Dynamic pharmacokinetic modifying anchors

Abstract

Novel oligonucleotide-based compounds comprising a (PK) -modifyinganchor are provided.SOLUTION: A compound is provided comprising a first oligonucleotide, wherein the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5 ' terminus, a 3 ' terminus; and a PK modifying anchor comprising an anchor oligonucleotide, an optional linker, and at least one polymer, wherein the anchor oligonucleotide comprises or consists of about 5 to about 15 nucleotides that are complementary to the first oligonucleotide and the polymer is at least about 2, 000Da.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 794,123, filed January 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Federally Funded Research This invention was made with government support awarded by the National Institutes of Health under grant numbers NS104022, HD086111 and OD020012. The government has certain rights in the invention.

[0003] The present disclosure relates to compounds containing novel oligonucleotide-based pharmacokinetic (PK)-modifying anchors that have useful applications for RNA interference (RNAi) and other gene therapy technologies. The PK-modifying anchors described herein are patterned to enable efficient modulation of the absorption, distribution, and clearance kinetics of therapeutic oligonucleotides, enhancing their tissue distribution. Efficient modulation of their absorption, distribution, and clearance kinetics can be achieved in blood / plasma, cerebrospinal fluid (CSF), and other relevant body / biological fluids and tissues. [Background technology]

[0004] Oligonucleotides are cleared extremely rapidly after cerebrospinal fluid (CSF) injection, with less than 1-2% of the injected dose being retained in the brain and spinal cord. One well-understood problem with the use of oligonucleotide therapeutics in central nervous system applications is rapid CSF clearance. In rodents, bolus injection is sufficient to support widespread oligonucleotide distribution in the cerebrum, with bulk CSF flow being the primary mechanism behind distribution. Rapid CSF clearance limits distribution of oligonucleotides to deep brain structures and is a major limitation of this platform for the treatment of many neurodegenerative disorders.

[0005] Similarly, when administered IV or SC, oligonucleotides are rapidly cleared systemically through renal filtration or by elimination via the reticuloendothelial system. Retention in secondary tissues other than the liver, kidney, bone marrow, and spleen is a real challenge in the field.

[0006] There remains a need for self-delivering siRNAs characterized by efficient RISC entry, minimal immune response and off-target effects, efficient cellular uptake without formulation, improved absorption, distribution and clearance kinetics, and efficient, specific or functional tissue distribution. Summary of the Invention

[0007] The present invention is based on the discovery of a novel platform, compounds containing a dynamic pharmacokinetic ("PK")-modifying anchor, which allows for efficient modulation of the absorption, distribution, and clearance kinetics of component therapeutic oligonucleotides in blood / plasma, CSF, and other body / biological fluids and tissues. Provided herein is a family of block copolymers (e.g., poloxamer 188, etc.) that function as non-immunogenic PK modifiers and are compatible with oligonucleotide compounds.

[0008] In one embodiment, the disclosure provides a compound comprising: a first oligonucleotide, wherein the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5' end, a 3' end; and a pharmacokinetic (PK)-modifying anchor comprising an anchor oligonucleotide, an optional linker, and at least one polymer, wherein the anchor oligonucleotide comprises or consists of about 5 to about 15 nucleotides complementary to the first oligonucleotide, and the polymer is at least about 2,000 Da.

[0009] In certain embodiments, the anchor oligonucleotide is about 5 to about 15 nucleotides in length, or about 5 to about 10 nucleotides in length.

[0010] In certain embodiments, the polymer is operably linked to the anchor oligonucleotide via an optional linker.

[0011] In certain embodiments, the compound has Formula (I): [ka] [In formula: O is the first oligonucleotide; L is a linker, present or absent; X c is selected from the group consisting of a hydrophobic moiety, a sugar, a peptide, an aptamer, and a nucleic acid, and is present or absent; Z is the anchor that modifies PK] Including those indicated by.

[0012] In certain embodiments, the first oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a synthetic miRNA, a synthetic mRNA, a single-stranded siRNA, and a modified CRISPR guide strand.

[0013] In certain embodiments, the ASO is a splice-switching ASO or an RNase H ASO.

[0014] In certain embodiments, the first oligonucleotide comprises complementarity to the target.

[0015] In certain embodiments, the first oligonucleotide comprises perfect complementarity with the target.

[0016] In certain embodiments, L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphodiester, phosphorothioate, phosphoramidate, amide, carbamate, or a combination thereof.

[0017] In certain embodiments, X cis selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, and endocannabinoids. c comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof. c has affinity for one or both of low density lipoproteins and medium density lipoproteins. c is a saturated or unsaturated moiety having less than three double bonds. c has an affinity for high density lipoproteins. In certain embodiments, X c is a polyunsaturated moiety having three or more double bonds.

[0018] In certain embodiments, the at least one polymer is selected from the group consisting of hydrophobic polycarbonates, polyesters, amphiphilic block copolymers, hydrophobic block polymers, polysaccharides, and polypeptides. In certain embodiments, the hydrophobic polycarbonate is poly(DTR carbonate). In certain embodiments, the polyester is selected from the group consisting of polyhydroxyalkanoates, polycaprolactones, poly(hydroxybuterate-hydroxyvalerate), polyglycolic acid, and polylactic acid. In certain embodiments, the amphiphilic block copolymer is selected from the group consisting of polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), acrylonitrile styrene acrylate, N-(2-hydroxypropyl) methacrylamide, and polyethylene glycol (PEG). In certain embodiments, the hydrophobic block copolymer is selected from the group consisting of poly(N,N-dimethylacrylamide), poly(N,N-diethylaniline), poly(diphenylamino), and poly(tetrahydrofurfuryl methacrylate). In certain embodiments, polysaccharide is selected from the group consisting of soluble polyglucose, insoluble polyglucose, cellulose, glycogen and amylopectin.In certain embodiments, polypeptide is polylysine, polyarginine, polyalanine, polyisoleucine, polymethionine, polyphenylalanine, polyvaline, polyproline and polyglycine, and any combination thereof.In certain embodiments, PEG has a molecular weight selected from the group consisting of about 10,000 Da, about 20,000 Da, about 40,000 Da, about 60,000 Da, about 80,000 Da and about 100,000 Da.

[0019] In certain embodiments, the PK-modifying anchor comprises more than one polymer, hi certain embodiments, the PK-modifying anchor comprises two, three, or four polymers.

[0020] In certain embodiments, the first oligonucleotide comprises 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides and the polymer is PEG.

[0021] In certain embodiments, the first oligonucleotide comprises one or more chemically modified nucleotides. In certain embodiments, the first oligonucleotide is completely chemically modified or partially chemically modified. In certain embodiments, the first oligonucleotide comprises one or more locked nucleic acids (LNA) or one or more peptide nucleic acids (PNA). In certain embodiments, the first oligonucleotide comprises one or more S-constrained ethyl (cET). In certain embodiments, the first oligonucleotide comprises about 50% 2'-methoxy-ribonucleotides, about 55% 2'-methoxy-ribonucleotides, about 60% 2'-methoxy-ribonucleotides, about 65% 2'-methoxy-ribonucleotides, about 70% 2'-methoxy-ribonucleotides, about 75% 2'-methoxy-ribonucleotides, about 80% 2'-methoxy-ribonucleotides, about 85% 2'-methoxy-ribonucleotides, about 90% 2'-methoxy-ribonucleotides, about 95% 2'-methoxy-ribonucleotides, about 96% 2'-methoxy-ribonucleotides, about 97% 2'-methoxy-ribonucleotides, about 98% 2'-methoxy-ribonucleotides, about 99% 2'-methoxy-ribonucleotides, or about 100% 2'-methoxy-ribonucleotides. In certain embodiments, the first oligonucleotide comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides. In certain embodiments, the nucleotides of the first oligonucleotide are joined via phosphodiester bonds, phosphorothioate bonds, or a combination of phosphodiester and phosphorothioate bonds.

[0022] In certain embodiments, (1) the first oligonucleotide comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides, wherein each nucleotide is a 2'-methoxy-ribonucleotide or a 2'-fluoro-ribonucleotide, and the nucleotides at positions 2 and 14 from the 5' end of the first oligonucleotide are not 2'-methoxyribonucleotides; and (2) the nucleotides of the first oligonucleotide are connected to adjacent nucleotides via phosphodiester or phosphorothioate bonds, wherein the nucleotides at positions 1-6 from the 3' end or 1-7 from the 3' end are connected to adjacent nucleotides via phosphorothioate bonds.

[0023] In certain embodiments, the compound comprises a second oligonucleotide comprising at least 12 contiguous nucleotides, a 5' end, a 3' end; wherein a portion of the first oligonucleotide is complementary to a portion of the second oligonucleotide.

[0024] In certain embodiments, the second oligonucleotide comprises a conjugate moiety, X c In certain embodiments, the second oligonucleotide comprises X c In certain embodiments, L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. c is attached at the 5' end, the 3' end, an internal position, or a mixture thereof, of the second oligonucleotide. c is attached at the 3' end of the second oligonucleotide. c is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, and endocannabinoids. c comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof.c has affinity for one or both of low density lipoproteins and medium density lipoproteins. c is a saturated or unsaturated moiety having less than three double bonds. c has an affinity for high density lipoproteins. In certain embodiments, X c is a polyunsaturated moiety having three or more double bonds.

[0025] In certain embodiments, the anchor oligonucleotide is perfectly complementary to the first oligonucleotide. In certain embodiments, the anchor oligonucleotide contains 1, 2, 3, or 4 mismatches compared to the first oligonucleotide.

[0026] In certain embodiments, the anchor oligonucleotide comprises one or more chemically modified nucleotides. In certain embodiments, the anchor oligonucleotide is fully chemically modified or partially chemically modified. In certain embodiments, the anchor oligonucleotide comprises one or more locked nucleic acids (LNA) or one or more peptide nucleic acids (PNA). In certain embodiments, the anchor oligonucleotide comprises one or more S-constrained ethyl (cET). In certain embodiments, the anchor oligonucleotide comprises alternating 2'-O-methyl ribonucleotides and 2'-fluoro ribonucleotides. In certain embodiments, the nucleotides of the anchor oligonucleotide comprise alternating 2'-O-methyl ribonucleotides and 2'-fluoro ribonucleotides and at least two adjacent phosphorothioate internucleotide linkages at the 5' and 3' ends. In certain embodiments, the nucleotides of the anchor oligonucleotide comprise alternating 2'-O-methyl ribonucleotides and 2'-fluoro ribonucleotides and comprise phosphorothioate internucleotide linkages at every nucleotide position. In certain embodiments, the anchor oligonucleotide comprises at least two adjacent 2',4'-restricted 2'O-ethyl bridged nucleic acids at the 5'-end and 3'-end.In certain embodiments, the anchor oligonucleotide comprises 2',4'-restricted 2'O-ethyl bridged nucleic acids at every nucleotide position, and phosphorothioate internucleotide linkages between each adjacent nucleotide.In certain embodiments, the anchor oligonucleotide comprises alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, and comprises at least two 2',4'-restricted 2'O-ethyl bridged nucleic acids at the 5'-end and 3'-end.In certain embodiments, the anchor oligonucleotide comprises peptide nucleic acids at every nucleotide position.

[0027] In certain embodiments, the anchor oligonucleotide is attached to the polymer at the 5' end, the 3' end, at an internal position, or a mixture thereof.

[0028] In certain embodiments, at least two oligonucleotides are crosslinked.

[0029] In certain embodiments, the compound further comprises a nanoparticle, an intercalating agent, a polycation, or a mixture thereof.

[0030] In certain embodiments, a) the first oligonucleotide is between 21 and 25 nucleotides in length; b) the second oligonucleotide is between 13 and 17 nucleotides in length; and c) the anchor oligonucleotide is between 5 and 8 nucleotides in length.

[0031] In certain embodiments, a) the first oligonucleotide is 21 nucleotides in length; b) the second oligonucleotide is 13 nucleotides in length; and c) the anchor oligonucleotide is 8 nucleotides in length.

[0032] In certain embodiments, a) the first oligonucleotide is 23 nucleotides in length; b) the second oligonucleotide is 15 nucleotides in length; and c) the anchor oligonucleotide is 8 nucleotides in length.

[0033] In certain embodiments, a) the first oligonucleotide is 25 nucleotides in length; b) the second oligonucleotide is 17 nucleotides in length; and c) the anchor oligonucleotide is 8 nucleotides in length.

[0034] In certain embodiments, the polymer comprises PEG.

[0035] In certain embodiments, the PK-modifying anchor affects the stability of the oligonucleotide therapeutic agent over time in parts of the body including the heart, kidney, liver, spleen, adrenal glands, pancreas, lung, blood, plasma, brain, or mixtures thereof, wherein the effect includes a change in the volume of distribution, area under the curve, clearance, half-life maximum concentration, bioavailability, or mixtures thereof.

[0036] In certain embodiments, the number of nucleotides in the first oligonucleotide is the same as the total number of nucleotides in the second oligonucleotide and anchor oligonucleotide.In certain embodiments, the number of nucleotides in the first oligonucleotide is greater than the total number of nucleotides in the second oligonucleotide and anchor oligonucleotide.In certain embodiments, the number of nucleotides in the first oligonucleotide is less than the total number of nucleotides in the second oligonucleotide and anchor oligonucleotide.

[0037] In one embodiment, the disclosure provides a compound comprising a first oligonucleotide, wherein the first oligonucleotide is 21 nucleotides in length, has a 5' end, a 3' end, and is complementary to a target; a second oligonucleotide, wherein the second oligonucleotide is 13 nucleotides in length, has a 5' end, a 3' end, and is complementary to nucleotides 1-13 of the first oligonucleotide; and an anchor oligonucleotide, wherein the anchor oligonucleotide is 8 nucleotides in length, has a 5' end, a 3' end, and is complementary to nucleotides 14-21 of the first oligonucleotide, wherein the second oligonucleotide is attached at its 3' end to a molecule comprising cholesterol, dichloroacetate, docosahexaenoic acid, or N-acetylgalactosamine, wherein the anchor oligonucleotide is attached to a polyethylene polymer having a molecular weight between 10,000 and 40,000 daltons, and wherein the first oligonucleotide is complementary to both the second oligonucleotide and the anchor oligonucleotide, forming an asymmetric duplex.

[0038] In certain embodiments, the compound further comprises a pharmaceutically active carrier.

[0039] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound described above and a pharmaceutically acceptable carrier.

[0040] In another aspect, the disclosure provides a method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a compound described above.

[0041] In one aspect, the disclosure provides a method of treating a patient with a disease or disorder, comprising administering an asymmetric oligonucleotide duplex comprising a first oligonucleotide, a second oligonucleotide, and an anchor oligonucleotide, wherein the anchor oligonucleotide comprises a pharmacokinetic-modifying moiety, and wherein the first oligonucleotide is capable of pairing with both the second oligonucleotide and the anchor oligonucleotide to form an asymmetric oligonucleotide duplex.

[0042] In one aspect, the disclosure provides a method of delivering a compound to the liver of a subject, the method comprising administering to the subject a compound comprising: a first oligonucleotide, wherein the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5' end, a 3' end, and comprises complementarity to a target; a second oligonucleotide comprising at least 12 contiguous nucleotides, a 5' end, a 3' end, wherein a portion of the first oligonucleotide is complementary to a portion of the second oligonucleotide, and the second oligonucleotide comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof; and a pharmacokinetic (PK)-modifying anchor comprising an anchor oligonucleotide, an optional linker, and at least one polymer, wherein the anchor oligonucleotide comprises about 5 to about 15 nucleotides that are complementary to the first oligonucleotide, and wherein the polymer is at least about 2,000 Da.

[0043] In one embodiment, the present disclosure provides an asymmetric duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand, wherein each oligonucleotide strand comprises at least one chemically modified nucleotide, the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety, the second oligonucleotide strand and the anchor oligonucleotide strand each comprise fewer nucleotides than the first oligonucleotide strand, and the first oligonucleotide strand can pair with both the second oligonucleotide strand and the anchor oligonucleotide strand to form the asymmetric duplex.

[0044] In certain embodiments, the first oligonucleotide strand comprises 10-50 nucleotides, the second oligonucleotide strand comprises 10-50 nucleotides, and the anchor oligonucleotide comprises 5-15 nucleotides.

[0045] In certain embodiments, the first oligonucleotide strand and the second oligonucleotide strand form siRNA or dsRNA with double-stranded region.In certain embodiments, the length of double-stranded region is about 10 to about 50 base pairs.In certain embodiments, the length of double-stranded region is about 10 to about 15, about 10 to about 20, about 10 to about 25 or about 10 to about 30 base pairs.In certain embodiments, the length of double-stranded region is 13, 14, 15, 16, 17 or 18 base pairs.

[0046] In certain embodiments, the first oligonucleotide strand is at least 16 nucleotides in length, the second oligonucleotide strand is at least 11 nucleotides in length, and the anchor oligonucleotide is about 5-15 nucleotides in length.In certain embodiments, the first oligonucleotide strand is about 21-23 nucleotides in length, the second oligonucleotide strand is about 13-16 nucleotides in length, and the anchor oligonucleotide is about 5-10 nucleotides in length.In certain embodiments, the first oligonucleotide strand is 21 nucleotides in length, the second oligonucleotide strand is 13 nucleotides in length, and the anchor oligonucleotide is 8 nucleotides in length.

