possibly protein.

[0557] SPAST for spastic paraplegia

[0558] SPASTIN mutations cause spastic paraplegia (SP) 4 with cognitive loss. This disease is a degenerative disorder of the lower motor nerves for which there is no disease-modifying therapy. The prevalence of SP is 5 per 100,000 population; SP4 is 45% of dominant SP. Targeting SPAST via human molecular genetics may be advantageous, 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 because nonsense and possibly dominant negative mutations cause abnormal microtubule metabolism and neurodegeneration. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that may be used include, for example, CSF SPAST mRNA and possibly protein.

[0559] Targeting KIF5A in spastic paraplegia

[0560] Mutations in kinesin family member 5A cause spastic paraplegia (SP) 10, with peripheral neuropathy and other impairments. This disease is a degenerative disorder of the lower motor nerves for which there is no disease-modifying therapy. The prevalence of SP is 5 per 100,000 people; SP10 is 1 per 1,000,000 people. Targeting KIF5A via human molecular genetics may be advantageous. For example, missense mutations in the amino terminus of KIF5A cause SP10; and KIF5A is expressed in the CNS and encodes a microtubule motor protein. The target tissue may be the spinal cord. The mechanism of this targeting may be that autosomal dominant missense mutations cause SP10, which may affect microtubules bound to motor nerves. Efficacy may be provided by the potential KD of the mutant allele. Regarding safety, KIF5A frameshift mutations cause refractory myoclonus in neonates, and splice site mutations may be associated with familial ALS through a LOF mechanism. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and possibly protein.

[0561] Targeting ATL1 in spastic paraplegia

[0562] Atlastin mutations cause spastic paraplegia 3A and sensory neuropathy 1D, hereditary sensory neuropathy (HSN). This disease is a degenerative disorder of the lower motor nerves for which there is 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 may be advantageous, for example, ATL1 point mutations cause familial SP. The target tissue may be the spinal cord. The mechanism of this targeting may be due to autosomal dominant expression of the dominant negative ATL1 protein, which causes SP3A; however, LOF mutations cause sensory neuropathy 1D. Efficacy has been shown with a 70% KD of a specific ATL1 allele. Regarding safety, heterozygous LOF mutations in ATL1 cause HSN1D. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF ATL1 mRNA and protein.

[0563] Targeting NIPA1 in spastic paraplegia

[0564] LOF NIPA1 mutations cause spastic paraplegia 6 with epilepsy and seizures. This disease is a degenerative disorder of the lower motor nerves for which there is no disease-modifying therapy. The prevalence of SP is 5 per 100,000 people; SP6 is a rare dominant form. Targeting NIPA1 via human molecular genetics may be advantageous, for example, where NIPA1 point mutations cause familial SP. The target tissue may be the spinal cord; or the CNS. The mechanism of this targeting may 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 may be used include, for example, CSF mRNA and possibly protein.

[0565] Dominantly inherited myotonic dystrophy is a disorder of the central nervous system (CNS), skeletal muscle, and myocardium that requires both CNS and systemic therapy. Targets include the MPKs of DM1.

[0566] Targeting DMPK of myotonic dystrophy 1

[0567] Effective therapies targeting myotonic dystrophy protein kinase require both CNS and systemic therapies. Myotonic dystrophy 1 (DM1) is a degenerative disorder of the muscle and CNS. It is a fatal disorder with no disease-modifying therapies. The prevalence of DM1 is 1 in 8,000 people worldwide. Targeting DMPK via human molecular genetics may be advantageous; for example, DMPK CTG repeat expansion causes familial DM1. Target tissues may be skeletal muscle, myocardium, or the CNS. The mechanism of this targeting may be due to autosomal dominant noncoding CTG repeats leading to aberrant RNA processing and dominant-negative effects; extreme expansions are expected to result in early-onset disease. Efficacy has been demonstrated with 70% of DMPKs, and ASO efficacy has been confirmed in mice. Safety has been demonstrated in KO and ASO KD mice. Possible diagnostics include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, blood and CSF mRNA and protein.

[0568] Targeting ZNF9 in myotonic dystrophy 2

[0569] Mutations in zinc finger protein 9 cause myotonic dystrophy 2 (DM2), a degenerative disorder of skeletal muscle. This is a severe disorder with no disease-modifying therapy. 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 may be advantageous, for example, a ZNF9 CTTG repeat expansion in intron 1 causes familial DM2. The target tissue may be skeletal muscle or cardiac muscle. The mechanism of this targeting may be because the autosomal dominant CTTG repeat expansion in intron 1 leads to abnormal RNA metabolism and a dominant negative effect. Efficacy has been shown with 70% of ZNF9. Safe KD 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 protein.

[0570] Dominantly inherited prion diseases are inherited, sporadic, and infectious PRNP disorders. Targets include PRNP.