[0047] In certain embodiments, the at least one chemically modified nucleotide comprises a 2'-O-methyl-ribonucleotide, a 2'-fluoro-ribonucleotide, a phosphorothioate internucleotide linkage, a locked nucleic acid, a 2',4'-constrained 2'O-ethyl bridged nucleic acid, a peptide nucleic acid, or a mixture thereof.

[0048] In certain embodiments, the second oligonucleotide strand comprises a ligand attached at the 5' end, at the 3' end, at an internal position, or at a mixture thereof.

[0049] In certain embodiments, the second-chain ligand comprises lipid, lipophilic substance, terpene, sugar, peptide, protein, alkyl chain, lectin, glycoprotein, hormone, drug, carbohydrate, antibody, aptamer, vitamin, cationic dye, bioactive conjugate, porphyrin, polycyclic aromatic hydrocarbon, synthetic polymer, or mixture thereof.In certain embodiments, the second-chain ligand comprises fatty acid, steroid, secosteroid, polyamine, ganglioside, nucleoside analog, endocannabinoid, omega-3 fatty acid, omega-6 fatty acid, omega-9 fatty acid, conjugated linoleic acid, saturated fatty acid, or mixture thereof. In certain embodiments, the second chain ligand comprises cholesterol, docosahexaenoic acid, conjugated phosphatidylcholine, N-acetylgalactosamine, dichloroacetic acid, epithelial cell adhesion molecule aptamer, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneal, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleolyl)lithocholic acid, O3-(oleolyl)cholenic acid, dimethoxytrityl, phenoxazine, or a mixture thereof.

[0050] In certain embodiments, the second strand further comprises a linker that attaches the ligand to the second strand.

[0051] In one embodiment, the disclosure provides an asymmetric duplex comprising a first oligonucleotide strand 21 nucleotides in length, a second oligonucleotide strand 13 nucleotides in length, and an anchor oligonucleotide strand 8 nucleotides in length, wherein each oligonucleotide strand comprises alternating 2'-fluoro-ribonucleotides and 2'-methoxyribonucleotides, the first strand comprising two adjacent phosphorothioate internucleotide linkages at the 5' end and eight adjacent phosphorothioate internucleotide linkages at the 3' end, and the second oligonucleotide strand comprising two adjacent phosphorothioate internucleotide linkages at the 5' end and two adjacent phosphorothioate internucleotide linkages at the 3' end. and a linker attached at its 3'-end to a molecule comprising cholesterol, dichloroacetate, docosahexaenoic acid, or N-acetylgalactosamine, wherein the anchor oligonucleotide strand comprises seven adjacent phosphorothioate internucleotide linkages at its 5'-end and a linker attached at its 3'-end to a pharmacokinetic-modifying moiety comprising a polyethylene glycol polymer having a molecular weight between 10,000 and 40,000 daltons, wherein the first oligonucleotide strand can pair with both the second oligonucleotide strand and the anchor oligonucleotide strand to form an asymmetric duplex.

[0052] In one aspect, the disclosure provides a method of treating a patient with a disease or disorder, comprising administering an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand, wherein the anchor oligonucleotide strand is a pharmacokinetic-modifying moiety, and wherein the first oligonucleotide strand is capable of pairing with both the second oligonucleotide strand and the anchor oligonucleotide strand to form an asymmetric oligonucleotide duplex.

[0053] In one embodiment, the present disclosure provides an asymmetric hairpin duplex comprising a hairpin oligonucleotide strand and an anchor oligonucleotide strand, wherein the hairpin oligonucleotide strand comprises an overhang and is capable of pairing with the anchor oligonucleotide strand to form an asymmetric hairpin duplex, and wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety.

[0054] In one aspect, the present disclosure provides a pharmaceutical composition comprising an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand, wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety, and the first oligonucleotide strand is capable of pairing with both the second oligonucleotide strand and the anchor oligonucleotide strand to form an asymmetric oligonucleotide duplex.

[0055] In one embodiment, the disclosure provides a universal anchor oligonucleotide capable of binding to an asymmetric oligonucleotide duplex, the universal anchor oligonucleotide being approximately 5-8 nucleotides in length and having a pharmacokinetic-modifying moiety at its 5' end, wherein at least one nucleotide comprises a chemical modification, the anchor oligonucleotide comprising a first oligonucleotide strand and a second oligonucleotide strand, wherein the sequence of the oligonucleotide anchor is complementary to a region at the 3' end of the first oligonucleotide strand.

[0056] In certain embodiments, the first strand and the anchor strand comprise a GC content of about 35 to about 100%. [Brief explanation of the drawings]

[0057] The above and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments considered in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the appropriate agency upon request and payment of the necessary fee.

[0058] [Figure 1] Schematic representation of the chemical structure of asymmetric siRNA according to certain exemplary embodiments. In a non-limiting example, the hydrophobically modified siRNA (hsiRNA) described herein is composed of an asymmetric duplex formed by a 21-oligonucleotide (21-mer) antisense strand and a 13-mer sense strand containing a hydrophobic cholesterol moiety. The asymmetric hsiRNA further comprises a complementary oligonucleotide anchor (e.g., an octamer) to which a pharmacokinetics (PK)-modifying polymer is attached. The complementary oligonucleotide anchor (e.g., an octamer) hybridizes with the complementary antisense strand. 2'-O-methyl is indicated in black, 2'-fluoro is indicated in gray, and phosphorothioate linkages are indicated by red dashes.

[0059] [Figure 2] Illustrated is the exemplary structure of asymmetric siRNA and the anchor containing complementary oligonucleotide.Antisense strand comprises overhang that can be paired with oligonucleotide anchor.In non-limiting examples, the present specification shows the following 21-mer antisense strand, which can be hybridized with 13-mer sense strand and 8-mer oligonucleotide anchor; 14-mer sense strand and 7-mer anchor; 15-mer sense strand and 6-mer anchor; or 16-mer sense strand and 5-mer anchor.In certain embodiments, the hybridized oligomer can contain 1, 2, 3 or more mismatches.See (Figure 24).

[0060] [Figure 3]Schematically depicted are exemplary embodiments of PK-modifying moieties, including hydrophilic polycarbonates, block copolymers (e.g., amphiphilic block copolymers, hydrophilic block copolymers, or poloxamers), polyethylene glycol, and polysaccharides (e.g., dextrin or chitosan).

[0061] [Figure 4] Schematic representation of two asymmetric siRNA duplexes linked together according to certain exemplary embodiments further described herein.As shown, a PK-modifying moiety is attached to each oligonucleotide anchor, so that when the oligonucleotide anchor binds with the siRNA duplex, the siRNA construct comprises two PK-modifying moieties.In this embodiment, the upper scaffold shows the use of a dynamic PK-modifying anchor, and the lower scaffold is made of a stably attached PK-modified moiety.Without intending to be limited by scientific theory, it is expected that considering the dynamic properties of the upper scaffold, this will enable improved distribution and retention in vivo.

[0062] [Figure 5] Schematic representations of exemplary configurations for attaching PK-modifying moieties to oligonucleotides are shown. Branching patterns allow multiple PK-modifying polymers to be attached to each oligonucleotide anchor. siRNAs and polymers that modify one, two, three, or four PKs are shown here.

[0063] [Figure 6] Schematic representation of asymmetric siRNA, either unconjugated or conjugated with lipid.Exemplary lipids include, but are not limited to, cholesterol, docosahexaenoic acid-conjugated phosphatidylcholine (PC-DHA), dichloroacetic acid (DCA), or epithelial cell adhesion molecule (EpCAM) aptamer.

[0064] [Figure 7] Schematic representation of cholesterol-conjugated siRNA and delivery systems. Suitable delivery systems include, but are not limited to, lipid nanoparticles, exosomes, microvesicles, etc.

[0065] [Figure 8A-8B] Figure 8A shows the blood / plasma circulation time and area under the curve of unconjugated (Figure 8A) and cholesterol-conjugated (Figure 8B) hsiRNA after intravenous injection. Tail vein injection of 20 mg / kg was performed in female FVB / N mice (approximately 9-12 weeks old).

[0066] [Figures 9A-9G] The effect of PK-modifying anchors on in vivo biodistribution is shown. Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric duplexes contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. A fully phosphorothioated 8-mer oligonucleotide anchor was used. Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). 48 hours later, the antisense strand was quantified using a peptide nucleic acid hybridization assay. The biodistribution of hsiRNA is shown in the liver (Figure 9A), spleen (Figure 9B), kidney (Figure 9C), adrenal gland (Figure 9D), heart (Figure 9E), pancreas (Figure 9F), and lung (Figure 9G).

[0067] [Figures 10A-10C] Figure 10 shows the effect of PK-modifying anchors on the delivery of hsiRNA compounds after intravenous injection, as measured by mRNA expression. mRNA expression was examined in the liver (Figure 10A), kidney (Figure 10B), and spleen (Figure 10C). Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). Tissues were collected 48 hours after injection, and mRNA expression was quantified using the QuantiGene b-DNA assay.

[0068] [Figures 11A-11C]The effect of PK-modifying anchors on the in vivo biodistribution of hSiRNA compounds in the central nervous system after intraventricular (IV) injection (Figures 11A and 11B) and intrathecal (IT) injection (Figure 11C) is shown. In Figure 11A, 4 nmoles (or approximately 250 μg) of hsiRNA was injected into the lateral ventricle, resulting in a concentration of approximately 2 nmoles / ventricle. In Figure 11B, 20 nmoles of hsiRNA was injected into the lateral ventricle, resulting in a concentration of approximately 10 nmoles / ventricle. The distribution of hsiRNA in the mouse brain is shown in Figures 11A and 11B. In Figure 11C, 10 nmoles of hsiRNA was injected intrathecally between L5 and L6. The distribution of hsiRNA in the mouse spine is shown in Figure 11C. Forty-eight hours after injection, mouse brain and spinal tissues were collected and stained with DAPI (nuclei, blue). Brains and tissues were imaged using a Leica DMi8 fluorescence microscope.

[0069] [Figure 12] 1 illustrates a hydrophobic polycarbonate polymer according to certain exemplary embodiments.

[0070] [Figure 13] 1 illustrates a polyester polymer according to certain exemplary embodiments.

[0071] [Figure 14] 1 illustrates an amphiphilic block copolymer according to certain exemplary embodiments.

[0072] [Figure 15] 1 illustrates a hydrophilic block copolymer according to certain exemplary embodiments.

[0073] [Figure 16] 1 illustrates a polysaccharide polymer according to certain exemplary embodiments.

[0074] [Figure 17] 1 shows the intrarenal distribution after IV administration.

[0075] [Figure 18]1 shows the liver distribution after IV administration.

[0076] [Figure 19] Splenic distribution after IV administration is shown.

[0077] [Figure 20] Shown is the intrarenal distribution after subcutaneous (SC) administration.

[0078] [Figure 21] Shows the distribution in the liver after SC administration.

[0079] [Figure 22] Distribution in the spleen after SC administration is shown.

[0080] [Figure 23] Shows the distribution in the skin after SC administration.

[0081] [Figure 24] Schematic representation of an exemplary configuration of an asymmetric siRNA and an anchor containing a mismatched complementary oligonucleotide for Tm optimization.

[0082] [Figure 25] Schematic representation of exemplary configurations of asymmetric siRNAs containing various chemical modifications.

[0083] [Figure 26] Schematic representation of a dynamic oligonucleotide anchor for use in delivery of other classes of nucleotides, e.g., ASOs (e.g., shown to be compatible with RNase H or splice switching), microRNAs, mRNAs, CRISPR guide strands, etc.

[0084] [Figure 27]Schematic representation of an exemplary configuration of an asymmetric siRNA and a complementary oligonucleotide-containing anchor. The circles represent fixed sequences of multiple dynamic oligonucleotide anchors that can be used in siRNA constructs targeting various different mRNAs. In certain embodiments, the length of the antisense strand increases to a total of 23 nucleotides. In certain embodiments, the nucleotides at positions 18 to 23 do not hybridize with the mRNA target. In certain embodiments, a fixed / conserved oligonucleotide anchor region is provided that can be used in various siRNAs targeting different mRNA targets. In certain embodiments, the 3' end of the antisense strand may or may not be fully complementary to the mRNA target. In certain embodiments, a 5-mer to 10-mer anchor is used.

[0085] [Figures 28A-28B]The graphs show that PK-modifying anchors dynamically improved the blood / plasma circulation time of parent hsiRNA compounds. The PK-modifying anchors broadened the area under the curve for unconjugated (Figure 28A) and cholesterol-conjugated hsiRNA (Figure 28B) after subcutaneous injection. The PK-modifying anchors delayed the time to peak and slowed the clearance rate of parent hsiRNA compounds. Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). The antisense strand was quantified by peptide nucleic acid (PNA) hybridization assay, as previously described in Godinho et al. (2017) Nucleic Acids Therapeutics. Briefly, this assay uses a Cy3-labeled PNA probe that hybridizes to the antisense strand, which is then quantified by HPLC. AUC was calculated using the model-independent trapezoidal method in GastroPlus Simulations Plus. Polyethylene glycol (PEG) was used as a model PK-modifying moiety, and a fully phosphorothioated octamer was used as a model anchor to modulate the circulation time of individual parent as21-s13 compounds. Both the length and chemical properties of the PK-modifying moiety and anchor may be adjusted depending on the purpose / target of delivery.

[0086] [Figure 29]This graph shows that PK-modifying anchors modulated the systemic in vivo biodistribution of parent hsiRNA compounds. PK-modifying anchors significantly affected the biodistribution of unconjugated (red) and cholesterol-conjugated (black) hsiRNA after subcutaneous injection. Overall, PK-modifying anchors improved the delivery of unconjugated oligos to most organs. Tail vein injections of 20 mg / kg were administered between the scapulae of female FVB / N mice (approximately 9-12 weeks old). Antisense strands were quantified 48 hours later by PNA hybridization assay. Polyethylene glycol (PEG) was used as a model PK-modifying moiety, and a fully phosphorothioated octamer was used as a model anchor to modulate the circulation time of individual parent as21-s13 compounds. Both the length and chemical properties of the PK-modifying moiety and anchor may be adjusted depending on the purpose / target of delivery.

[0087] [Figure 30] The results of gel shift assays using PK-modifying anchors of 10 kDa, 20 kDa, and 40 kDa are shown. Anchors were bound to asymmetric siRNA duplexes with or without cholesterol conjugation. The antisense strand of the asymmetric siRNA duplex was 21 nucleotides in length, the sense strand was 13 nucleotides in length, and the anchor complementary to the tail of the antisense strand was 8 nucleotides in length. The asymmetric siRNA duplexes were Cy3-labeled at the 5' end of the sense strand.

[0088] [Figure 31] Representative siRNA constructs used to measure blood concentration and tissue distribution profiles when administered systemically via intravenous and subcutaneous administration are shown.

[0089] [Figures 32A-32F]Figure 32 shows the blood concentration profiles of PK-modifying anchors paired with the panel of asymmetric siRNA duplex structures shown in Figure 31. Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric duplexes contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. A fully phosphorothioated 8-mer oligonucleotide anchor was used. Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). 48 hours later, the antisense strand was quantified using a peptide nucleic acid hybridization assay. The blood concentration levels of siRNA are shown for unconjugated siRNA (Figure 32A), GalNAc-conjugated siRNA (Figure 32B), DHA-conjugated siRNA (Figure 32C), biantennary siRNA (Figure 32D), cholesterol-conjugated siRNA (Figure 32E), and DCA-conjugated siRNA (Figure 32F).

[0090] [Figures 33A-33F] Figure 33 shows the tissue distribution profiles of PK-modifying anchors paired with the panel of asymmetric siRNA duplex structures shown in Figure 31. Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric duplexes contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. A fully phosphorothioated 8-mer oligonucleotide anchor was used. Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). 48 hours later, the antisense strand was quantified using a peptide nucleic acid hybridization assay. The blood levels of siRNA are shown for unconjugated siRNA (Figure 33A), GalNAc-conjugated siRNA (Figure 33B), DHA-conjugated siRNA (Figure 33C), biantennary siRNA (Figure 33D), cholesterol-conjugated siRNA (Figure 33E), and DCA-conjugated siRNA (Figure 33F).

[0091] [Figure 34A-34B]Figure 31 shows the blood concentration profiles of PK-modifying anchors paired with unconjugated siRNA (Figure 34A) or biantennary siRNA (Figure 34B). Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric duplex contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. A fully phosphorothioated 8-mer oligonucleotide anchor was used. Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). 48 hours later, the antisense strand was quantified using a peptide nucleic acid hybridization assay.

[0092] [Figure 35A-35B] Figure 31 shows the tissue distribution profiles of PK-modifying anchors paired with unconjugated siRNA (Figure 35A) or biantennary siRNA (Figure 35B). Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric duplex contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. A fully phosphorothioated 8-mer oligonucleotide anchor was used. Subcutaneous injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). 48 hours later, the antisense strand was quantified using a peptide nucleic acid hybridization assay.

[0093] [Figure 36] 1 shows the delivery scheme for aptamer-siRNA chimeras with PK-modifying anchors. Subcutaneous injections of 20 mg / kg were performed in tumor-bearing Balb-c mice. The mice had 4T1E breast cancer cell line tumors and P815 mastocytoma tumors.

[0094] [Figure 37A-37B]The blood concentration profile (Figure 37A) and tissue distribution profile (Figure 37B) of the PK-modifying anchor paired with the aptamer-siRNA chimera shown in Figure 36 are shown. The aptamer binds to the EPCAM receptor and is delivered to 4T1E tumors. Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric duplex contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. A fully phosphorothioated 8-mer oligonucleotide anchor was used. The aptamer was conjugated to the 3' end of the sense strand. Subcutaneous injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks old). 48 hours later, the antisense strand was quantified using a peptide nucleic acid hybridization assay.

[0095] [Figures 38A-38C] Figure 38 shows the tissue distribution profile of PK-modifying anchors paired with unconjugated siRNA or biantennary siRNA. The siRNA was delivered via intravenous or subcutaneous administration. Polyethylene glycol (PEG) was used as the PK-modifying polymer. Figure 38A shows liver distribution, Figure 38B shows spleen distribution, and Figure 38C shows kidney distribution.

[0096] [Figure 39] Figure 1 shows the tissue distribution profile in mouse placenta of PK-modifying anchors paired with unconjugated siRNA. siRNA was delivered via subcutaneous administration. Two doses of 20 mg / kg were delivered, as indicated in the timeline. Pregnant female FVB / N mice (approximately 9-12 weeks old, 4 mice / group) were used, and tissues were collected 48 hours after the last injection. Peptide nucleic acid (PNA) hybridization assays were used to quantify antisense.

[0097] [Figure 40]This study demonstrates the efficacy of sFlt-1 mRNA silencing in selected tissues using a PK-modifying anchor paired with unconjugated siRNA. siRNA was delivered via subcutaneous administration. Two doses of 20 mg / kg were delivered, as indicated in the timeline. Pregnant female FVB / N mice (approximately 9-12 weeks old, 6-8 mice per group) were used. Branched DNA (bDNA) was used for mRNA quantification. Target mRNA, sFlt-1, levels were measured in placenta, liver, and kidney tissues. Mouse weight profiles were measured, demonstrating that the use of the PK-modifying anchor did not cause acute systemic toxicity. A panel of blood chemistries and complete blood counts was also measured, demonstrating that the use of the PK-modifying anchor did not cause acute systemic toxicity.

[0098] [Figure 41] This figure shows the delivery of GalNAc-conjugated siRNA to the liver via intravenous or subcutaneous administration. The distribution of the 21-13-8 and 25-17-8 siRNA-PK-modified anchors was compared. The 25-17-8 siRNA-PK-modified anchor contained a 25-nucleotide conserved sequence from nucleotide positions 18 to 25 of the antisense strand, which constitutes the antisense strand tail of the asymmetric siRNA. This 8-nucleotide conserved sequence was complementary to the 8-nucleotide anchor.

[0099] [Figure 42] This figure shows the results of gel shift assays using 8-nucleotide or 6-nucleotide PK-modifying anchors paired with asymmetric siRNA targeting HTT-mRNA. The siRNA has a 21-nucleotide antisense strand and a 13-nucleotide sense strand. A 40 kDa PEG anchor was used. The siRNA / anchor molar ratios were 1:1, 1:2, and 1:4.

[0100] [Figure 43]This figure shows the results of gel shift assays using 8-nucleotide, 7-nucleotide, 6-nucleotide, or 5-nucleotide PK-modifying anchors paired with asymmetric siRNA targeting sFlt-1 mRNA. The siRNAs have a 21-nucleotide antisense strand and a 13-nucleotide sense strand. A 40 kDa PEG anchor was used. The siRNA / anchor molar ratios were 1:1, 1:2, and 1:4.

[0101] [Figure 44] Gel shift assays using 7-, 6-, or 5-nucleotide PK-modifying anchors paired with asymmetric siRNAs targeting sFlt-1 mRNA are shown. The 7-nucleotide anchor was paired with a 14-nucleotide sense strand. The 6-nucleotide anchor was paired with a 15-nucleotide sense strand. The 5-nucleotide anchor was paired with a 16-nucleotide sense strand. A 40 kDa PEG anchor was used. 1:1, 1:2, and 1:4 siRNA / anchor molar ratios were used.

[0102] [Figure 45]Schematic representation of the design of anchor universal sequences for modifying PK. In option 1, a 6-nucleotide universal sequence starting at nucleotide position 18 of a 23-nucleotide antisense strand is engineered into the antisense strand. In the first example of option 1, a 17-nucleotide sense strand is used with a 6-nucleotide anchor sequence that is complementary to the 6-nucleotide universal sequence of the antisense strand. In the second example of option 1, a 15-nucleotide sense strand is used with an 8-nucleotide anchor sequence that is complementary to the 6-nucleotide universal sequence of the antisense strand and the other two nucleotides (which will vary depending on the target sequence selected for the antisense strand) are complementary to nucleotides at positions 16 and 17 of the antisense strand. In option 2, an 8-nucleotide universal sequence starting at nucleotide position 18 of a 25-nucleotide antisense strand is engineered into the antisense strand. In option 2, a 17-nucleotide sense strand is used with an 8-nucleotide anchor sequence that is complementary to the 8-nucleotide universal sequence of the antisense strand.

[0103] [Figures 46A-46B] The mRNA silencing effect of target Htt mRNA is shown. A 23-nucleotide antisense strand sequence (Figure 46A) and a 25-nucleotide antisense strand sequence (Figure 46B) were used. Dose-response was performed by incubating HeLa cells for 72 hours. A bDNA assay was used for mRNA evaluation. Results were normalized to HPRT or PPIB. DETAILED DESCRIPTION OF THE INVENTION

[0104] The present disclosure relates to therapeutic oligonucleotides (e.g., therapeutic siRNAs) containing pharmacokinetic (PK)-modifying anchors. The therapeutic oligonucleotides provided herein containing PK-modifying anchors effectively regulate absorption, distribution, and clearance kinetics in relevant body / biological fluids (e.g., cerebrospinal fluid and plasma) and other tissues. The PK-modifying anchors enable functional delivery to a range of tissues, such as the heart, kidney, liver, spleen, adrenal gland, pancreas, lung, blood (e.g., plasma), and brain tissue.

[0105] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the case of potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, the singular includes the plural and the plural includes the singular. The use of "or" means "and / or" unless specifically stated otherwise. The use of the word "comprising," as well as other terms such as "comprises" and "included," is not limiting. As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise. Thus, for example, "protein" includes a plurality of protein molecules.

[0106] In general, the nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and the laboratory operations and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0107] In order that the disclosure may be more readily understood, selected terms are defined below.

[0108] As used herein, the term "pharmacokinetics modifier" or "PK modifier" refers to a compound that can be used to modify the concentration of a therapeutic agent (e.g., an RNAi agent) over time. In certain embodiments, a PK modifier affects the stability of a therapeutic agent in one or more locations of a subject (e.g., in the heart, kidney, liver, spleen, adrenal gland, pancreas, lung, blood (e.g., plasma), and / or brain tissue). Modified PK parameters include volume of distribution (V d ), area under the curve (AUC), clearance (CL), half-life (t 1 / 2 ), maximum concentration (C max ), bioavailability (F), etc.

[0109] As used herein, the terms "pharmacokinetic-modifying anchor," "PK-modifying anchor," or "Z" refer to a construct comprising an oligonucleotide anchor attached to a polymer via an optional linker. The oligonucleotide anchor of Z can be complementary to an oligonucleotide, for example, to an overhang of a double-stranded nucleic acid sequence or a portion of a single-stranded nucleic acid sequence. The polymer can be attached to an oligonucleotide, for example, to an overhang of a double-stranded nucleic acid sequence or a portion of a single-stranded oligonucleotide, via hybridization of the oligonucleotide anchor. The polymer portion of Z can include a PK-modifying moiety.

[0110] In certain embodiments, a polymer described herein (eg, a PK-modifying polymer) is attached directly (eg, without a separate linker) to the oligonucleotide anchor.

[0111] In certain embodiments, oligonucleotide anchor is connected to polymer through linker, which provides functional group, and connects polymer to oligonucleotide anchor.In certain embodiments, said linker can be, for example, the alkyl chain of about 1 carbon to about 25 carbon, or for example, the well-defined propylene or ethylene glycol chain of about 1 to about 25 units.Exemplary linker is: [ka] is.

[0112] In certain embodiments, the oligonucleotide anchor of Z has a GC content of between about 35% and about 100% when hybridized to a target oligonucleotide. In certain embodiments, the oligonucleotide anchor of Z has a GC content of about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% when hybridized to a target oligonucleotide.

[0113] In certain embodiments, the target oligonucleotide comprises a fixed or conserved region at its 3' end that is complementary to the fixed or conserved region of the oligonucleotide anchor. In certain embodiments, the fixed or conserved region of the target oligonucleotide is fully complementary, partially complementary, or not complementary to the target mRNA.

[0114] In certain exemplary embodiments, Z comprises more than one polymer. In certain exemplary embodiments, Z comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polymers. In certain exemplary embodiments, Z comprises 2, 3, 4, or more polymers.

[0115] In certain exemplary embodiments, Z is a polymer moiety having a molecular weight ranging from about 2,000 Daltons (Da) to about 100,000 Da, including all values ​​therebetween. In certain exemplary embodiments, the molecular weight of the polymer is about 2,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 6,500 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about 8,500 Da, about 9,000 Da, about 9,500 Da, or about 10,000 Da, including all values ​​therebetween. In certain exemplary embodiments, the molecular weight of the polymer is about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da, about 45,000 Da, about 50,000 Da, about 55,000 Da, about 60,000 Da, about 65,000 Da, about 70,000 Da, about 75,000 Da, about 80,000 Da, about 85,000 Da, about 90,000 Da, about 95,000 Da, or about 100,000 Da, including all values ​​therebetween. In certain exemplary embodiments, the molecular weight of the polymer is about 2,000 Da, about 4,500 Da, about 10,000 Da, about 20,000 Da, about 40,000 Da, or about 100,000 Da.

[0116] In certain exemplary embodiments, suitable polymers may include one or any combination of hydrophilic polycarbonates, polyethylene glycols (PEGs), block copolymers (e.g., including amphiphilic or hydrophilic block copolymers), poloxamers, polysaccharides (e.g., including dextrins or chitosan), and poly(lactic-co-glycolic acid) (PLGA). An exemplary embodiment of a suitable PK-modifying moiety is shown in Figure 3.

[0117] In certain exemplary embodiments, the PK-modifying polymer is a hybrid polymer containing multiple types of polymer subunits. An exemplary hybrid polymer is a PEG-polypeptide. (De Marre A et al.: Synthesis, characterization, and in vitro biodegradation of poly(ethylene glycol) modified poly[5N-(2-hydroxyethyl-L-glutamine]. J Bioact Compat Polym 1996, 11:85-99. 76. Chen C, Wang Z, Li Z: Thermoresponsive polypeptides from pegylated poly-L-glutamates. Biomacromolecules 2011, 12:2859-2863)

[0118] In certain exemplary embodiments, the polymer used in Z is PEG, e.g., PEG-4, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-12, PEG-14, PEG-16, PEG-18, PEG-20, PEG-32, PEG-33, PEG-40, PEG-45, PEG-55, PEG-60, PEG-75, PEG-80, PEG-90, PEG-100, PEG-135, PEG-150, PEG-180, PEG-200, PEG-2 ... PEG-240, PEG-350, PEG-400, PEG-500, PEG-600, PEG-800, PEG-1000, PEG-1500, PEG-2000, PEG-4000, PEG-5000, PEG-6000, PEG-7000, PEG-8000, PEG-9000, PEG-14,000, PEG-20,000, PEG-23,000, PEG-25,000, PEG-45,000, PEG-65,000, PEG-90,000, and the like.

[0119] In certain exemplary embodiments, the polymer used in Z comprises a poloxamer. Suitable poloxamers include, but are not limited to, poloxamer 118, poloxamer 188, poloxamer 288, poloxamer 338, poloxamer 407, poloxamine 1107, or poloxamine 1307. Commercially available poloxamers Synperonics (Croda Healthcare), Pluronics (BASF), and Kolliphor (BASF) are also suitable.

[0120] In certain exemplary embodiments, the polymer used in Z comprises a hydrophobic polycarbonate, such as, for example, a tyrosine-derived polycarbonate (Figure 12).

[0121] In certain exemplary embodiments, the polymer used in Z includes polyesters such as, for example, polyhydroxyalkanoates (PHAs), polycaprolactones (PCLs), poly(hydroxybuterates-hydroxyvalerates), polyglycolic acids (PGAs), polylactic acids (PLAs), and the like (Figure 13).

[0122] In certain exemplary embodiments, the polymer used in Z comprises an amphiphilic block copolymer (e.g., poly(2-ethyl-2-oxazoline) (i.e., Aquazol), polyvinylpyrrolidone, acrylonitrile styrene acrylate, N-(2-hydroxypropyl) methacrylamide, polyethylene glycol, etc.) (Figure 14) or a hydrophilic block polymer (e.g., poly(DMA), poly(DEA), poly(DPA), tetrahydrofurfuryl methacrylate, poloxamer (e.g., poloxamer 188, poloxamer 407, poloxamer 338), etc. (Figure 15).

[0123] In certain exemplary embodiments, the polymer used in Z includes a polysaccharide, such as polyglucose (e.g., soluble starch, insoluble starch), small cellulose, chitin, glycogen, amylose, amylopectin, etc. (Figure 16).

[0124] In certain exemplary embodiments, the polymer used in Z comprises a polypeptide, such as polylysine, polyarginine, or other positively charged or hydrophobic amino acids (e.g., polyalanine, polyisoleucine, polymethionine, polyphenylalanine, polyvaline, polyproline, polyglycine, etc., and any combination thereof).

[0125] In certain exemplary embodiments, the melting temperature (Tm) of the nucleotide anchor is optimized to reduce the clearance rate of the associated oligonucleotide. In certain exemplary embodiments, the Tm of the anchor is between about 37°C and about 70°C, including all values ​​therebetween. In certain exemplary embodiments, the Tm is about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C or about 70°C, including all values ​​therebetween. In certain exemplary embodiments, the Tm is between about 37°C and about 40°C, including all values ​​therebetween. In certain exemplary embodiments, the Tm is between about 40°C and about 45°C, including all values ​​therebetween. In certain exemplary embodiments, the Tm is between about 45°C and about 50°C, including all values ​​therebetween. In certain exemplary embodiments, the Tm is between about 50°C and about 55°C, including all values ​​therebetween. In certain exemplary embodiments, the Tm is between about 55°C and about 60°C, including all values ​​therebetween.

[0126] In certain exemplary embodiments, multiple PK-modifying polymers can be attached to a single-stranded oligonucleotide, a partially double-stranded oligonucleotide, or a fully double-stranded nucleic acid duplex. An exemplary embodiment is shown in FIG. 5, which illustrates various configurations useful for attaching a PK-modifying polymer to an oligonucleotide anchor. In certain exemplary embodiments, the PK-modifying polymer can be attached to both the 5' and 3' ends of the oligonucleotide anchor. In certain exemplary embodiments, both the 3' and 5' ends of the oligonucleotide anchor contain multiple PK-modifying polymers. Certain exemplary embodiments include one, two, or three PK-modifying polymers attached to the 3' end, the 5' end, or both the 3' and 5' ends of the polynucleotide anchor.

[0127] In certain exemplary embodiments, Z regulates the delivery of branched oligonucleotides, in which two or more double-stranded oligonucleotides are linked together.In certain embodiments, the PK-modifying polymer is attached to the oligonucleotide anchor of the double-stranded oligonucleotide.In such a configuration, two or more PK-modifying anchors can be attached to the linked double-stranded oligonucleotide.

[0128] As used herein, the term "L" refers to a linker. L can be selected from the group consisting of an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, and any combination thereof. In certain embodiments, L is linked to O via a second oligonucleotide. In certain embodiments, L is a divalent linker. In certain embodiments, L is a trivalent linker.

[0129] In certain embodiments, L is a trivalent linker L1, also referred to herein as C7: [ka]

[0130] In another particular embodiment, L is a divalent linker L2: [ka]

[0131] In another particular embodiment, L is [ka] is a trivalent or bivalent linker selected from the group consisting of:

[0132] As used herein, "X c" refers to a moiety that has affinity for low density lipoproteins and / or medium density lipoproteins. In certain embodiments, X c is a saturated or unsaturated moiety with fewer than three double bonds. c The term " may refer to a tethered ligand, as described below in the "Tethered Ligands" section of this disclosure. In certain exemplary embodiments, X c contains an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof.

[0133] In certain exemplary embodiments, X c has an affinity for high density lipoproteins. In a related embodiment, X c is a polyunsaturated moiety having three or more double bonds (e.g., having 3, 4, 5, 6, 7, 8, 9, or 10 double bonds). c is a polyunsaturated moiety having three double bonds. c is a polyunsaturated moiety having four double bonds. c is a polyunsaturated moiety having five double bonds. c is a polyunsaturated moiety with six double bonds.

[0134] In certain exemplary embodiments, X c is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, ganglioside and nucleoside analogs, and endocannabinoids.

[0135] In certain exemplary embodiments, X cis a neuromodulatory lipid, such as an endocannabinoid. Non-limiting examples of endocannabinoids include, but are not limited to, anandamide, arachidonylethanolamine, 2-arachidonylglyceryl ether (noladin ether), 2-arachidonylglycerol, and N-arachidonyldopamine.

[0136] In certain exemplary embodiments, X c is an omega-3 fatty acid. Non-limiting examples of omega-3 fatty acids include, but are not limited to, hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), cearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA, thymnodonic acid), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA, clupanodonic acid), docosahexaenoic acid (DHA, cervonic acid), tetracosapentaenoic acid, and tetracosahexaenoic acid (nisinic acid).