[0571] PRNP targeting myotonic prion disease

[0572] Myotonic prion diseases are dominantly inherited prion diseases, including PRNP-related 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 such diseases is 1 in 1,000,000 people. Targeting PRNP via human molecular genetics can be advantageous, for example, PRNP mutations cause familial and sporadic prion diseases. The target tissue can be the CNS. The mechanism of this targeting can be due to neurotoxicity caused by mid-folding of the autosomal dominant protein. Efficacy has been shown with a PRNP KD of 70%; and PRNP polymorphisms appear to have a protective effect against kuru. Regarding safety, PRNP KO mice have been reported to be healthy. Possible diagnostics include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0573] Targeting glycogen synthase in Lafora's myoclonic spastic epilepsy

[0574] Mutations in the laforin (EPM2A) gene cause AR myoclonic epilepsy, an inherited progressive seizure disorder. This disease is a fatal disorder characterized by seizures and cognitive impairment, 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 may be advantageous, for example, mutations cause AR familial myoclonic epilepsy in Lafora. The target tissue may be the CNS. The mechanism of this targeting may be because autosomal recessive dysfunction of laforin leads to misfolding of glycogen and foci of seizures. Efficacy has been shown by a 70% KD of the glycogen synthase GYS1. Regarding safety, GYS1 deficiency leads to glycogen deficiency in skeletal and cardiac muscle; surviving GYS1 mice have muscle defects. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and protein.

[0575] In some embodiments, the present invention provides compounds that target genes for diseases including, but not limited to, age-related macular degeneration (AMD) (dry and wet), birdshot chorioretinopathy, retinitis pigmentosa 4, Fuch's dystrophy, hATTR amyloidosis, hereditary and sporadic glaucoma, and Stargardt's disease.

[0576] In some embodiments, the present invention provides compounds that target VEGF in wet (or exudative) AMD.

[0577] In some embodiments, the present invention provides compounds that target C3 of dry (or non-exudative) AMD.

[0578] In some embodiments, the present invention provides compounds that target CFB of dry (or non-exudative) AMD.

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

[0580] In some embodiments, the present invention provides compounds that target ROCK2 in glaucoma.

[0581] In some embodiments, the present invention provides compounds that target ADRB2 in glaucoma.

[0582] In some embodiments, the present invention provides compounds that target CA2 in glaucoma.

[0583] In some embodiments, the present invention provides compounds that target CRYGC in cataracts.

[0584] In some embodiments, the present invention provides compounds that target PPP3CB in dry eye syndrome.

[0585] ligand

[0586] In certain embodiments, the compound of the present invention is further modified by covalent attachment of one or more conjugate groups. In general, the conjugate group regulates one or more characteristics of the compound of the present invention attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cellular uptake, charge and removal. Conjugate groups are commonly used in chemical technology, and are directly or via optional linking moieties or linking groups connected to the parent compound, such as oligomeric compounds. The preferred list of conjugate 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, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes.

[0587] In some embodiments, the compound further comprises a targeting ligand that targets a receptor that mediates delivery to a specific CNS tissue. These targeting ligands can be conjugated in combination with a lipophilic moiety to achieve specific intrathecal and systemic delivery.

[0588] Exemplary targeting ligands that target receptors that mediate delivery to CNS tissues are peptide ligands such as angiopoietin-2, lipoprotein receptor-related protein (LRP) ligands, bEnd.3 cell-binding ligands; transferrin receptor (TfR) ligands (which can utilize the iron transport system in the brain and transport cargo into the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transporters, and LDL receptor ligands.

[0589] In some embodiments, the compound further comprises a targeting ligand that targets and mediates delivery to receptors of specific ocular tissues. These targeting ligands can be conjugated in combination with lipophilic moieties to achieve specific eye delivery (e.g., intravitreal delivery) and systemic delivery. Exemplary targeting ligands for targeting and mediating delivery to receptors of ocular tissues are lipophilic ligands, such as all-trans retinol (which targets retinoic acid receptors); 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).

[0590] Preferred conjugate groups suitable for use in the present invention include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Letters, 1994, 4, 1053); Res. [Nucleic Acids Research], 1992, 20, 533); aliphatic chains, for example, dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. [Journal of the European Molecular Biology Association], 1991, 10, 111; Kabanov et al., FEBS Lett. [Letter of the Federation of European Biochemical Societies], 1990, 259, 327; Svinarchuk et al., Biochimie [Biochemistry], 1993, 75, 49); phospholipids, for example, di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glyceryl-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. [Tetrahedron Letters], 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0591] In general, a wide variety of entities (eg, ligands) can be coupled to the oligomeric compounds described herein. The ligands can include naturally occurring molecules, or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazene, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, Thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamate, polyaspartic acid, aptamers, asialofetuin, hyaluronic acid, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-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, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-o-(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptides (e.g., alpha-helical peptides, amphipathic peptides, RGD peptides, cell-penetrating peptides, endosomal lytic / membrane fusion peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, polyamino groups, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, forest, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin 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 alpha (TNFα), interleukin-1β, interferon gamma, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell permeabilizing agents (e.g., helical cell permeabilizing agents).