[0137] In another embodiment, X c is an omega-6 fatty acid. Non-limiting examples of omega-6 fatty acids include, but are not limited to, linoleic acid, gamma-linolenic acid (GLA), eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), docosadienoic acid, adrenic acid, docosapentaenoic acid (osbond acid), tetracosatetraenoic acid, and tetracosapentaenoic acid.

[0138] In another embodiment, X c is an omega-9 fatty acid. Non-limiting examples of omega-9 fatty acids include, but are not limited to, oleic acid, eicosenoic acid, mead acid, erucic acid, and nervonic acid.

[0139] In another embodiment, X cis a conjugated linoleic acid. Non-limiting examples of conjugated linoleic acids include, but are not limited to, α-calendic acid, β-calendic acid, jacaronic acid, α-eleostearic acid, β-eleostearic acid, catarpic acid, and punicic acid.

[0140] In another embodiment, X c is a saturated fatty acid. Non-limiting examples of saturated fatty acids include, but are not limited to, caprylic acid, capric acid, docosanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid.

[0141] In another embodiment, X c is an acid selected from the group consisting of lumelenic acid, α-parinaric acid, β-parinaric acid, boseopentaenoic acid, pinolenic acid and podocarpic acid.

[0142] In another embodiment, X c is selected from the group consisting of docosanoic acid (DCA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). c is docosanoic acid (DCA). In another particular embodiment, X c In another particular embodiment, X is DHA. c is the EPA.

[0143] In another embodiment, X c is a secosteroid. In certain embodiments, X c is calciferol. c is a steroid other than cholesterol.

[0144] In another embodiment, X cis selected from the group consisting of alkyl chains, vitamins, peptides, and bioactive conjugates (including, but not limited to, glycosphingolipids, polyunsaturated fatty acids, secosteroids, steroid hormones, and steroid lipids).

[0145] In another embodiment of the oligonucleotide, X c is characterized by a cLogP value in a range selected from -10 to -9, -9 to -8, -8 to -7, -7 to -6, -6 to -5, -5 to -4, -4 to -3, -3 to -2, -2 to -1, -1 to 0, 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, and 9 to 10.

[0146] As used herein, the term "O" refers to an oligonucleotide of at least 16 consecutive nucleotides, wherein the oligonucleotide has a 5'-end, a 3'-end, and complementarity to the target. In one embodiment, the oligonucleotide has sufficient complementarity to the target to hybridize. In certain embodiments, the complementarity is >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%. In one embodiment, the oligonucleotide has complete complementarity to the target. In another embodiment, the oligonucleotide has 1, 2, 3, 4 or more mismatches with the target.

[0147] In one embodiment, O comprises one or more chemically modified nucleotides. In a particular embodiment, the oligonucleotide comprises alternating 2'-methoxy-nucleotides and 2'-fluoro-nucleotides. In another particular embodiment, the nucleotides at positions 1 and 2 from the 3'-end of the oligonucleotide are connected to adjacent nucleotides via phosphorothioate bonds. In yet another particular embodiment, the nucleotides at positions 1 and 2 from the 3'-end of the oligonucleotide and the nucleotides at positions 1 and 2 from the 5'-end of the oligonucleotide are connected to adjacent nucleotides via phosphorothioate bonds. In yet another particular embodiment, the oligonucleotide comprises a 2'-fluoro modification at each nucleotide at positions 2 and 14 from the 5'-end, and a 2'-methoxy modification at each other nucleotide position.

[0148] In one embodiment, O has complete homology to the target. In a specific embodiment, the target is a mammalian or viral mRNA. In another specific embodiment, the target is an intron region of the mRNA.

[0149] In one embodiment, O comprises an asymmetric duplex. The length of the sense strand and the antisense strand of the asymmetric duplex can vary. In certain exemplary embodiments, the asymmetric duplex contains at least 16 consecutive nucleotides in its antisense strand and at least 12 consecutive nucleotides in its sense strand. In some embodiments, the asymmetric duplex contains a 21-mer oligonucleotide antisense strand and a 13-mer, 14-mer, 15-mer, or 16-mer oligonucleotide sense strand. In some embodiments, the asymmetric duplex contains a 22-mer oligonucleotide antisense strand and a 13-mer, 14-mer, 15-mer, or 16-mer oligonucleotide sense strand. In some embodiments, the asymmetric duplex contains a 23-mer oligonucleotide antisense strand and a 13-mer, 14-mer, 15-mer, or 16-mer oligonucleotide sense strand. The length of the oligonucleotide anchor can vary with respect to the length of the oligonucleotide sense strand. In some embodiments, the sense strand is a 13-mer, 14-mer, 15-mer, or 16-mer oligonucleotide, and the oligonucleotide anchor is an 8-mer, 7-mer, 6-mer, or 5-mer oligonucleotide (see Figure 2). In certain embodiments, the hybridized oligomer can contain one, two, three, or more mismatches (see Figure 24).

[0150] In some embodiments, the asymmetric double strand comprises 23-mer oligonucleotide antisense strand and 13-mer, 14-mer, 15-mer or 16-mer oligonucleotide sense strand.The length of oligonucleotide anchor can vary with respect to the length of the oligonucleotide sense strand.In some embodiments, the sense strand is 13-mer, 14-mer, 15-mer or 16-mer oligonucleotide, and the oligonucleotide anchor is 10-mer, 9-mer, 8-mer, 7-mer, 6-mer or 5-mer oligonucleotide.

[0151] In certain embodiments, O is a therapeutic RNA, e.g., an ASO, a ssRNA, etc., and the oligonucleotide anchor is a 15-mer, a 14-mer, a 13-mer, a 12-mer, an 11-mer, a 10-mer, a 10-mer, a 9-mer, an 8-mer, a 7-mer, a 6-mer, or a 5-mer oligonucleotide.

[0152] As used herein in the context of oligonucleotide sequences, "A" represents a nucleoside containing the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside containing the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside containing the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside containing the base cytosine (e.g., cytidine or a chemically modified derivative thereof).

[0153] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxynucleotides. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide but still retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; and the 8-position for adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or as known in the art) nucleotides; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0154] Nucleotide analogs may also contain modifications to the sugar portion of the nucleotide. For example, the 2'OH-group may be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0155] The term "complementary" refers to the relationship between nucleotides exhibiting Watson-Crick base pairing, or to the formation of double-stranded nucleic acids by oligonucleotides hybridized via Watson-Crick base pairing. The term "complementarity" refers to the state in which an oligonucleotide (e.g., a sense strand or an antisense strand) is partially or completely complementary to another oligonucleotide. An oligonucleotide described herein as complementary to a second oligonucleotide may be 100%, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% complementary to the second oligonucleotide.

[0156] As used herein in the context of oligonucleotide sequences, "A" represents a nucleoside containing the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside containing the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside containing the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside containing the base cytosine (e.g., cytidine or a chemically modified derivative thereof).

[0157] As used herein, the term "3' end" refers to the end of a nucleic acid that contains an unmodified hydroxyl group on the 3' carbon of the ribose ring.

[0158] As used herein, the term "5' end" refers to the end of a nucleic acid containing a phosphate group attached to the 5' carbon of the ribose ring.

[0159] As used herein, the term "nucleoside" refers to a molecule composed of a heterocyclic base and its sugar.

[0160] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxyl group.

[0161] An RNAi agent, e.g., an siRNA, having a strand that is "sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has sufficient sequence to induce destruction of the target mRNA by RNAi.

[0162] As used herein, the term "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material or culture when produced by recombinant techniques, or that is substantially free of chemical precursors or other compounds when chemically synthesized.

[0163] The term "discriminatory RNA silencing" refers to the ability of an RNA molecule to substantially inhibit the expression of a "first" or "target" polynucleotide sequence without substantially inhibiting the expression of a "second" or "non-target" polynucleotide sequence, for example, when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence encoding a regulatory region of the target gene (e.g., a promoter or enhancer element). In other embodiments, the target polynucleotide sequence is a target mRNA encoded by the target gene.

[0164] As used herein, the term "siRNA" refers to small interfering RNA that induces the RNA interference (RNAi) pathway. siRNA molecules vary in length (generally between 18-30 base pairs) and can contain varying degrees of complementarity to their target mRNA. The term "siRNA" encompasses duplexes of two separate strands, as well as single strands that can form hairpin structures containing a duplex region.

[0165] As used herein, the term "antisense strand" refers to the strand of an siRNA duplex that contains a degree of complementarity to a target gene or mRNA and that contains complementarity to the sense strand of the siRNA duplex.

[0166] As used herein, the term "sense strand" refers to the strand of an siRNA duplex that contains complementarity to the antisense strand of the siRNA duplex.

[0167] As used herein, the term "overhang" or "tail" refers to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides at the 3' end of one or both of the sense and antisense strands that are single-stranded, i.e., do not base pair with the other strand of the siRNA duplex (i.e., do not form a duplex).

[0168] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a nucleic acid (e.g., RNA) that has sufficient sequence complementarity to the target RNA (e.g., SNP-containing mRNA or SNP-containing pre-mRNA) to effectively block a region of the target RNA, for example, to inhibit the translation of the target mRNA and / or the splicing of the target pre-mRNA.Antisense oligonucleotides with a "sufficiently complementary sequence to the target RNA" mean that the antisense agent has a sufficient sequence to mask the binding site of a protein that would otherwise regulate splicing, and / or that the antisense agent has a sufficient sequence to mask the binding site of a ribosome, and / or that the antisense agent has a sufficient sequence to alter the three-dimensional structure of the targeted RNA to prevent splicing and / or translation.

[0169] In certain exemplary embodiments, the siRNA of the invention is asymmetric. In certain exemplary embodiments, the siRNA of the invention is symmetric.

[0170] In certain exemplary embodiments, the siRNA of the invention comprises a duplex region of about 8-20 nucleotides or nucleotide analogs in length, about 10-18 nucleotides or nucleotide analogs in length, about 12-16 nucleotides or nucleotide analogs in length, or about 13-15 nucleotides or nucleotide analogs in length (e.g., a duplex region of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs).

[0171] In certain exemplary embodiments, the siRNA of the present invention comprises 1 or 2 overhangs.In certain embodiments, each of the overhangs of the siRNA comprises at least about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 consecutive nucleotides.In certain embodiments, each of the overhangs of the siRNA of the present invention is about 4, about 5, about 6 or about 7 nucleotides in length.In certain embodiments, the overhang of sense strand and the overhang of antisense strand have the same number of nucleotides in length.In other embodiments, the overhang of sense strand has fewer nucleotides than the overhang of antisense strand.In other embodiments, the overhang of antisense strand has fewer nucleotides than the overhang of sense strand.

[0172] In certain exemplary embodiments, siRNA of the present invention comprises sense strand and / or antisense strand, each of which has about 10, about 15, about 20, about 25 or about 30 nucleotides in length.In certain embodiments, siRNA of the present invention comprises sense strand and / or antisense strand, each of which has about 15 to about 25 nucleotides in length.In certain embodiments, siRNA of the present invention comprises sense strand and antisense strand, each of which has about 20 nucleotides in length.In certain embodiments, the sense strand and antisense strand of siRNA are the same length.In other embodiments, the sense strand and antisense strand of siRNA are different in length.

[0173] In certain exemplary embodiments, the siRNA of the present invention has a total length (from the 3' end of the antisense strand to the 3' end of the sense strand) of about 20, about 25, about 30, about 35, about 40, about 45, about 50 or about 75 nucleotides. In certain exemplary embodiments, the siRNA of the present invention has a total length of about 15 to about 35 nucleotides. In other exemplary embodiments, the siRNA of the present invention has a total length of about 20 to about 30 nucleotides. In other exemplary embodiments, the siRNA of the present invention has a total length of about 22 to about 28 nucleotides. In certain embodiments, the siRNA of the present invention has a total length of about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 nucleotides.

[0174] As used herein, the term "chemically modified nucleotide" or "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including ribonucleotides or deoxyribonucleotides that do not occur in nature. Exemplary nucleotide analogs are modified at any position to alter the specific chemical properties of the nucleotide, but still retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; and the 8-position for adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or as known in the art) nucleotides; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310. Exemplary chemical modifications are shown in Figure 25.

[0175] Nucleotide analogs may also contain modifications to the sugar portion of the nucleotide. For example, the 2'OH-group may be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0176] As used herein, the term " metabolically stabilized " refers to the RNA molecule that contains 2'-ribose modification, so that natural 2'-hydroxyl group is replaced with 2'-O-methyl group or 2'-fluoro group.In certain embodiments, the double-stranded region of siRNA comprises 1 or 2 2'-fluoro modification, and / or comprises at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93% or at least about 94% 2'-methoxy modification.In certain exemplary embodiments, antisense strand comprises 2 2'-fluoro modification and at least about 90%, at least about 91%, at least about 92%, at least about 93% or at least about 94% 2'-methoxy modification. In certain exemplary embodiments, the sense strand comprises at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% 2'-methoxy modifications. In certain exemplary embodiments, the sense strand comprises no 2'-fluoro modifications and at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% 2'-methoxy modifications. In certain exemplary embodiments, single-stranded RNA is provided that comprises two 2'-fluoro modifications and at least about 90%, at least about 91%, at least about 92%, at least about 93%, or at least about 94% 2'-methoxy modifications. In certain exemplary embodiments, single-stranded RNAs are provided that contain no 2'-fluoro modifications and at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% 2'-methoxy modifications.

[0177] As used herein, the term "phosphorothioate" refers to the phosphate group of nucleotide that is modified by replacing one or more oxygens of the phosphate group with sulfur.Phosphorothioate further comprises cationic counterions (for example, sodium, potassium, calcium, magnesium, etc.).The term "phosphorothioated nucleotide" refers to the nucleotide that is linked with another nucleotide by one or two phosphorothioate bonds.In certain embodiments, the single-stranded tail of the siRNA of the present invention comprises or is composed of phosphorothioated nucleotide.

[0178] In some embodiments, the compounds, oligonucleotides, and nucleic acids described herein may be modified to include one or more internucleotide linkages as provided in Figure 3. In certain embodiments, the compounds, oligonucleotides, and nucleic acids described herein include one or more internucleotide linkages selected from phosphodiester and phosphorothioate.

[0179] It is understood that certain internucleotide linkages provided herein, including, for example, phosphodiester and phosphorothioate, contain a formal charge of −1 at physiological pH, which may be balanced by a cationic moiety, e.g., an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium, or an ammonium or guanidinium ion.

[0180] As used herein, the term "lipid formulation" refers to a liposome formulation, for example, liposomes can be used to form nanoparticles with nucleic acid to promote the internalization of nucleic acid into cells.Without being bound by theory, suitable liposomes for use are those that can easily fuse with the phospholipid bilayer of cell membrane, thereby allowing nucleic acid to penetrate into cells.In one embodiment, the compound comprises nanoparticles, intercalators, polycations, or mixtures thereof.

[0181] In certain embodiments, compounds of the present disclosure have Formula (I): [ka] [In the formula, the solid line "-" indicates "X c ", "L", "O", and "Z" represent the means of interaction between them. In certain embodiments, the interaction is via base pair complementarity, such as base pair complementarity between oligonucleotide O and anchor oligonucleotide of anchor Z modifying PK. In certain embodiments, the interaction is via base pair complementarity between oligonucleotide O and anchor oligonucleotide of anchor Z modifying PK. c and L, or between L and O.

[0182] Pharmaceutical Compositions and Methods of Administration In one aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds, oligonucleotides, or nucleic acids described herein and a pharmaceutically acceptable carrier.In one embodiment, the pharmaceutical composition comprises one or more double-stranded chemically modified nucleic acids comprising a pharmacokinetics-modifying anchor described herein and a pharmaceutically acceptable carrier.In a specific embodiment, the pharmaceutical composition comprises one double-stranded chemically modified nucleic acid comprising a pharmacokinetics-modifying anchor described herein and a pharmaceutically acceptable carrier.In another specific embodiment, the pharmaceutical composition comprises two double-stranded chemically modified nucleic acids comprising a pharmacokinetics-modifying anchor described herein and a pharmaceutically acceptable carrier.

[0183] The present invention relates to the use of the above-mentioned agents for the above-mentioned therapeutic treatments. Therefore, the modulators (e.g., RNAi agents) of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include nucleic acid molecules, proteins, antibodies, or regulatory compounds and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the active compound, their use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0184] The pharmaceutical composition of the present invention is formulated to be compatible with its intended administration route.Examples of administration routes include parenteral, for example, intravenous (IV), intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular, oral (for example, inhalation), transdermal (topical), intravitreal, intraarticular, intranasal, intravaginal, rectal, sublingual and transmucosal administration.In certain exemplary embodiments, the pharmaceutical composition of the present invention is delivered to cerebrospinal fluid (CSF) by administration routes including, but not limited to, intrastriatal (IS) administration, intracerebroventricular (ICV) administration and intrathecal (IT) administration (for example, administration via pump, infusion, etc.). Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antibacterial agents such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and agents for adjusting isotonicity such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be placed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0185] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if soluble in water) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be appropriate to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by incorporating into the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0186] Sterile injectable solution can be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the above-mentioned components as needed, followed by sterile filtration.Generally, dispersion is prepared by blending the active compound into a sterile vehicle that contains the dispersion medium as a base and other required components from the above-mentioned components.For the case of sterile powder for preparing sterile injectable solution, its exemplary preparation method is vacuum drying and freeze-drying, thereby obtaining the powder of active ingredient and any additional desired components from the solution that has been previously sterile-filtered.