[0592] Peptide and peptide mimetic ligands include ligands with naturally occurring or modified peptides, such as D or L peptides; α, β or γ peptides; N-methyl peptides; aza peptides; peptides with 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 mimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into a defined three-dimensional structure similar to a natural peptide. The peptide or peptide mimetic ligand 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.

[0593] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainine, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentini-1, and caerin.

[0594] As used herein, the term "endosomal lytic ligand" refers to a molecule with an endosomal lytic property. Endosomal lytic ligands promote the dissolution of the compositions of the present invention or its components and / or transport to the cytoplasm of cells from intracellular compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes or other vesicles. Some exemplary endosomal lytic ligands include but are not limited to imidazoles, polyimidazoles or oligoimidazoles, straight or branched polyethyleneimines (PEI), straight and branched polyamines (e.g., spermine, cationic straight and branched polyamines), polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, straight or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimetics, pH sensitive peptides, natural and synthetic membrane fusion lipids, natural and synthetic cationic lipids.

[0595] Exemplary endosomal lysis / membrane fusion peptides include, but are not limited to, AALEAALEALAEALEALAEAAAAGGC (GALA) (SEQ ID NO: 3); AALAEALAEALAEALAEALAEALAEALAAAAGGC (EALA) (SEQ ID NO: 4); ALEALAEALEALEALAEA (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); GLF EAI EGFI ENGW EGnIDG KGLF EAI EGFI ENGW EGnIDG (INF-5, n is norleucine) (SEQ ID 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 ID NO:19); and CHK6HC (SEQ ID NO:20).

[0596] Without wishing to be bound by theory, membrane fusion lipids fuse with the membrane and thus destabilize the membrane. Membrane fusion lipids typically have a small head group and an unsaturated acyl chain. Exemplary membrane fusion lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z, 9Z, 28Z, 31Z)-triacontac-6,9,28,31-tetraene-19-ol (Di-Lin), N-methyl (2,2-bis((9Z, 12Z)-octadeca-9,12-dienyl)-1,3-dioxolane-4-yl)methylamine (DLin-k-DMA) and N-methyl-2-(2,2-bis((9Z, 12Z)-octadeca-9,12-dienyl)-1,3-dioxolane-4-yl)ethylamine (also referred to herein as XTC).

[0597] Synthetic polymers with endosomolytic activity suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the contents of which are incorporated herein by reference in their entirety.

[0598] Exemplary cell penetrating 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 (transit peptide) (SEQ ID NO: 25); KLALKLALKALKAALKLA (amphipathic model peptide) (SEQ ID NO: 26); RRRRRRRRR (Arg9) (SEQ ID NO: 27); KFFKFFKFFK (bacterial cell wall penetrating peptide) (SEQ ID NO: 28); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO: NO:29); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO:30); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO:31); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO:32); RRRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-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).

[0599] Exemplary cationic groups include, but are not limited to, protonated amino groups derived from, for example, O-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy groups, such as O(CH2); nAmine (e.g., amine═NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); amino (e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH 2 CH 2 NH) n CH2CH2-amine (amine=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0600] As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, body region or compartment, such as a cell, tissue or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.

[0601] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent trehalose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of more than one monosaccharide unit. Such monosaccharide subunits may be linked to each other or to a backbone molecule via glycosidic bonds.

[0602] Many folic acid and folic acid analogs suitable for use as ligands in the present invention are described in US Pat. Nos. 2,816,110; 5,552,545; 6,335,434; and 7,128,893, the contents of which are incorporated herein by reference in their entirety.

[0603] As used herein, the terms "PK modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and thyroxine transporter binding ligands (e.g., tetraiodothyronine acetate, 2,4,6-triiodophenol, and flufenamic acid). Also known to comprise the oligomeric compound of key between multiple thiophosphate sugars and serum albumin, so short oligomeric compound, for example comprise about 5 to 30 nucleotide (for example, 5 to 25 nucleotide, preferably 5 to 20 nucleotide, for example 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 nucleotide) and in backbone, comprise the oligonucleotide of multiple thiophosphate bonds and also be applicable to the present invention as part (for example, as PK regulates part).PK regulates oligonucleotide and can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15 or more thiophosphate and / or dithiophosphate bond.In certain embodiments, PK regulates all nucleotides in oligonucleotide and key is thiophosphate and / or dithiophosphate bond.In addition, the fit of combining serum components (for example serum albumin) also regulates part and is applicable to the present invention as PK. Binding to serum components (eg, serum proteins) can be predicted from an albumin binding assay, such as that described in Oravcova et al., Journal of Chromatography B (1996), 677: 1-27.

[0604] 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 may have different properties. For example, a ligand may have targeting properties, endosomal lytic activity, or PK modulating properties. In preferred embodiments, all ligands have different properties.

[0605] When a monomer is incorporated into a component of a compound of the present invention (e.g., a compound of the present invention or a joint), a ligand or a tethered ligand may be present on the monomer. In certain embodiments, after a "precursor" monomer has been incorporated into a component of a compound of the present invention (e.g., a compound of the present invention or a joint), a ligand may be incorporated into the "precursor" monomer via coupling. For example, a monomer having a tether (i.e., no associated ligand) such as an amino-terminated tether, such as a monomer-joint-NH2, may be incorporated into a component of a compound of the present invention (e.g., a compound of the present invention or a joint). In subsequent operations, i.e., after a precursor monomer is incorporated into a component of a compound of the present invention (e.g., a compound of the present invention or a joint), a ligand with an electrophilic group, such as a pentafluorophenyl ester or an aldehyde group, may then be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the precursor monomer.

[0606] In another example, a monomer with a chemical group suitable for participating in a click chemistry reaction, such as an azide- or alkyne-terminated tether / linker, can be incorporated. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand with a complementary chemical group (e.g., an alkyne or azide group) can be attached to the precursor monomer by coupling the alkyne and azide groups together.

[0607] In some embodiments, the ligand can be conjugated to a nucleobase, a sugar moiety, or an internucleoside bond of a compound of the present invention. Conjugation to a purine nucleobase or a derivative thereof can occur at any position including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is attached to the conjugate moiety. Conjugation to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. When the ligand is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bond interactions required for base pairing.

[0608] Conjugation with the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms that can be attached to the sugar moiety of the conjugate moiety include 2', 3' and 5' carbon atoms. The 1' position can also be attached to the conjugate moiety, such as in abasic residues. The internucleoside bond can also carry the conjugate moiety. For phosphorus-containing bonds (such as phosphodiester, thiosulfate, dithiophosphate, aminophosphoryl ester, etc.), the conjugate moiety can be directly attached to the phosphorus atom or the O, N or S atom combined with the phosphorus atom. For internucleoside bonds (such as PNA) containing amines or amides, the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atoms of the amine or amide.

[0609] There are many methods for preparing oligonucleotide conjugates. Generally speaking, the oligonucleotide is attached to the conjugate portion by contacting a reactive group on the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, and the like) with a reactive group on the conjugate portion. In some embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0610] For example, the electrophilic group can be a carbonyl-containing functional group and the nucleophilic group can be an amine or a thiol. Methods for conjugating nucleic acids and related oligomeric compounds with and without linking groups are fully 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.

[0611] The ligand can be attached to the compound 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 typically a bond, that can be used and adapted to incorporate a carrier monomer into a main chain (e.g., a phosphate or modified phosphate of an oligonucleotide, such as a sulfur-containing main chain). A "tether attachment point" (TAP) refers to an atom of a carrier monomer that connects a selected portion, such as a carbon atom or a heteroatom (different from the atom that provides the main chain attachment point). The selected portion can be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. Optionally, the selected portion is connected to the carrier monomer by an inserted tether. Thus, the carrier will typically include a functional group, such as an amino group, or typically provide a bond that is suitable for incorporating or tethering another chemical entity (e.g., a ligand) to the constituent atoms.

[0612] Representative U.S. patents that teach the preparation of nucleic acid conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782;

[0613] 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 which are incorporated herein by reference in their entirety.

[0614] 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.

[0615] Because the ligand can be conjugated to the iRNA agent via a linker or carrier, and because the linker or carrier can contain a branched linker, the iRNA agent can then be attached via the same or different backbone attachment points of the carrier, or contain multiple ligands via a branched linker. For example, the branch point of the branched linker can be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom, or a group exhibiting such multivalence. In certain embodiments, the branch point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC; wherein Q is independently H or an optionally substituted alkyl group at each occurrence. In other embodiments, the branch point is glycerol or a glycerol derivative.

[0616] Evaluation of candidate iRNAs

[0617] We can assess candidate iRNA agents, such as modified RNA, for selected properties by exposing the agent or modified molecule and control molecule to appropriate conditions and assessing the presence of the selected property. For example, resistance to degradation agents can be assessed as follows. Candidate modified RNA (and control molecule, typically unmodified form) can be exposed to degradation conditions, such as exposure to an environment comprising a degradation agent (such as a nuclease). For example, we can use biological samples, such as biological samples similar to the environment that may be encountered for therapeutic purposes, such as blood or cell parts, such as cell-free homogenates or broken cells. Subsequently, the resistance of candidate and control to degradation can be assessed by any of a variety of methods. For example, candidate and control can be labeled with, for example, radioactive or enzyme labels or fluorescent labels (such as Cy3 or Cy5) before exposure. Control and modified RNA can be incubated with degradation agents and optional control (e.g., inactivated, such as heated inactivated) degradation agents. Physical parameters, such as size, of modified molecules and control molecules are subsequently measured. It can be determined by physical methods, such as by polyacrylamide gel electrophoresis or fractionation columns, to assess whether the molecule retains its original length, or functionally. Alternatively, the length of the unlabeled modified molecule can be determined using Northern blot assays.