[0187] The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are particularly suitable. Compounds that exhibit toxic side effects can also be used, although care must be taken to design a delivery system that delivers such compounds to the site of targeted infected tissues in order to minimize the potential for damage to uninfected cells, thereby reducing side effects.

[0188] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. In certain embodiments, the dosage of such compounds falls within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the present invention, a therapeutically effective dose can be initially estimated from cell culture assays. Doses can be formulated in animal studies to achieve a circulating plasma concentration range that includes the EC50 (i.e., the concentration of the test compound that achieves half of the maximum response) determined in cell culture. Doses can also be formulated by confirming the gene silencing effect on the tissue concentration of the oligonucleotide in animal studies. Such information can be used to more accurately determine useful doses in humans. Plasma concentrations can be measured, for example, by high-performance liquid chromatography.

[0189] Treatment method As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding same) to a patient with a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder, or the application or administration of a therapeutic agent to a tissue or cell line isolated from a patient, with the intent to cure, relieve, alleviate, palliate, alter, modify, ameliorate, improve, or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition to a disease.

[0190] In one embodiment, a method for preventing the above-mentioned disease or disorder in a subject is provided, comprising administering a therapeutic agent (e.g., an RNAi agent or vector, or a transgene encoding the same) to the subject.Subjects at risk of the disease can be identified, for example, by any diagnostic or prognostic assay described herein, or a combination thereof.Administering a prophylactic agent can be performed before the onset of the characteristic symptoms of the disease or disorder, so that the disease or disorder is prevented or, alternatively, its progression is delayed.

[0191] Design of siRNA molecules In some embodiments, the siRNA molecule of the present invention is a duplex consisting of a sense strand and a complementary antisense strand, and the antisense strand is sufficiently complementary to target mRNA to mediate RNAi.In particular exemplary embodiments, the siRNA molecule has a length of about 10-50 or more nucleotides, that is, each strand comprises 10-50 nucleotides (or nucleotide analogs).In particular exemplary embodiments, the siRNA molecule has a length of about 16-30 nucleotides, for example, each length is about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 nucleotides, and one of the strands is sufficiently complementary to the target region. The strands can be aligned such that there are at least about 1, 2, or 3 bases that are not aligned at the ends of the strands (i.e., no complementary bases on the opposite strand) so that when the strands are annealed, an overhang of about 1, 2, or 3 residues is created at one or both ends of the duplex. The strands can be aligned such that there are about 5, 6, 7, or 8 bases that are not aligned at the ends of the strands and form an overhang. The siRNA molecule can have a length of about 10 to 50 or more nucleotides, i.e., each strand can contain about 10 to about 50 nucleotides (or nucleotide analogs). In particularly exemplary embodiments, the siRNA molecule has each strand of about 16 to about 30, e.g., about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30, nucleotides in length, wherein one of the strands is substantially complementary to the target sequence and the other strand is identical or substantially identical to the first strand.

[0192] Generally, siRNAs can be designed using methods known in the art, for example, by using the following protocol:

[0193] 1. The siRNA should be specific to the target sequence. The first strand should be complementary to the target sequence, and the other strand is substantially complementary to the first strand. In another embodiment, the target sequence is outside the coding region of the target gene. Exemplary target sequences are selected from the 5'-untranslated region (5'-UTR) or intron region of the target gene. Cleavage of mRNA at these sites should eliminate translation of the corresponding protein. Target sequences from other regions of the target gene are also suitable for targeting. The sense strand is designed based on the target sequence. Furthermore, siRNAs with a low G / C content (35-55%) may be more active than siRNAs with a G / C content higher than 55%. Thus, in one embodiment, the present invention encompasses nucleic acid molecules with a G / C content of 35-55%.

[0194] 2. The sense strand of the siRNA is designed based on the sequence of the selected target site. In particular exemplary embodiments, the sense strand comprises about 10 to about 20 nucleotides, for example, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In particular exemplary embodiments, the sense strand comprises about 13, about 14, about 15, or about 16 nucleotides. However, those skilled in the art will understand that siRNAs less than about 10 nucleotides or more than about 20 nucleotides in length can also function to mediate RNAi. Therefore, siRNAs of such lengths are within the scope of the present invention, provided they retain the ability to mediate RNAi. Longer RNA silencing agents have been demonstrated to induce interferon or protein kinase R (PKR) responses in certain mammalian cells, which may be undesirable. In certain embodiments, the RNA silencing agents of the present invention do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNA silencing agents may be useful, for example, in cell types that are unable to generate a PKR response or in situations where the PKR response is downregulated or suppressed by alternative means.

[0195] The siRNA molecule of the present invention has sufficient complementarity with target sequence so that the siRNA can mediate RNAi.Generally, the nucleotide sequence of the siRNA provided is sufficiently identical to the target sequence portion of target gene, so that the RISC-mediated cleavage of the target gene is achieved.Therefore, in certain exemplary embodiments, the sense strand of siRNA is designed to have a sequence that is sufficiently identical to the target portion.For example, the sense strand can have 100% identity with the target site.However, 100% identity is not required.The identity between the sense strand and the target RNA sequence can be more than about 80%, for example, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identity. The present invention has the advantage that it can tolerate certain sequence changes to enhance the efficiency and specificity of RNAi.In one embodiment, the sense strand has about 4, about 3, about 2, about 1 or about 0 mismatched nucleotides with the target region, for example, the target region that has at least one base pair difference between wild type and mutant allele, for example, the target region comprises gain-of-function mutation, and the other strand is the same or substantially identical to the first strand.In addition, the siRNA sequence that has one or two nucleotides slightly inserted or deleted can also be effective in mediating RNAi.Alternatively, the siRNA sequence that has nucleotide analogues substituted or inserted can be effective in inhibiting.

[0196] Sequence identity can be determined using sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules at that position are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), and optionally penalizes the score for the number of gaps introduced and / or the length of the introduced gaps.

[0197] Comparing sequences and determining the percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, alignment is performed over specific portions of the aligned sequences where there is sufficient identity, but not over portions where there is a lower degree of identity (i.e., local alignment). A non-limiting example of a local alignment algorithm used to compare sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77. Such an algorithm is incorporated into the BLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10.

[0198] In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison, gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity is determined over the entire length of the aligned sequences (i.e., a global alignment). A non-limiting example of a mathematical algorithm used for global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0199] 3. The antisense or guide strand of siRNA usually differs in length from the sense strand and contains complementary nucleotides. In one embodiment, the strands of siRNA can be paired to have a 3' overhang of about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides. The overhang can comprise (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang can comprise (or consist of) deoxyribonucleotides, such as dTs, or nucleotide analogs, or other suitable non-nucleotide materials. The overhanging nucleotides can be either RNA or DNA. As described above, it is desirable to select a target region where the mutant:wild-type mismatch is a purine:purine mismatch.

[0200] 4. Compare potential targets to the appropriate genome database (human, mouse, rat, etc.) using any method known in the art and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searching is known as BLAST, which is available at the National Center for Biotechnology Information website.

[0201] 5. Select one or more sequences that meet the evaluation criteria

[0202] Further general information about the design and use of siRNAs can be found in "The siRNA User Guide," available at the Max-Plank-Institut fur Biophysikalishe Chemie website.

[0203] Alternatively, siRNA can be functionally defined as a nucleotide sequence (or oligonucleotide sequence) capable of hybridizing to a target sequence (e.g., hybridization in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours; followed by washing). Further exemplary hybridization conditions include hybridization in 1xSSC at 70°C or 1xSSC, 50% formamide at 50°C, followed by washing in 0.3xSSC at 70°C, or hybridization in 4xSSC at 70°C or 4xSSC, 50% formamide at 50°C, followed by washing in 1xSSC at 67°C. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5-10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined by the following formula: For hybrids less than 18 base pairs in length, the Tm (°C) is 2(# of A+T bases) + 4(# of G+C bases). For hybrids 18 and 49 base pairs in length, the Tm (°C) is 81.5 + 16.6(log10[Na+]) + 0.41(%G+C) - (600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] in 1x SSC is 0.165M). Further examples of stringent conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, FM Ausubel et al. (eds.), John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are incorporated herein by reference.

[0204] Negative control siRNA will have the same nucleotide composition as selected siRNA, but will not have significant sequence homology with the appropriate genome.Such negative control can be designed by randomly scrambling the nucleotide sequence of selected siRNA.Homology search can be carried out to confirm that negative control lacks homology with any other gene in the appropriate genome.In addition, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0205] 6. To verify the effectiveness of siRNA in disrupting target mRNA, siRNA may be incubated with target cDNA in a Drosophila-based in vitro mRNA expression system. 32 Newly synthesized target mRNA radiolabeled with P is detected by autoradiography on an agarose gel. The presence of cleavage of the target mRNA indicates mRNA nuclease activity. Suitable controls include omitting the siRNA and using a non-target cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but does not have significant sequence homology to the appropriate target gene. Such a negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology to any other genes in the appropriate genome. Additionally, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0206] siRNA can be designed to target any of the above-mentioned target sequences.The siRNA comprises the antisense strand that is sufficiently complementary with target sequence to mediate the silencing of target sequence.In certain embodiments, the RNA silencing agent is siRNA.

[0207] The site of siRNA-mRNA complementarity that results in optimal mRNA specificity and maximal mRNA cleavage is selected.

[0208] siRNA-like molecules The siRNA-like molecules of the present invention have a sequence that is "sufficiently complementary" to the target sequence of target mRNA (i.e., have a sequence strand) to direct gene silencing by either RNAi or translational repression. siRNA-like molecules are designed in the same way as siRNA molecules, but the degree of sequence identity between sense strand and target RNA is approximately the same as that observed between miRNA and its target. Generally, as the degree of sequence identity between miRNA sequence and its corresponding target gene sequence decreases, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi increases. Therefore, in an alternative embodiment, when post-transcriptional gene silencing by translational repression of target gene is desired, the miRNA sequence is partially complementary to the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementary sites) dispersed in target mRNA (for example, in the 3'-UTR of target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003).Because the mechanism of translational repression is cooperative, in certain embodiments, multiple (for example, 2, 3, 4, 5 or 6) complementary sites can be targeted.

[0209] The ability of an siRNA-like duplex to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at the complementary site. In one embodiment, when gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementary site, so that the duplex formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003). In another embodiment, 2, 3, 4, 5, or 6 consecutive or non-consecutive non-identical nucleotides are introduced. The non-identical nucleotides may be selected so that they form wobble base pairs (e.g., G:U) or mismatch base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the "bulge" is concentrated at nucleotide positions 12 and 13 from the 5' end of the miRNA molecule (e.g., antisense strand).

[0210] Modified RNA silencing agents In certain embodiments of the present invention, the above-mentioned RNA silencing agent of the present invention (or any part thereof) can be modified to further improve the activity of the agent.For example, the above-mentioned RNA silencing agent can be modified with any of the following modifications.Modifications can be partially used to further enhance target discrimination, enhance the stability of the agent (for example, prevent degradation), promote cellular uptake, enhance targeting efficiency, improve the binding efficacy (for example, with the target), improve the patient's tolerance to the agent, and / or reduce toxicity.

[0211] 1) Modifications that enhance target discrimination In certain embodiments, the RNA silencing agent of the present invention may be replaced with a destabilizing nucleotide to enhance single nucleotide target discrimination (see U.S. Patent Application No. 11 / 698,689 (filed January 25, 2007) and U.S. Provisional Patent Application No. 60 / 762,225 (filed January 25, 2006), both of which are incorporated herein by reference). Such modifications may be sufficient to abolish the specificity of the RNA silencing agent for non-target mRNAs (e.g., wild-type mRNAs) without significantly affecting the specificity of the RNA silencing agent for target mRNAs (e.g., gain-of-function mutant mRNAs).

[0212] In certain embodiments, the RNA silencing agent of the present invention is modified by introducing at least one universal nucleotide into its antisense strand.Universal nucleotide comprises a base moiety that can form base pairs indiscriminately with any of the four conventional nucleotide bases (e.g., A, G, C, U).Universal nucleotide can be used because it has relatively little effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA.Exemplary universal nucleotides include those having an inosine base moiety or an inosine analog base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In certain embodiments, the universal nucleotide is an inosine residue or a naturally occurring analogue thereof.

[0213] In certain embodiments, the RNA silencing agent of the present invention is modified by introducing at least one destabilizing nucleotide at the nucleotide within 5 nucleotides from specificity-determining nucleotide (i.e., the nucleotide that recognizes disease-related polymorphism).For example, destabilizing nucleotide can be introduced at the position of 5, 4, 3, 2 or 1 nucleotides from the specificity-determining nucleotide.In exemplary embodiments, destabilizing nucleotide can be introduced at the position that is 3 nucleotides from the specificity-determining nucleotide (i.e., there are two stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide).In the RNA silencing agent having two strands or strand portions (such as siRNA and shRNA), destabilizing nucleotide can be introduced into the strand or strand portion that does not contain specificity-determining nucleotide.In certain embodiments, destabilizing nucleotide is introduced into the same strand or strand portion that contains specificity-determining nucleotide.

[0214] 2) Modifications to enhance efficacy and specificity In certain embodiments, the RNA silencing agent of the present invention can be modified according to asymmetric design rules to promote the enhancement of efficacy and specificity in the mediation of RNAi (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705).This modification selects the sense strand and facilitates the antisense strand of siRNA (for example, the siRNA designed by the method of the present invention, or the siRNA produced by shRNA) to enter RISC, so that the antisense strand preferentially induces the cleavage or translational suppression of target mRNA, thus increasing or improving the efficiency of targeted cleavage and silencing. In certain embodiments, the asymmetry of the RNA silencing agent is enhanced by weakening the base pairing strength between the 5' end of the antisense strand (AS5') and the 3' end of the sense strand (S3') of the RNA silencing agent relative to the binding strength or base pairing strength between the 3' end of the antisense strand (AS3') and the 5' end of the sense strand (S'5) of the RNA silencing agent.

[0215] In one embodiment, the asymmetry of the RNA silencing agent of the present invention can be strengthened so that there are fewer G:C base pairs between the 5' end of the antisense strand portion and the 3' end of the sense strand portion than between the 3' end of the antisense strand portion and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present invention can be strengthened so that there is at least one mismatched base pair between the 5' end of the first or antisense strand portion and the 3' end of the sense strand portion. In certain embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the asymmetry of the RNA silencing agent of the present invention can be strengthened so that there is at least one wobble base pair, for example, G:U, between the 5' end of the first or antisense strand portion and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present invention can be strengthened by having at least one base pair that contains rare nucleotide, such as inosine (I).In certain embodiments, the base pair is selected from the group consisting of I:A, I:U and I:C.In yet another specific embodiment, the asymmetry of the RNA silencing agent of the present invention can be strengthened by having at least one base pair that contains modified nucleotide.

[0216] 3) RNA silencing agents with enhanced stability The RNA silencing agent of the present invention can be modified to improve stability in serum or in cell culture growth medium.To enhance stability, 3'-residues can be stabilized against degradation, for example, they can be selected to consist of purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, the substitution of pyrimidine nucleotides with modified analogs, for example, the substitution of uridine with 2'-deoxythymidine, can be tolerated and does not affect the efficiency of RNA interference.

[0217] In certain embodiments, the present invention features an RNA silencing agent that can include a first, second, and third strand, wherein any of the first, second, and third strands can be modified by replacing an internal nucleotide with a modified nucleotide to enhance in vivo stability compared to the corresponding unmodified RNA silencing agent.As defined herein, an "internal nucleotide" is one that is present at any position other than the 5'-end or 3'-end of a nucleic acid molecule, polynucleotide, or oligonucleotide.Internal nucleotides can be within a single-stranded molecule, or within a double-stranded or double-stranded molecule.In one embodiment, the sense strand and / or antisense strand are modified by replacing at least one internal nucleotide.In another embodiment, the sense strand and / or antisense strand are modified by replacing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more internal nucleotides. In another embodiment, the sense and / or antisense strands are modified by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more internal nucleotide substitutions. In yet another embodiment, the sense and / or antisense strands are modified by all internal nucleotide substitutions.

[0218] In certain embodiments, RNA silencing agent (for example, any combination of the first oligonucleotide, the second oligonucleotide and the third oligonucleotide) can optionally contain at least one modified nucleotide analogue.This nucleotide analogue can be located at the position where the target-specific silencing activity, for example, RNAi-mediated activity or translational repression activity, is not substantially affected at the 5'-end and / or 3'-end region of siRNA molecule.In particular, this end can be stabilized by incorporating modified nucleotide analogue.

[0219] Exemplary nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., modifications to the phosphate-sugar backbone in any of the first, second, and / or third oligonucleotides). For example, the phosphodiester linkage of natural RNA may be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphodiester group connecting adjacent ribonucleotides is replaced with a modified group, such as a phosphorothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH, NHR, NR, or ON, where R is C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0220] In certain embodiments, the modification is 2'-fluoro, 2'-amino and / or 2'-thio modification.Specific exemplary modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and / or 5-amino-allyl-uridine.In certain embodiments, 2'-fluoro ribonucleotides are all uridines and cytidines.Further exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine and 5-fluoro-uridine. 2'-deoxy-nucleotide and 2'-ohm nucleotide can also be used in the portion of the modified RNA-silencing agent of the present invention.More modified residues include deoxy-abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin.In certain exemplary embodiments, the 2' portion is a methyl group, so that the linking portion is a 2'-O-methyl oligonucleotide.