[0618] Functional assays can also be used to evaluate candidate agents. Functional assays can be applied initially or after an earlier non-functional assay (e.g., an assay for resistance to degradation) to determine whether modification changes the ability of a molecule to silence gene expression. For example, cells (e.g., mammalian cells, such as mice or human cells) can be co-transfected with a plasmid expressing a fluorescent protein (e.g., GFP) and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00 / 44914). For example, compared to control cells (e.g., a control without adding an agent and / or a control with unmodified RNA) that do not include candidate dsiRNA in the transfection, the ability of modified dsiRNA homologous to GFP mRNA to suppress GFP expression can be determined by monitoring the reduction in cell fluorescence. The efficacy of candidate agents to gene expression can be assessed by comparing cell fluorescence in the presence of modified and unmodified dssiRNA compounds.

[0619] In another functional assay, a candidate dssiRNA compound homologous to an endogenous mouse gene (e.g., a maternally expressed gene, such as c-mos) can be injected into immature mouse oocytes to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01 / 36646). The phenotype of the oocyte (e.g., the ability to maintain stagnation in metaphase II) can be monitored as an indicator of the agent's ability to inhibit expression. For example, cleavage of c-mos mRNA by a dssiRNA compound will cause the oocyte to exit metaphase arrest and induce parthenogenesis (Colledge et al. Nature [Nature] 370: 65-68, 1994; Hashimoto et al. Nature [Nature], 370: 68-71, 1994). Compared to a negative control, the effect of the modified agent on target RNA levels can be verified by measuring the reduction in the level of target mRNA by Northern blotting, or by measuring the reduction in the level of target protein by Western blotting. The control can include cells to which no agent is added and / or cells to which unmodified RNA is added.

[0620] Physiological effects

[0621] The siRNA compounds described herein can be designed to make it easier to determine therapeutic toxicity by the complementarity of siRNA to human and non-human animal sequences. By these methods, siRNA can be composed of sequences that are completely complementary to nucleic acid sequences from humans and nucleic acid sequences from at least one non-human animal (e.g., non-human mammals, such as rodents, ruminants, or primates). For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, bonobo, chimpanzee, rhesus monkey, or cynomolgus monkey. The sequence of the siRNA compound can be complementary to sequences within homologous genes (e.g., oncogenes or tumor suppressor genes) of non-human mammals and humans. By determining the toxicity of the siRNA compound in non-human mammals, we can infer the toxicity of the siRNA compound in humans. For more onerous toxicity testing, siRNA can be complementary to humans and more than one (e.g., two or three or more) non-human animals.

[0622] The methods described herein can be used to correlate any physiological effect of siRNA compounds on humans, for example, any unwanted effect, such as a toxic effect, or any positive or desired effect.

[0623] Increased cellular uptake of siRNA

[0624] Described herein are various siRNA compositions containing covalently attached conjugates that increase cellular uptake and / or intracellular targeting of the siRNA.

[0625] Additionally provided are methods of the present invention comprising administering an siRNA compound and an agent that affects siRNA uptake into cells. The agent can be administered before, after, or simultaneously with the administration of the siRNA compound. The agent can be covalently or non-covalently linked to the siRNA compound. The agent can be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The agent can have a transient effect on cells. The agent can increase siRNA compound uptake into cells, for example, by disrupting the cellular cytoskeleton, for example, by disrupting microtubules, microfilaments, and / or intermediate filaments. The agent can be, for example, taconazole, vincristine, vinblastine, cytochalasin, nocodazole, jeppenlide, latrunculin A, phalloidin, svenholide A, indanoxin, or meseven. The agent can also increase siRNA compound uptake into a given cell or tissue, for example, by activating an inflammatory response. Exemplary agents with such effects include tumor necrosis factor alpha (TNFα), interleukin-1β, CpG motifs, interferon gamma, or more generally, agents that activate toll-like receptors.

[0626] siRNA production

[0627] siRNA can be produced in large quantities, for example, by a variety of methods. Exemplary methods include organic synthesis and RNA cleavage, such as in vitro cleavage.

[0628] Organic Synthesis. siRNA can be prepared by synthesizing single-stranded RNA molecules or each strand of a double-stranded RNA molecule separately, followed by annealing the component strands.

[0629] 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 a 1.5 molar excess of phosphoramidite nucleotides. To make RNA strands, ribonucleotide amino esters are used. Standard cycles of monomer addition can be used to synthesize 21 to 23 nucleotide strands of siRNA. Typically, two complementary strands are prepared separately and subsequently annealed, for example, after release from a solid support and deprotection.

[0630] Organic synthesis can be used to produce discrete siRNA species. Can accurately specify the complementarity of species and specific target gene. For example, species can be complementary with the region comprising polymorphism (such as single nucleotide polymorphism). In addition, can accurately determine the position of polymorphism. In certain embodiments, polymorphism is located in internal region, for example from at least 4,5,7 or 9 nucleotides of one or both ends.