[0221] In exemplary embodiments, the RNA silencing agent of the present invention (for example, any combination of the first oligonucleotide, the second oligonucleotide and the third oligonucleotide) comprises locked nucleic acid (LNA).LNA comprises a sugar-modified nucleotide that is resistant to nuclease activity (highly stable) and has single nucleotide discrimination against mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42: 7967-7975; Petersen et al. (2003) Trends Biotechnol 21: 74-81).These molecules have a 2'-O, 4'-C-ethylene bridged nucleic acid, which can be modified, for example, with 2'-deoxy-2''-fluorouridine. Furthermore, LNAs enhance the specificity of oligonucleotides by constraining the sugar moiety in a 3'-endo conformation, thereby pre-organizing the nucleotides for base pairing and increasing the melting temperature of the oligonucleotide by up to 10°C per base.

[0222] In another exemplary embodiment, the RNA silencing agent of the present invention (for example, any combination of the first oligonucleotide, the second oligonucleotide and the third oligonucleotide) comprises peptide nucleic acid (PNA). PNA comprises a modified nucleotide in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety, which has the ability to form a polyamide backbone, which is highly resistant to nuclease digestion and gives the molecule improved binding specificity (Nielsen et al., Science, (2001), 254: 1497-1500).

[0223] In another exemplary embodiment, the RNA silencing agent of the present invention (e.g., any combination of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide) comprises a phosphorodiamidate morpholino oligomer (PMO). PMOs comprise modified nucleotides that have a standard nucleobase linked to a methylene morpholine ring via a phosphorodiamidate group instead of a phosphate (Summerton et al., (1997) Antisense & Nucleic Acid Drug Development. 7 (3): 187-95).

[0224] Also provided is a ribonucleotide with modified nucleobase, i.e., ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.The base may be modified to block the activity of adenosine deaminase.Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine, and they are suitable.It should be noted that the above-mentioned modifications may be combined.

[0225] In other embodiments, crosslinking can be used to modify the pharmacokinetics of the RNA silencing agent, for example, to increase its half-life in the body.Thus, the present invention encompasses RNA silencing agents that have two complementary strands of nucleic acid, where the two strands are crosslinked.The present invention also encompasses RNA silencing agents that are conjugated or unconjugated (e.g., at the 3' end) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), etc.Modifying siRNA derivatives in this way can improve cellular uptake or enhance the cell targeting activity of the resulting siRNA derivative compared to the corresponding siRNA, making it useful for tracking the siRNA derivative in cells, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

[0226] Other exemplary modifications include: (a) 2' modifications, such as providing a 2'OMe moiety in the sense or antisense strand, particularly at U in the sense strand, or providing a 2'OMe moiety in the 3' overhang, for example, at the 3' end (3' end means the 3' atom of the molecule, or up to the 3' portion, for example, up to the 3' P or 2' position, as indicated by the context); (b) modifications of the backbone, for example, replacing O with S in the phosphate backbone, for example, providing a phosphorothioate modification of U or A, or both, particularly in the antisense strand, for example, replacing P with S; (c) replacing U with a C5 amino linker; (d) replacing A with G (the sequence change is preferably located in the sense strand, not the antisense strand); and (e) modifications at the 2', 6', 7', or 8' position. Exemplary embodiments are those in which one or more of these modifications are present in the sense strand but not in the antisense strand, or in which the antisense strand is largely free of such modifications. Still other exemplary modifications include the use of a methylated P in the 3' overhang, e.g., at the 3' end; a combination of 2' modifications, e.g., providing a 2'OMe moiety and modifying the backbone, e.g., replacing P with S, e.g., providing a phosphorothioate modification, or the use of a methylated P in the 3' overhang, e.g., at the 3' end; modifications with 3' alkyls; modifications in the 3' overhang, e.g., with a baseless pyrrolidone at the 3' end; modifications with naproxen, ibuprofen, or other moieties that inhibit degradation at the 3' end.

[0227] 4) Modifications to enhance cellular uptake In other embodiments, the RNA silencing agent (e.g., any combination of the first strand oligonucleotide, the second oligonucleotide, and the third oligonucleotide) can be modified with a chemical moiety, for example, to enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the present invention encompasses RNA silencing agents that are conjugated (e.g., at the 3' end of the sense strand) or unconjugated to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. Conjugation can be achieved by methods known in the art, for example, using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describing nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describing nucleic acids linked to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describing nucleic acids linked to nanoparticles).

[0228] In certain embodiments, the RNA silencing agent of the present invention is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0229] 5) Linked Ligand Other entities can be linked to the RNA silencing agents of the present invention. For example, ligands can be linked to RNA silencing agents to improve stability, thermodynamics of hybridization with target nucleic acids to target specific tissues or cell types, or cell permeability, for example, via endocytosis-dependent or -independent mechanisms. Ligands and associated modifications can also increase sequence specificity, thereby reducing off-site labeling. The linked ligand can include one or more modified bases or sugars that can function as intercalators. These are typically located in internal regions, such as the bulge, of the RNA silencing agent / target duplex. Intercalators can be aromatic, such as polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators can have stacking capabilities and can include systems with two, three, or four fused rings. The universal bases described herein can be included in the ligand. In one embodiment, a cleavage group can be included that cleaves the target nucleic acid, thereby contributing to the inhibition of the target gene. The cleavage group can be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, polypeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, for example, Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote selective cleavage of target RNA at the bulge site by free metal ions such as Lu(III). In some embodiments, a peptide ligand can be linked to an RNA silencing agent, for example, to promote cleavage of target RNA at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (eg, via an amino acid derivative) to promote target RNA cleavage.The linked ligand can be an aminoglycoside ligand, which allows the RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of an acridine analog can enhance sequence specificity. For example, neomycin B has a higher affinity for RNA than DNA, but lower sequence specificity. The acridine analog, neo-5-acridine, has a higher affinity for the HIV Rev-response element (RRE). In some embodiments, a guanidine analog (guanidinoglycoside) of the aminoglycoside ligand is linked to the RNA silencing agent. In guanidinoglycosides, the amine group of amino acids is replaced with a guanidine group. The attachment of guanidine analogs can enhance the cell permeability of RNA silencing agents. The linking ligand can be a polyarginine peptide, peptoid, or peptidomimetic, which can enhance the cellular uptake of oligonucleotide agents.

[0230] Exemplary ligands are bound, for example, covalently bound, to the carrier that is conjugated with the ligand, either directly or indirectly through an intermediate tether.In exemplary embodiments, the ligand is bound to the carrier through an intermediate tether.In exemplary embodiments, the ligand changes the distribution, targeting or life span of the RNA silencing agent that it is incorporated into.In exemplary embodiments, the ligand provides enhanced affinity to selected target, for example, molecule, cell or cell type, compartment, for example, cell or organ compartment, body tissue, organ or region, for example, compared with the absence of such ligand.

[0231] Exemplary ligands can improve transport, hybridization, and specificity properties, and may also improve nuclease resistance of the resulting natural or modified RNA silencing agent or polymer molecule containing any combination of the monomers and / or natural or modified ribonucleotides described herein. Generally, ligands can include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; crosslinkers; moieties that confer nuclease resistance; and natural or unnatural nucleobases. Common examples include lipophilic substances, lipids, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein-binding agents, integrin-targeting molecules, polycationic agents, peptides, polyamines, and peptidomimetics. Ligands can include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids, or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymers, poly(L-lactide-co-glycolized) copolymers, divinyl ether-maleic anhydride copolymers, N-(2-hydroxypropyl)methacrylamide copolymers (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethanes, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazines.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0232] Ligands can also include targeting groups, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specialized cell types such as kidney cells. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, transferrin-mimetic peptides, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidium aminoglycosides, artificial endonucleases, lipophilic molecules such as cholesterol (and its thio analogs), cholic acid, cholanic acid, lithocholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, bis-, or tris-fatty acid esters, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C19 , or C 20 fatty acids) and their ethers, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide) amide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu complex of tetraazamacrocycle), dinitrophenyl, HRP, or AP.

[0233] The ligand can be a protein, e.g., a glycoprotein or peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specific cell type, such as a cancer cell, endothelial cell, or bone cell. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. The ligand can be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0234] The ligand can be a substance, such as a drug, that can enhance the uptake of RNA silencing agents into cells, for example, by disrupting the cytoskeleton of cells, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of cells.The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.The ligand can enhance the uptake of RNA silencing agents into cells, for example, by activating inflammatory response.Exemplary ligands with such effects include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or gamma interleukin.In one embodiment, the ligand is a lipid or lipid-based molecule.In certain embodiments, such lipid or lipid-based molecule binds to serum protein, for example, human serum albumin (HSA).The HSA-binding ligand distributes the conjugate to target tissue, for example, non-renal target tissue of the body. For example, target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, and / or (c) be used to adjust the binding with serum protein, for example, HSA.Lipid-based ligand can be used to adjust, for example, control the binding of conjugate to target tissue.For example, lipid or lipid-based ligand that binds more strongly to HSA is less likely to target the kidney, and therefore less likely to be removed from the body.Lipid or lipid-based ligand that binds less strongly to HSA can be used to target conjugate to the kidney.In certain embodiments, lipid-based ligand binds to HSA.Lipid-based ligand can bind to HSA with sufficient affinity, so that conjugate is distributed to tissues other than the kidney.However, in certain embodiments, the affinity is not so strong that the HSA-ligand binding cannot be reversed. In another specific embodiment, the lipid-based ligand binds weakly or not at all to HSA, so that the conjugate is distributed to the kidney. Other moieties that target kidney cells can also be used instead of or in addition to the lipid-based ligand.

[0235] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E, and K.Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0236] In another embodiment, the ligand is a cell-permeating agent, for example, a helical cell-permeating agent.The agent can be amphipathic.An exemplary agent is a peptide such as Tat or Antennapedia.If the agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide binding, and the use of D-amino acids.In certain embodiments, the helical agent is an alpha-helical agent, which can optionally have a lipophilic phase and a lipophobic phase.

[0237] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. The peptide moiety can be an L-peptide or a D-peptide. Alternatively, the peptide moiety can include a hydrophobic membrane translocating sequence (MTS). The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as peptides identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In an exemplary embodiment, the peptide or peptidomimetic linked to the RNA silencing agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or an RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to enhance stability or direct structural properties. Any of the following structural modifications can be utilized:

[0238] All references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated herein by reference in their entirety for all purposes, as are the documents cited therein. The disclosure will utilize, unless otherwise indicated, conventional techniques of immunology, molecular biology, and cell biology, which are well known in the art.

[0239] This disclosure also incorporates by reference all such techniques known in the fields of molecular biology and drug delivery.

[0240] [Example] Example 1. Oligonucleotides containing anchors that modify pharmacokinetics (PK) for cerebrospinal fluid and systemic delivery 1.1 PK-modifying anchors A major challenge in the field of therapeutic oligonucleotides is that non-serum-bound oligonucleotides are cleared from the cerebrospinal fluid (CSF) and blood / plasma within minutes of injection. This rapid clearance is a major factor limiting the delivery of oligonucleotides to tissues beyond the liver and kidney. In the central nervous system, bulk CSF flow is the primary mechanism behind oligonucleotide distribution through the brain. Oligonucleotides are rapidly removed from the central nervous system, which limits their distribution in organisms with large, complex brains (including humans). The PK-modifying molecular anchors disclosed herein are designed to enable efficient modulation of the absorption, distribution, and clearance kinetics of therapeutic oligonucleotides and enhance their tissue distribution. Efficient modulation of absorption, distribution, and clearance kinetics can be achieved in blood / plasma, cerebrospinal fluid (CSF), and other relevant body / biological fluids and tissues. The PK-modifying molecular anchors disclosed herein modulate CSF clearance kinetics and enhance the efficacy of therapeutic oligonucleotides in all brain regions, regardless of the administration site.

[0241] The described PK-modifying anchors dynamically adjust the size of therapeutic oligonucleotides, resulting in modulation of clearance kinetics relative to tissue uptake and distribution. The dynamic nature of this concept is achieved through optimization of the anchor's size and chemical composition. PK-modifying anchors of optimal size for modulation of CSF clearance rate and systemic clearance are described herein. Additionally, a panel of non-immunogenic polymers (including poloxamer 188) and block polymers that function as pharmacokinetic-modifying moieties are described herein.

[0242] 1.2 PK-modifying anchors dynamically improved the blood / plasma circulation time of hsiRNA compounds As shown in Figure 2, the effect of PK-modifying anchors on the blood / plasma circulation time of hydrophobically modified siRNA (hsiRNA) was tested. It was determined that PK-modifying molecular anchors enhanced the circulation time and area under the curve of unconjugated (Figure 8A) and cholesterol-conjugated (Figure 8B) siRNA after intravenous injection. Polyethylene glycol (PEG) was used as a model PK-modifying polymer. An 8-mer oligonucleotide with a phosphorothioated backbone was used as a model oligonucleotide anchor. The PK-modifying anchors were hybridized to asymmetric hsiRNA duplexes containing a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. Increasing the length of the PEG moiety significantly improved the circulation time of the hsiRNA compounds.

[0243] Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks of age). Antisense strands were quantified using a peptide nucleic acid (PNA) hybridization assay previously described in Godinho et al., 2017 (Nucleic Acids Therapeutics). Briefly, this assay uses a Cy3-labeled PNA probe that hybridizes to the antisense strand, followed by quantification by HPLC. Area under the curve (AUC) was calculated using the model-independent trapezoidal method with GastroPlus Simulations Plus.

[0244] 1.3 PK-modifying anchors regulated the systemic in vivo biodistribution of hsiRNA compounds As shown in Figure 9, the effect of PK-modifying anchors on the biodistribution of hsiRNA was examined. hsiRNA localization was examined in the liver (Figure 9A), spleen (Figure 9B), kidney (Figure 9C), adrenal gland (Figure 9D), heart (Figure 9E), pancreas (Figure 9F), and lung (Figure 9G). PEG was used as a model PK-modifying polymer. An 8-mer oligonucleotide with a phosphorothioated backbone was used as a model oligonucleotide anchor. The PK-modifying anchors were hybridized to asymmetric hsiRNA duplexes containing a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand.

[0245] Many embodiments of pharmacokinetic-modifying anchors and oligonucleotide anchors are possible. The length and chemical properties of the anchor can be adjusted depending on the purpose and targeted delivery. As shown in Figure 9, pharmacokinetic-modifying anchors include: The biodistribution of unconjugated and cholesterol-conjugated siRNA after intravenous injection was significantly affected. Figure 9 shows a positive correlation between increasing the length of the PEG moiety and improving delivery of unconjugated oligonucleotides to most organs.

[0246] A 20 mg / kg tail vein injection was performed in female FVB / N mice (approximately 9-12 weeks old). After 48 hours, the antisense strand was quantified using a PNA hybridization assay.

[0247] 1.4 PK-modifying anchors enabled efficient and potent gene silencing after systemic administration The ability of PK-modifying anchors to deliver hsiRNA compounds to the liver (Figures 10A, 18), kidney (Figures 10B, 17), and spleen (Figures 10C, 19) after intravenous administration and subsequent gene silencing was tested. PK-modifying anchors enhanced the delivery of hsiRNA compounds after intravenous administration. Productive gene silencing was observed after 48 hours. The addition of larger PEG moieties did not interfere with gene silencing, indicating (without being bound by scientific theory) that RNA-induced silencing complex (RISC) loading and activity were comparable to that of hsiRNA alone.

[0248] Tail vein injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks of age). Tissues were collected 48 hours after injection, and mRNA was quantified using the QuantiGene b-DNA assay as described in Coles et al., 2015.

[0249] The PK-modifying anchors also delivered hsiRNA compounds to the kidney (Figure 20), liver (Figure 21), spleen (Figure 22), and skin (Figure 23) after subcutaneous administration.

[0250] 1.5 PK-modifying anchors regulated the in vivo biodistribution of hsiRNA compounds within the central nervous system after intracerebroventricular and intrathecal injection The effect of various hsiRNA constructs on in vivo biodistribution was examined in mice after intracerebroventricular injection (Figures 11A and 11B) or intrathecal injection (Figure 11C). In Figure 11A, 4 nanomoles (or approximately 250 μg) of hsiRNA was injected into the lateral ventricle, resulting in a concentration of approximately 2 nanomoles / ventricle. In Figure 11B, 20 nanomoles of hsiRNA was injected into the lateral ventricle, resulting in a concentration of approximately 10 nanomoles / ventricle. The distribution of hsiRNA in the mouse brain is shown in Figures 11A and 11B. In Figure 11C, 10 nanomoles of hsiRNA was injected intrathecally between L5 and L6. The distribution of hsiRNA in the mouse spine is shown in Figure 11C.

[0251] hsiRNA constructs were prepared starting with a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. Various attachments tested included cholesterol alone, cholesterol-anchor alone, cholesterol-2000 Da PK-modifying anchor, cholesterol-4500 Da PK-modifying anchor, cholesterol-free anchor alone, cholesterol-free 2000 Da PK-modifying anchor, cholesterol-free 4500 Da PK-modifying anchor, and cholesterol-free hsiRNA alone. Forty-eight hours after injection, mouse brain and spinal cord tissues were collected and stained with DAPI (nuclei, blue). Brains and tissues were imaged using a Leica DMi8 fluorescence microscope.