[0631] 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 by in vitro cleavage, the following method can be used:

[0632] In vitro transcription. dsiRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, HiScribe TM The RNAi Transcription Kit (New England Biolabs) provides vectors and methods for generating dsiRNAs from nucleic acid segments that are cloned into the vector at positions flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands of the dsiRNA. The templates are transcribed in vitro by the addition of T7 RNA polymerase and dsiRNA is generated. Similar methods using PCR and / or other RNA polymerases (e.g., T3 or SP6 polymerase) may also contain toxins that may contaminate recombinant enzyme preparations.

[0633] In vitro cleavage. In one embodiment, the RNA produced by this method is carefully purified to remove terminal siRNAs, which are cleaved into siRNAs in vitro using, for example, Dicer or similar RNase III-based activity. For example, dsiRNAs can be incubated in vitro extracts from Drosophila or using purified components such as purified RNases or RISC complexes (RNA-induced silencing complexes). See, for example, Ketting et al. Genes Dev. 2001 Oct 15; 15(20): 2654-9 and Hammond Science. 2001 Aug 10; 293(5532): 1146-50.

[0634] DsiRNA cleavage generally produces multiple siRNA species, each a specific 21 to 23 nt fragment of the source dsiRNA molecule. For example, there can be siRNAs that include sequences complementary to overlapping regions and adjacent regions of the source dsiRNA molecule.

[0635] Regardless of the synthesis method, the siRNA formulation can be prepared in a solution (e.g., aqueous solution and / or organic solution) suitable for the formulation. For example, the siRNA formulation can be precipitated and redissolved in double distilled water and lyophilized. The dried siRNA can then be resuspended in a solution suitable for the desired formulation method.

[0636] Making double-stranded iRNA agents conjugated to lipophilic moieties

[0637] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to the compound via a nucleobase, a sugar moiety, or an internucleoside linkage.

[0638] Conjugation with a purine nucleobase or a derivative thereof can occur at any position including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7- or 8-positions of a purine nucleobase are attached to the conjugate 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 a pyrimidine nucleobase can be substituted with a conjugate moiety. When the lipophilic moiety is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization (i.e., does not interfere with the hydrogen bond interactions required for base pairing). In one embodiment, the lipophilic monomer containing the lipophilic moiety can be conjugated to the nucleobase via a linker containing an alkyl, alkenyl or amide bond.

[0639] Conjugation to the sugar portion of the nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar portion to which the lipophilic moiety can be attached include the 2', 3', and 5' carbon atoms. The lipophilic moiety can also be attached to the 1' position, such as in abasic residues. In one embodiment, the lipophilic moiety can be conjugated to the sugar portion via a 2'-O modification with or without a linker.

[0640] Internucleoside linkages may also carry lipophilic moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorothioate diester, phosphoamino acid, etc.), the lipophilic moiety may be attached directly to the phosphorus atom or to an O, N, or S atom bonded to the phosphorus atom. For internucleoside linkages containing amines or amides (e.g., PNA), the lipophilic moiety may be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0641] There are many methods for preparing oligonucleotide conjugates. Generally speaking, the oligonucleotide is attached to the conjugate portion by contacting a reactive group on the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, and the like) with a reactive group on the conjugate portion. In some embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0642] For example, the electrophilic group can be a carbonyl-containing functional group and the nucleophilic group can be an amine or a thiol. Methods for conjugating nucleic acids and related oligomeric compounds with and without linking groups are fully 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.

[0643] In one embodiment, a first (complementary) RNA strand and a second (sense) RNA strand can be synthesized separately, wherein one of the RNA strands comprises a pendant lipophilic moiety, and the first and second RNA strands can be mixed to form dsRNA. The step of synthesizing the RNA strands preferably involves solid-phase synthesis, wherein the individual nucleotides are joined end-to-end by forming internucleotide 3'-5' phosphodiester bonds in successive synthesis cycles.

[0644] In one embodiment, the lipophilic molecule with phosphoramidite groups is coupled to the 3' end or 5' end of the first (complementary) or second (righteousness) RNA chain in the final synthesis cycle. In the solid phase synthesis of RNA, nucleotides are initially in the form of nucleoside phosphoramidites. In each synthesis cycle, another nucleoside phosphoramidite is connected with the-OH group of the nucleotide previously incorporated. 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 nucleoside phosphoramidites. Conventional RNA synthesizers can be used for synthesis to be carried out in an automated and standardized manner. The synthesis of the lipophilic molecule with phosphoramidite groups can include the phosphitylation of free hydroxyl groups to produce phosphoramidite groups.