[0252] The PK-modifying anchor enabled unique diffusion and retention of highly lipophilic conjugates in mouse brain after intracerebroventricular injection (Figure 11A). Fixing larger PEG moieties to the hsiRNA-cholesterol conjugated compound improved penetration in the brain parenchyma. The PK-modifying anchor enabled unique diffusion and retention of highly lipophilic conjugates in mouse spinal cord after intrathecal administration (Figure 11B). As observed in brain tissue, fixing larger PEG moieties to the hsiRNA-cholesterol conjugated compound improved penetration in the spinal cord parenchyma.

[0253] 1.6 PK-modifying anchors dramatically improved blood / plasma circulation time and systemic in vivo biodistribution after subcutaneous injection The PK-modifying anchor broadened the area under the curve of unconjugated (Figure 28A) and cholesterol-conjugated (Figure 28B) hsiRNA after subcutaneous injection. The PK-modifying anchor significantly affected the biodistribution of unconjugated (red) and cholesterol-conjugated (black) hsiRNA after subcutaneous injection (Figure 29).

[0254] Example 2. Conjugated oligonucleotides containing anchors that modify pharmacokinetics (PK) 2.1 PK-modifying anchors The PK-modifying anchor was paired with different conjugated asymmetric siRNAs, as shown in Figure 31. Specifically, siRNAs with a 21-nucleotide antisense strand and a 13-nucleotide sense strand were conjugated with one of cholesterol, DCA, DHA, or GalNAc. Di-branched siRNA compounds were also tested, in which a linker connects two siRNAs at the 3' end of the sense strand. Each conjugated asymmetric siRNA was paired with a PK-modifying anchor containing a 40 kDa PEG moiety, in which all internucleotide linkages were phosphorothioate.

[0255] 2.2 PK-modifying anchors dynamically improved the blood / plasma circulation time of intravenously injected siRNA compounds The effect of PK-modifying anchors on the blood / plasma circulation time of conjugated siRNAs was tested, as shown in Figure 32. PK-modifying molecular anchors were measured to enhance the circulation time and area under the curve of unconjugated siRNA (Figure 32A), GalNAc-conjugated siRNA (Figure 32B), DHA-conjugated siRNA (Figure 32C), di-siRNA (Figure 32D), cholesterol-conjugated siRNA (Figure 32E), and DCA-conjugated siRNA (Figure 32F) after intravenous injection.

[0256] A 20 mg / kg tail vein injection was administered to female FVB / N mice (approximately 9-12 weeks of age). Antisense strands were quantified using a peptide nucleic acid (PNA) hybridization assay previously described in Godinho et al., 2017 (Nucleic Acids Therapeutics). Briefly, this assay uses a Cy3-labeled PNA probe that hybridizes to the antisense strand, followed by quantification by HPLC.

[0257] 2.3 PK-modifying anchors modulated the systemic in vivo biodistribution of intravenously administered siRNA compounds As shown in Figure 33, the effect of PK-modifying anchors on the biodistribution of the conjugated siRNA of Example 2.2 was tested. siRNA localization was tested in the pancreas, lung, heart, adrenal gland, spleen, kidney, muscle, and liver. As in Example 2.2, unconjugated siRNA (Figure 33A), GalNAc-conjugated siRNA (Figure 33B), DHA-conjugated siRNA (Figure 33C), di-siRNA (Figure 33D), cholesterol-conjugated siRNA (Figure 33E), and DCA-conjugated siRNA (Figure 33F) were also tested. The results show that PK-modifying anchors enhance the biodistribution of conjugated siRNA across multiple tissues. PK-modifying anchors also reduce the accumulation of conjugated siRNA in the kidney. Because the kidney is a clearance tissue, kidney avoidance may increase the serum half-life and biodistribution of conjugated siRNA.

[0258] These results also demonstrate the unexpected enhancement of liver delivery that the PK-modifying anchor confers on GalNAc-conjugated siRNA. As shown in Figure 33B, the amount of siRNA with a GalNAc-conjugated PK-modifying anchor is more than double that of siRNA without a GalNAc-conjugated PK-modifying anchor. GalNAc conjugates are known to promote liver delivery of siRNA, but the addition of a PK-modifying anchor promotes higher liver delivery than GalNAc conjugates and may be useful for enhancing the therapeutic efficacy of GalNAc-conjugated siRNA.

[0259] 2.4 PK-modifying anchors dynamically improved the blood / plasma circulation time of subcutaneously administered siRNA compounds As shown in Figure 34, the effect of PK-modifying anchors on the blood / plasma circulation time of unconjugated siRNA (Figure 34A) and di-siRNA (Figure 34B) after subcutaneous injection was measured.

[0260] Subcutaneous injections of 20 mg / kg were administered to female FVB / N mice (approximately 9-12 weeks of age). Antisense strands were quantified using a peptide nucleic acid (PNA) hybridization assay as previously described in Godinho et al., 2017 (Nucleic Acids Therapeutics).

[0261] 2.5 PK-modifying anchors modulated the systemic in vivo biodistribution of subcutaneously administered siRNA compounds As shown in Figure 35, the effect of PK-modifying anchors on the biodistribution of the siRNA of Example 2.4 was examined. The localization of the siRNA was examined in the pancreas, lung, heart, adrenal gland, spleen, kidney, muscle, and liver. As in Example 2.4, unconjugated siRNA (Figure 35A) and di-siRNA (Figure 35B) were also examined.

[0262] 2.6 PK-modifying anchors modulated the blood / plasma circulation time and systemic in vivo biodistribution of subcutaneously administered aptamer-siRNA chimeric compounds The effect of PK-modifying anchors on the blood / plasma circulation time (Fig. 37A) and biodistribution (Fig. 37B) of aptamer-siRNA chimeras was examined, as shown in Figures 36 and 37. An aptamer that binds to the EPCAM receptor was conjugated to the 3' end of the siRNA sense strand.

[0263] Subcutaneous injections of 20 mg / kg were administered to tumor-bearing Balb-c mice. The mice contained both 4T1E and P815 cell-derived tumors. Sense strands were quantified using a peptide nucleic acid (PNA) hybridization assay, as previously described in Godinho et al., 2017 (Nucleic Acids Therapeutics). Tissues were collected for the assay 48 hours after injection. In this assay, the EPCAM-binding aptamer-siRNA conjugate was internalized by 4T1E tumors, which express the EPCAM receptor, while P815 served as a negative control. As shown in Figures 37A and 37B, the PK-modifying anchor enhanced circulation time and improved delivery to target tumors by 2- to 4-fold compared to adaptor-siRNA conjugates without the PK-modifying anchor.

[0264] 2.7 PK-modifying anchors modulated the systemic in vivo biodistribution of intravenously and subcutaneously administered dibranched siRNA compounds As shown in Figure 38, the effect of PK-modifying anchors on the biodistribution of unconjugated siRNA and di-siRNA was measured compared to intravenous and subcutaneous injection. siRNA localization was examined in the liver (Figure 38A), spleen (Figure 38B), and kidney (Figure 38C). The results show that PK-modifying anchors enhanced delivery of unconjugated and di-siRNA parent asymmetric siRNA to the liver and other secondary distribution organs after SC and IV administration. PK-modifying anchors also reduced renal clearance of both siRNA scaffolds. Experiments were performed in triplicate and are shown with fluorescent tissue images for each of the three mice used in each condition.

[0265] 2.8 PK-modifying anchors regulated systemic in vivo biodistribution to the placenta As shown in Figure 39, the effect of PK-modifying anchors on the biodistribution of unconjugated siRNA into the mouse placenta was measured. Two separate 20 mg / kg subcutaneous injections were administered to pregnant female FVB / N mice (approximately 9-12 weeks old, 4 mice per group). Tissues were collected 48 hours after the latter injection. Antisense strands were quantified using a peptide nucleic acid (PNA) hybridization assay, as previously described in Godinho et al., 2017 (Nucleic Acids Therapeutics). Results showed that the PK-modifying anchor enhanced placental distribution by 10-fold compared to siRNA without the PK-modifying anchor.

[0266] 2.9 PK-modifying anchors enable gene silencing in the placenta As shown in Figure 40, the effect of PK-modifying anchors on target mRNA silencing in mouse placenta by unconjugated siRNA was measured. The experiment was performed as described in Example 2.9. The siRNA used targeted sFlt-1 mRNA. The relative level of sFlt-1 mRNA was measured using a branched DNA (bDNA) assay. The results show that target mRNA silencing using PK-modifying anchors was comparable to siRNA without PK-modifying anchors. To demonstrate that PK-modifying anchors do not cause acute systemic toxicity, the body weight profile (% body weight gain), blood chemistry, and complete blood counts of mice were measured. The results demonstrated that the body weight profile, blood chemistry, and complete blood counts of mice were comparable between saline-injected controls and mice injected with PK-modifying anchors. These results demonstrated that PK-modifying anchors do not cause acute systemic toxicity.

[0267] 2.10 PK-modifying anchors regulated the in vivo liver biodistribution of GalNAc-conjugated siRNA As shown in Figure 41, the effect of PK-modifying anchors on the liver biodistribution of GalNAc-conjugated siRNA was measured. The assay utilized two different types of asymmetric siRNA. The first was an siRNA with a 21-nucleotide antisense strand, a 13-nucleotide sense strand, and an 8-nucleotide anchor (designated 21-13-8). The second was an siRNA with a 25-nucleotide antisense strand, a 17-nucleotide sense strand, and an 8-nucleotide anchor (designated 25-17-8). The results demonstrated that PK-modifying anchors enhance liver distribution in both 21-13-8 siRNA format and 25-17-8 siRNA format, both in intravenous and subcutaneous delivery, compared with siRNA without PK-modifying anchors. This enhancement was particularly strong for hepatocytes in the liver. These results also demonstrate the unexpected enhancement of liver delivery that PK-modifying anchors confer on GalNAc-conjugated siRNA. As shown in Example 2.3 above, GalNAc conjugates are known to enhance liver delivery of siRNA, but the addition of a PK-modifying anchor promotes enhanced liver delivery beyond GalNAc conjugates and may be useful for enhancing the therapeutic efficacy of GalNAc-conjugated siRNA.

[0268] Example 3. Development of a conserved universal oligonucleotide anchor sequence The experiments described above in Examples 1 and 2 were performed using asymmetric siRNAs targeting sFlt-1 mRNA. The siRNAs were in a 21-13 (antisense strand length - sense strand length) format. Thus, there was an 8-nucleotide-long antisense tail to which an 8-nucleotide anchor sequence could be attached. The tail sequence of the asymmetric siRNA targeting sFlt-1 mRNA utilized a tail sequence rich in G / C nucleotides (G / C nucleotide content was 87.5%). siRNAs with sequences poor in G / C nucleotides were tested to determine whether the PK-modifying anchor could successfully bind to their target antisense strand tail sequence. The sFlt-1 siRNA sequences and the siRNA sequences targeting Htt mRNA are shown below: HTT10150: Antisense strand, 21-nucleotide: [ka] PK-modifying anchor, an 8-nucleotide with a 40 kDa PEG moiety: [ka] PK-modifying anchor, a 6-nucleotide with a 40 kDa PEG moiety: [ka] sFLT1-2283: Antisense strand, 21-nucleotide: [ka] Sense strand, 13-nucleotide: [ka] PK-modifying anchor, an 8-nucleotide with a 40 kDa PEG moiety: [ka]

[0269] In the above sequence, "V" corresponds to a 5'-vinylphosphonate moiety, "m" corresponds to a 2'-O-methyl modification, "f" corresponds to a 2'-fluoro modification, "#" corresponds to a phosphorothioate internucleotide linkage, "40k" corresponds to a 40kDa PEG moiety, and the bold / underlined portion of the antisense sequence corresponds to the 8-nucleotide tail to which the PK-modifying anchor is attached. As described above, the G / C content of the sFlt-1 antisense strand tail and the corresponding anchor is 87.5%, while the G / C content of the Htt antisense strand tail and the corresponding anchor is 25%. As shown in Figure 42, when used with a 6-nucleotide or 8-nucleotide anchor in Htt siRNA, there was no detectable shift of siRNA in the gel shift assay. This was true when the molar ratio of siRNA to anchor was 1:1, 1:2, and 1:4. The results indicate that tail sequences with poor G / C content are insufficient to achieve anchor binding to the tail sequence.

[0270] In an effort to develop conserved universal anchor and antisense tail sequences, we varied the length of the anchor and adjacent sense strand to measure the effect on anchor-to-tail binding. As shown in Figure 43, 21-13 siRNA was utilized with 8-nucleotide, 7-nucleotide, 6-nucleotide, and 5-nucleotide anchors. The 7-, 6-, and 5-nucleotide anchors each contain a gap (1-, 2-, and 3-nucleotide gaps, respectively) between the adjacent sense strand and anchor. When a gap remains between the sense strand and oligonucleotide anchor, binding efficiency decreases due to the loss of the positive effect of coaxial stacking. The 7-nucleotide anchor required a fourfold molar amount to achieve complete shift in gel electrophoresis assays. As shown in Figure 44, this decrease in binding efficiency could be alleviated by increasing the length of the sense strand and closing the gap between the sense strand and anchor. A 7-nucleotide anchor, a 6-nucleotide anchor, and a 5-nucleotide anchor were used with a 14-nucleotide sense strand, a 15-nucleotide sense strand, and a 16-nucleotide sense strand, respectively. Gel-shift assays demonstrated that the use of a 14-nucleotide sense strand and a 7-nucleotide anchor reduced the decrease in binding efficiency compared to the combination of a 13-nucleotide sense strand / 7-nucleotide anchor.

[0271] Several options were tested to design universal PK-modifying anchor sequences. In option 1, a 6-nucleotide universal sequence was incorporated into the antisense strand starting at nucleotide position 18 of the 23-nucleotide antisense strand. In a first example of option 1, a 17-nucleotide sense strand was used with a 6-nucleotide anchor sequence that was complementary to the 6-nucleotide universal sequence of the antisense strand. In a second example of option 1, a 15-nucleotide sense strand was used with an 8-nucleotide anchor sequence that was complementary to the 6-nucleotide universal sequence of the antisense strand and the other two nucleotides were complementary to the nucleotides at positions 16 and 17 of the antisense strand, which would vary depending on the target sequence selected for the antisense strand. In option 2, an 8-nucleotide universal sequence was incorporated into the antisense strand starting at nucleotide position 18 of the 25-nucleotide antisense strand. In option 2, a 17-nucleotide sense strand was used with an 8-nucleotide anchor sequence that was complementary to the 8-nucleotide universal sequence of the antisense strand (Figure 45).

[0272] The three alternative universal PK-modifying anchor sequence solutions described above were tested against an Htt mRNA target. The mRNA silencing efficacy of the target Htt mRNA was measured. A dose-response was performed in HeLa cells with a 72-hour incubation. bDNA was used for mRNA evaluation. Results were normalized to HPRT or PPIB. All 23-nucleotide-based antisense sequences functioned as perfect complementary controls, except for those with a conserved region located from nucleotide 16 (data points boxed "P3 Chol HTT 23-15 16nt (8 nts)" in Figure 46A). Furthermore, all 25-nucleotide-based antisense sequences functioned as perfect complementary controls. The results demonstrate that the use of a conserved universal sequence can be utilized for any siRNA, thus overcoming any issues associated with tail sequences with poor G / C content (Figures 46A and 46B).

[0273] In addition to the in vitro silencing experiments described above, the effect of anchors modifying PK with conserved sequences on biodistribution was measured. The assay utilized two different types of asymmetric siRNA. The first siRNA was an siRNA targeting sFlt-1 mRNA with a 21-nucleotide antisense strand, a 13-nucleotide sense strand, and an 8-nucleotide anchor (designated 21-17-8). The 25-17-8 siRNA utilized the above-described conserved universal tail sequence, starting at position 18 of the antisense strand. Each siRNA had a GalNAc conjugate for liver delivery. The results showed that anchors modifying PK with conserved universal tail sequences also enhanced liver distribution compared to siRNAs without conserved sequences for both intravenous and subcutaneous delivery (Figure 41).

Claims

1. a first oligonucleotide, wherein the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5' end, and a 3' end; a pharmacokinetic (PK)-modifying anchor comprising an anchor oligonucleotide, an optional linker, and at least one polymer, wherein the anchor oligonucleotide comprises or consists of about 5 to about 15 nucleotides that are complementary to the first oligonucleotide, and the polymer is at least about 2,000 Da; Contains, compounds.

2. 2. The compound of claim 1, wherein the anchor oligonucleotide is about 5 to about 15 nucleotides in length, or about 5 to about 10 nucleotides in length.

3. 10. The compound of claim 1, wherein the polymer is operably linked to the anchor oligonucleotide via an optional linker.

4. Formula (I): 【Chemistry 1】 [In the formula: O is a first oligonucleotide; L is a linker, present or absent; X ｃ is selected from the group consisting of a hydrophobic moiety, a sugar, a peptide, an aptamer, and a nucleic acid, and is present or absent; and Z is an anchor that modifies PK. The compound of claim 1,

5. The first oligonucleotide of claim 1, selected from the group consisting of an antisense oligonucleotide (ASO), a synthetic miRNA, a synthetic mRNA, a single-stranded siRNA, and a modified CRISPR guide strand.

6. The first oligonucleotide of claim 5 , wherein the ASO is a splice-switching ASO or an RNAase H ASO.

7. A first oligonucleotide according to any one of claims 1 to 6, which has complementarity to a target.

8. The first oligonucleotide according to any one of claims 1 to 6, which has perfect complementarity to the target.

9. 5. The compound of claim 4, wherein L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

10. X ｃ 5. The compound of claim 4, wherein is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, and endocannabinoids.