[0645] In general, oligonucleotides can be synthesized using protocols known in the art, for example, as described in Caruthers et al., Methods in Enzymology (1992) 211:3-19; 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. Pat. No. 6,001,311; each of which is incorporated herein by reference in its entirety. In general, the synthesis of oligonucleotides involves conventional nucleic acid protecting and coupling groups, such as a dimethoxytrityl group at the 5' end and a phosphoramidite at the 3' end. In one non-limiting example, small-scale synthesis is performed using nucleoside phosphoramidites sold by ChemGenes Corporation (Ashland, Mass.) on an Expedite 8909 RNA synthesizer sold by Applied Biosystems, Inc. (Weiterstadt, Germany). Alternatively, the synthesis can be performed on a 96-well plate synthesizer, such as that produced by Protogene, Inc. (Palo Alto, Calif.), or by methods such as those described in Usman et al., J. Am. Chem. Soc. (1987) 109:7845; Scaringe et al., Nucl. Acids Res. (1990) 18:5433; Wincott et al., Nucl. Acids Res. (1990) 23:2677-2684; and Wincott et al., Methods Mol. Bio. (1997) 74:59, each of which is incorporated herein by reference in its entirety.

[0646] The nucleic acid molecules of the present invention can be synthesized separately and joined together after synthesis, 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 can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, which is incorporated herein by reference in its entirety) and resuspended in water.

[0647] Pharmaceutical composition

[0648] In one aspect, the invention features a pharmaceutical composition comprising an siRNA compound, e.g., a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an ssiRNA compound, or a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof), comprising a nucleotide sequence that is complementary (e.g., substantially and / or exactly complementary) to a target RNA. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the siRNA compound (a) is 19-25 nucleotides long, e.g., 21-23 nucleotides, (b) is complementary to the endogenous target RNA, and optionally, (c) comprises at least one 1-5 nt long 3' overhang. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

[0649] In one example, the pharmaceutical composition includes an siRNA compound mixed with a local delivery agent. The local delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In some embodiments, the liposomes are cationic liposomes.

[0650] In another aspect, the pharmaceutical composition comprises 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 a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof)) mixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-10 Alkyl ester, monoglyceride, diglyceride or a pharmaceutically acceptable salt thereof.

[0651] In another embodiment, the topical permeation enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glutamic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, tauro-24,25-dihydro-sodium fusidate, diol dihydrofusidate sodium, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.

[0652] In another embodiment, the penetration enhancer is a chelating agent, which can be EDTA, citric acid, salicylate, N-acyl derivatives of collagen, laureth-9, N-aminoacyl derivatives of β-diketone, or mixtures thereof.

[0653] In another embodiment, the penetration enhancer is a surfactant, such as an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-hexadecyl ether, perfluorochemical emulsion or a mixture thereof.

[0654] In another embodiment, the penetration enhancer can be selected from the group consisting of unsaturated cyclic ureas, 1-alkyl-alkanones, 1-alkenylaziridine-propanones, steroidal anti-inflammatory agents and mixtures thereof. In another embodiment, the penetration enhancer can be a diol, pyrrole, azone or terpene.

[0655] In one aspect, the present invention is characterized in that it is a pharmaceutical composition in a form suitable for oral delivery, which includes 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 a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or its precursor)). 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., to treat colon cancer), etc. The oral delivery form can be a tablet, capsule, or gel capsule. In one embodiment, the siRNA compound of the pharmaceutical composition regulates the expression of cell adhesion proteins, regulates cell proliferation rate, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes 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 can be acetic acid phthalate, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate phthalate.

[0656] In another embodiment, the oral dosage form of the pharmaceutical composition includes a permeation enhancer. The permeation enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

[0657] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example, the excipient is polyethylene glycol. In another example, the excipient is precirol.

[0658] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin, dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0659] In one aspect, the invention features a pharmaceutical composition comprising an siRNA compound and a delivery vehicle. In one embodiment, the siRNA compound (a) is 19-25 nucleotides in length, e.g., 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and optionally, (c) comprises at least one 1-5 nucleotide long 3' overhang.

[0660] In one embodiment, the delivery vehicle can deliver 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 a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof)) to a cell via a local administration route. The delivery vehicle can be a microvesicle. In one example, the microvesicle is a liposome. In some embodiments, the liposome is a cationic liposome. In another example, the microvesicle is a micelle. In one aspect, the present invention features a pharmaceutical composition in an injectable 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 a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof)). 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, sterile solutions may include diluents such as water; physiological saline solution; fixed oils, polyethylene glycol, glycerol, or propylene glycol.

[0661] In one aspect, the invention features a pharmaceutical composition in an oral dosage form that includes 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 a DNA encoding an 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 a tablet, a capsule, and a gel capsule. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents the tablet, capsule, or gel capsule from dissolving in the stomach of a mammal. In some embodiments, the enteric material is a coating. The coating can be acetic acid phthalate, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, such as a penetration enhancer described herein.

[0662] In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example, the excipient is polyethylene glycol. In another example, the excipient is precirol.

[0663] In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin, dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0664] In one aspect, the invention features a pharmaceutical composition in a rectal dosage form that includes an siRNA compound, e.g., a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an ssiRNA compound, or a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

[0665] In one aspect, the invention features a pharmaceutical composition in a vaginal dosage form that includes an siRNA compound, e.g., a double-stranded siRNA compound or an ssiRNA compound (e.g., a precursor, e.g., a larger siRNA compound that can be processed into an ssiRNA compound, or a DNA encoding an siRNA compound (e.g., a double-stranded siRNA compound or an ssiRNA compound or a precursor thereof). In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

[0666] In one aspect, the invention features a pharmaceutical composition in a pulmonary or nasal dosage form that includes 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 a 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, a powder, or an implantable solid.

[0667] Treatment methods and delivery routes

[0668] Another aspect of the invention relates to a method of reducing the expression of a target gene in a cell, the method comprising contacting the cell with a compound of the invention. In one embodiment, the cell is an extrahepatic cell.

[0669] Another aspect of the present invention relates to a method of reducing the expression of a target gene in a subject, comprising administering to the subject a compound of the present invention.

[0670] Another aspect of the present 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 a double-stranded RNAi agent of the present invention, thereby treating the subject. Exemplary CNS disorders treatable by the methods of the present invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar diseases, prion diseases, and Lafora's disease.

[0671] The compounds of the present invention can be delivered to a subject by a variety of routes, depending on the type of gene targeted and the type of disorder to be treated. In some embodiments, the compound is administered extrahepatically, such as ocularly (e.g., intravitreally) or intrathecally or intracerebroventricularly.

[0672] In one embodiment, the compound is administered intrathecally or intraventricularly. By administering the double-stranded iRNA agent intrathecally or intraventricularly, the method can reduce target gene expression in brain or spinal tissue (e.g., cortex, cerebellum, cervical, lumbar and thoracic spine).

[0673] 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 administered directly (e.g., intravitreally) to one or more eyes. By intravitreally administering a double-stranded iRNA agent, this method can reduce the expression of the target gene in ocular tissue.

[0674] For ease of illustration, the formulations, compositions, and methods in this section are primarily discussed with respect to modified siRNA compounds. However, it will be understood that these formulations, compositions, and methods can be practiced with other siRNA compounds (e.g., unmodified siRNA compounds), and such practice is within the present invention. Compositions comprising iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

[0675] The iRNA molecules of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more iRNA species and a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, they are contemplated for use in the composition. Supplementary active compounds may also be incorporated into the composition.

[0676] The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ocular, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal, intraventricular, or intracerebroventricular administration.

[0677] The route of administration and site can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be a logical choice. Lung cells can be targeted by administering iRNA in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with iRNA and mechanically introducing the DNA.

[0678] Preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical 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.

[0679] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges or lozenges. In the case of tablets, usable carriers 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 administra...

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; R2′ is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; B is a modified or unmodified nucleobase; W is alkyl; and R, R', and R" are each independently H or alkyl.

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%, at least 60%, or at least 70% 2'-OMe modified nucleotides.

10. The compound of claim 1, wherein the lipophilic monomer is selected from the group consisting of: wherein R and R' are each independently H, methyl, ethyl, isopropyl, or tert-butyl.

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, LDL receptor ligand, and carbohydrate-based 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 or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

23. A method for reducing the expression of a target gene in a cell, the method comprising contacting the cell with 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; R2′ is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; W is alkyl; and R, R', and R" are each independently H or alkyl.

24. The method of claim 23, wherein the cell is not a hepatocyte.

25. The method of claim 23, wherein the ow The measured lipophilicity of the lipophilic monomer is greater than zero.

26. The method of claim 25, wherein the hydrophobicity of the compound as measured by the unbound fraction in a plasma protein binding assay of a double-stranded iRNA agent exceeds 0.

2.

27. The method of claim 26, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin.

28. A method of reducing the expression of a target gene in a subject, the method comprising administering to the subject 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; R2′ is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; W is alkyl; and R, R', and R" are each independently H or alkyl.

29. The method of claim 28, wherein the compound is administered extrahepatically.

30. The method of claim 29, wherein the compound is administered intrathecally or intracerebroventricularly.

31. The method of claim 28, wherein the method reduces expression of the target gene in brain or spinal tissue.

32. The method of claim 31, wherein the brain or spinal tissue is selected from the group consisting of: cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine.

33. The method of claim 31, wherein the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, and TTR.

34. The method of claim 28, wherein the compound is administered directly to one or both eyes of the subject.

35. The method of claim 28, wherein the method reduces expression of the target gene in ocular tissue.

36. A method of treating a subject suffering from a CNS disorder, the method comprising: Administering a therapeutically effective amount of a compound of any one of claims 1-22 to the subject, thereby treating the subject.

37. The method of claim 36, wherein the CNS disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar disease, prion disease, and Lafora's disease.

38. 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 containing a lipophilic moiety, the lipophilic moiety being conjugated to the sense strand and / or antisense strand via a carrier selected from the group consisting of: in: R is the lipophilic part, R2' is H, OH, F, OMe, O-methoxyalkyl, O-allyl, ON-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl; and B is a modified or unmodified nucleobase.

39. 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 containing a lipophilic moiety conjugated to an internal position of the sense strand and / or antisense strand via a carrier selected from the group consisting of: in: R is the lipophilic moiety; 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; and B is a modified or unmodified nucleobase.

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