11. X ｃ The compound of claim 4, wherein comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof.

12. X ｃ The compound of claim 4, wherein said compound has affinity for one or both of low density lipoproteins and medium density lipoproteins.

13. X ｃ is a saturated or unsaturated moiety having less than three double bonds.

14. X ｃ The compound of claim 4, wherein said compound has an affinity for high density lipoproteins.

15. X ｃ 5. The compound of claim 4, wherein is a polyunsaturated moiety having three or more double bonds.

16. 16. The compound of any one of claims 1 to 15, wherein at least one polymer is selected from the group consisting of hydrophobic polycarbonates, polyesters, amphiphilic block copolymers, hydrophobic block polymers, polysaccharides, and polypeptides.

17. 17. The compound of claim 16, wherein the hydrophobic polycarbonate is poly(DTR carbonate).

18. 17. The compound of claim 16, wherein the polyester is selected from the group consisting of polyhydroxyalkanoates, polycaprolactones, poly(hydroxybuterates-hydroxyvalerates), polyglycolic acids, and polylactic acids.

19. 17. The compound of claim 16, wherein the amphiphilic block copolymer is selected from the group consisting of polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), acrylonitrile styrene acrylate, N-(2-hydroxypropyl) methacrylamide, and polyethylene glycol (PEG).

20. 17. The compound of claim 16, wherein the hydrophobic block copolymer is selected from the group consisting of poly(N,N-dimethylacrylamide), poly(N,N-diethylaniline), poly(diphenylamino), and poly(tetrahydrofurfuryl methacrylate).

21. 17. The compound of claim 16, wherein the polysaccharide is selected from the group consisting of soluble polyglucose, insoluble polyglucose, cellulose, glycogen, and amylopectin.

22. 17. The compound of claim 16, wherein the polypeptide is polylysine, polyarginine, polyalanine, polyisoleucine, polymethionine, polyphenylalanine, polyvaline, polyproline, and polyglycine, and any combination thereof.

23. 20. The compound of claim 19, wherein the PEG has a molecular weight selected from the group consisting of about 10,000 Da, about 20,000 Da, about 40,000 Da, about 60,000 Da, about 80,000 Da, and about 100,000 Da.

24. The compound of any one of claims 1 to 23, wherein the PK-modifying anchor comprises two or more polymers.

25. 25. The compound of claim 24, wherein the PK-modifying anchor comprises two, three, or four polymers.

26. 26. The compound of any one of claims 1 to 25, wherein the first oligonucleotide comprises 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides and the polymer is PEG.

27. The compound of any one of claims 1 to 26, wherein the first oligonucleotide comprises one or more chemically modified nucleotides.

28. 28. The compound of claim 27, wherein the first oligonucleotide is fully chemically modified or partially chemically modified.

29. 28. The compound of claim 27, wherein the first oligonucleotide comprises one or more locked nucleic acids (LNA) or one or more peptide nucleic acids (PNA).

30. 28. The compound of claim 27, wherein the first oligonucleotide comprises one or more S-constrained-ethyl (cET).

31. 28. The compound of claim 27, wherein the first oligonucleotide comprises about 50% 2'-methoxyribonucleotides, about 55% 2'-methoxyribonucleotides, about 60% 2'-methoxyribonucleotides, about 65% 2'-methoxyribonucleotides, about 70% 2'-methoxyribonucleotides, about 75% 2'-methoxyribonucleotides, about 80% 2'-methoxyribonucleotides, about 85% 2'-methoxyribonucleotides, about 90% 2'-methoxyribonucleotides, about 95% 2'-methoxyribonucleotides, about 96% 2'-methoxyribonucleotides, about 97% 2'-methoxyribonucleotides, about 98% 2'-methoxyribonucleotides, about 99% 2'-methoxyribonucleotides, or about 100% 2'-methoxyribonucleotides.

32. The compound of any one of claims 1 to 31, wherein the first oligonucleotide comprises alternating 2'-methoxyribonucleotides and 2'-fluoro-ribonucleotides.

33. 33. The compound of any one of claims 1 to 32, wherein the nucleotides of the first oligonucleotide are joined via phosphodiester linkages, phosphorothioate linkages, or a combination of phosphodiester and phosphorothioate linkages.

34. (1) the first oligonucleotide comprises alternating 2'-methoxyribonucleotides and 2'-fluoro-ribonucleotides, wherein each nucleotide is a 2'-methoxyribonucleotide or a 2'-fluoro-ribonucleotide, and the nucleotides at positions 2 and 14 from the 5' end of the first oligonucleotide are not 2'-methoxyribonucleotides; and (2) The compound of any one of claims 1 to 33, wherein a nucleotide of the first oligonucleotide is connected to an adjacent nucleotide via a phosphodiester or phosphorothioate bond, and wherein the nucleotide at positions 1-6 from its 3' end or at positions 1-7 from its 3' end is connected to an adjacent nucleotide via a phosphorothioate bond.

35. 35. The compound of any one of claims 1 to 34, further comprising a second oligonucleotide comprising at least 12 contiguous nucleotides, a 5' end, and a 3' end; wherein a portion of the first oligonucleotide is complementary to a portion of the second oligonucleotide.

36. 36. The compound of claim 35, wherein the second oligonucleotide comprises Xc of the conjugate moiety.

37. The second oligonucleotide is linked to X using the linker L. ｃ 36. The compound of claim 35, wherein the compound is bound to:

38. 36. The compound of claim 35, wherein L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

39. X ｃ is attached at the 5' end, the 3' end, an internal position, or a mixture thereof, of the second oligonucleotide.

40. X ｃ is attached at the 3' end of the second oligonucleotide.

41. X ｃ 36. The compound of claim 35, wherein is selected from the group consisting of a fatty acid, a steroid, a secosteroid, a lipid, a ganglioside, a nucleoside analog, and an endocannabinoid.

42. X ｃ 36. The compound of claim 35, wherein comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof.

43. X ｃ 36. The compound of claim 35, wherein said compound has affinity for one or both of low density lipoprotein and medium density lipoprotein.

44. X ｃ is a saturated or unsaturated moiety having less than three double bonds.

45. X ｃ 36. The compound of claim 35, wherein said compound has an affinity for high density lipoproteins.

46. X ｃ is a polysaturated moiety having three or more double bonds.

47. 47. The compound of any one of claims 1 to 46, wherein the anchor oligonucleotide is perfectly complementary to the first oligonucleotide.

48. 47. The compound of any one of claims 1 to 46, wherein the anchor oligonucleotide contains 1, 2, 3 or 4 mismatches relative to the first oligonucleotide.

49. 49. The compound of any one of claims 1 to 48, wherein the anchor oligonucleotide comprises one or more chemically modified nucleotides.

50. 50. The compound of claim 49, wherein the anchor oligonucleotide is fully chemically modified or partially chemically modified.

51. 50. The compound of claim 49, wherein the anchor oligonucleotide comprises a locked nucleic acid (LNA) or one or more peptide nucleic acids (PNAs).

52. 50. The compound of claim 49, wherein the anchor oligonucleotide comprises one or more S-constrained ethyls (cETs).

53. 53. The compound of any one of claims 1 to 52, wherein the anchor oligonucleotide comprises alternating 2'-O-methyl ribonucleotides and 2'-fluoro-ribonucleotides.

54. 53. The compound of any one of claims 1 to 52, wherein the anchor oligonucleotide comprises alternating 2'-O-methyl ribonucleotides and 2'-fluoro ribonucleotides and contains at least two adjacent phosphorothioate internucleotide linkages at the 5' and 3' ends.

55. 53. The compound of any one of claims 1 to 52, wherein the anchor oligonucleotide comprises alternating 2'-O-methyl ribonucleotides and 2'-fluoro ribonucleotides and contains phosphorothioate internucleotide linkages at every nucleotide position.

56. 54. The compound of any one of claims 1 to 53, wherein the anchor oligonucleotide comprises at least two adjacent 2',4'-constrained 2'O-ethyl bridged nucleic acids at the 5' and 3' ends.

57. 57. The compound of any one of claims 1 to 56, wherein the anchor oligonucleotide comprises a 2',4'-constrained 2'O-ethyl bridged nucleic acid at every nucleotide position with a phosphorothioate internucleotide linkage between each adjacent nucleotide.

58. 58. The compound of any one of claims 1 to 57, wherein the anchor oligonucleotide comprises at least two 2',4'-tethered 2'O-ethyl bridged nucleic acids at the 5' and 3' ends, alternating 2'-O-methyl ribonucleotides and 2'-fluoro ribonucleotides.

59. the anchor oligonucleotide comprises a peptide nucleic acid at every nucleotide position; 59. The compound of any one of claims 1 to 58.

60. 60. The compound of any one of claims 1 to 59, wherein the anchor oligonucleotide is attached to the polymer at the 5' end, the 3' end, an internal position, or a mixture thereof.

61. 61. The compound of any one of claims 1 to 60, wherein at least two oligonucleotides are crosslinked.

62. 62. The compound of any one of claims 1 to 61, further comprising a nanoparticle, an intercalating agent, a polycation, or a mixture thereof.

63. a) the length of the first oligonucleotide is between 21 and 25 nucleotides; b) the second oligonucleotide is between 13 and 17 nucleotides in length; and c) the anchor oligonucleotide is between 5 and 8 nucleotides in length; The compound according to any one of claims 35 to 62.

64. a) the first oligonucleotide is 21 nucleotides in length; b) the second oligonucleotide is 13 nucleotides in length; and c) the anchor oligonucleotide is 8 nucleotides in length; The compound according to any one of claims 35 to 62.

65. a) the first oligonucleotide is 23 nucleotides in length; b) the second oligonucleotide is 15 nucleotides in length; and c) the anchor oligonucleotide is 8 nucleotides in length; The compound according to any one of claims 35 to 62.

66. a) the first oligonucleotide is 25 nucleotides in length; b) the second oligonucleotide is 17 nucleotides in length; and c) the anchor oligonucleotide is 8 nucleotides in length; The compound according to any one of claims 35 to 62.

67. 67. The compound of any one of claims 63 to 66, wherein the polymer comprises PEG.

68. 67. The compound of any one of claims 1-66, wherein the PK-modifying anchor affects the stability of the oligonucleotide therapeutic over time in a part of the body including the heart, kidney, liver, spleen, adrenal gland, pancreas, lung, blood, plasma, brain, or a mixture thereof, wherein the effect comprises a change in volume of distribution, area under the curve, clearance, half-life maximum concentration, bioavailability, or a mixture thereof.

69. 69. The compound of any one of claims 35 to 68, wherein the number of nucleotides in the first oligonucleotide comprises the same number of nucleotides in the second oligonucleotide and the anchor oligonucleotide combined.

70. 69. The compound of any one of claims 35 to 68, wherein the number of nucleotides in the first oligonucleotide comprises a greater number of nucleotides than in the second oligonucleotide and the anchor oligonucleotide combined.

71. 69. The compound of any one of claims 35 to 68, wherein the number of nucleotides in the first oligonucleotide comprises fewer nucleotides than in the second oligonucleotide and anchor oligonucleotide combined.

72. a first oligonucleotide, the length of which is 21 nucleotides from the 5' end to the 3' end, and which is complementary to the target; a second oligonucleotide, the second oligonucleotide having a length of 13 nucleotides from the 5' end to the 3' end, and complementary to nucleotides 1-13 of the first oligonucleotide; and an anchor oligonucleotide (wherein the anchor oligonucleotide has a length of 8 nucleotides including the 5' and 3' ends, and is complementary to nucleotides 14-21 of the first oligonucleotide); wherein the second oligonucleotide is linked at its 3' end to a molecule comprising cholesterol, dichloroacetate, docosahexaenoic acid, or N-acetylgalactosamine; wherein the anchor oligonucleotide is attached to a polyethylene glycol polymer comprising a molecular weight between 10,000 and 40,000 daltons; wherein the first oligonucleotide contains complementarity to both the second oligonucleotide and the anchor oligonucleotide to form an asymmetric duplex; compound.

73. 74. The compound of any one of claims 1 to 73, further comprising a pharmaceutically active carrier.

74. 75. A pharmaceutical composition comprising a compound according to any one of claims 1 to 74 and a pharmaceutically acceptable carrier.

75. 76. A method for treating a disease or disorder in a patient in need thereof, comprising administering to said patient a compound according to any one of claims 1 to 75.

76. A method for treating a patient with a disease or disorder, comprising administering an asymmetric oligonucleotide duplex comprising a first oligonucleotide, a second oligonucleotide, and an anchor oligonucleotide, wherein the anchor oligonucleotide comprises a pharmacokinetic-modifying moiety, and the method may comprise pairing the first oligonucleotide with both the second oligonucleotide and the anchor oligonucleotide to form an asymmetric oligonucleotide duplex.

77. 1. A method of delivering a compound to the liver of a subject, comprising administering to the subject: a first oligonucleotide, wherein the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5' end, a 3' end, and comprises complementarity to the target; a second oligonucleotide comprising at least 12 consecutive nucleotides, a 5' end, and a 3' end, wherein a portion of the first oligonucleotide is complementary to a portion of the second oligonucleotide, and the second oligonucleotide comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof; a pharmacokinetic (PK)-modifying anchor comprising an anchor oligonucleotide, an optional linker, and at least one polymer, wherein the anchor oligonucleotide comprises about 5 to about 15 nucleotides complementary to the first oligonucleotide, and the polymer is at least about 2,000 Da; A method comprising administering a compound comprising:

78. An asymmetric duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand, each of which comprises at least one chemically modified nucleotide, wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety, the second oligonucleotide strand and the anchor oligonucleotide strand each comprise fewer nucleotides than the first oligonucleotide strand, and the first oligonucleotide strand is formed by pairing with both the second oligonucleotide strand and the anchor oligonucleotide strand.

79. 80. The asymmetric duplex of claim 79, wherein the first oligonucleotide strand comprises 10 to 50 nucleotides, the second oligonucleotide strand comprises 10 to 50 nucleotides, and the anchor oligonucleotide comprises 5 to 15 nucleotides.

80. 80. The asymmetric duplex of claim 79, wherein the first oligonucleotide strand and the second oligonucleotide strand form an siRNA or dsRNA having a double-stranded region.

81. 80. The asymmetric duplex of claim 79, wherein the duplex region is from about 10 to about 50 base pairs in length.

82. 80. The asymmetric duplex of claim 79, wherein the length of the duplex region is about 10 to about 15, about 15 to about 20, about 10 to about 25, or about 10 to about 30 base pairs.

83. 80. The asymmetric duplex of claim 79, wherein the duplex region is 13, 14, 15, 16, 17, or 18 base pairs.

84. 80. The asymmetric duplex of claim 79, wherein the first oligonucleotide strand is at least 16 nucleotides in length, the second oligonucleotide strand is at least 11 nucleotides in length, and the anchor oligonucleotide is about 5-15 nucleotides in length.

85. 80. The asymmetric duplex of claim 79, wherein the first oligonucleotide strand is about 21-23 nucleotides in length, the second oligonucleotide strand is about 13-16 nucleotides in length, and the anchor oligonucleotide is about 5-10 nucleotides in length.

86. 80. The asymmetric duplex of claim 79, wherein the first oligonucleotide strand is 21 nucleotides in length, the second oligonucleotide strand is 13 nucleotides in length, and the anchor oligonucleotide is 8 nucleotides in length.

87. 80. The asymmetric duplex of claim 79, wherein the at least one chemically modified nucleotide comprises a 2'-O-methyl-ribonucleotide, a 2'-fluoro-ribonucleotide, a phosphorothioate internucleotide linkage, a locked nucleic acid, a 2',4'-constrained 2'O-ethyl bridged nucleic acid, a peptide nucleic acid, or a mixture thereof.

88. 80. The asymmetric duplex of claim 79, wherein the second oligonucleotide strand comprises a ligand attached at the 5' end, at the 3' end, at an internal position, or a mixture thereof.

89. 90. The asymmetric duplex of claim 89, wherein the ligand of the second strand comprises a lipid, a lipophile, a terpene, a sugar, a peptide, a protein, an alkyl chain, a lectin, a glycoprotein, a hormone, a drug, a carbohydrate, an antibody, an aptamer, a vitamin, a cationic dye, a bioactive conjugate, a porphyrin, a polycyclic aromatic hydrocarbon, a synthetic polymer, or a mixture thereof.

90. 90. The asymmetric duplex of claim 89, wherein the ligand of the second strand comprises a fatty acid, a steroid, a secosteroid, a polyamine, a ganglioside, a nucleoside analog, an endocannabinoid, an omega-3 fatty acid, an omega-6 fatty acid, an omega-9 fatty acid, conjugated linoleic acid, a saturated fatty acid, or a mixture thereof.

91. 90. The asymmetric duplex of claim 89, wherein the ligand of the second strand comprises cholesterol, docosahexaenoic acid, conjugated phosphatidylcholine, N-acetylgalactosamine, dichloroacetic acid, an epithelial cell adhesion molecule aptamer, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, a geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleolyl)lithocholic acid, O3-(oleolyl)cholenoic acid, dimethoxytrityl, phenoxazine, or a mixture thereof.

92. 90. The asymmetric duplex of claim 89, wherein the second strand further comprises a linker that attaches the ligand to the second strand.

93. 1. An asymmetric duplex comprising a first oligonucleotide strand 21 nucleotides in length, a second oligonucleotide strand 13 nucleotides in length, and an anchor oligonucleotide strand 8 nucleotides in length, wherein each oligonucleotide strand comprises alternating 2'-fluoro-ribonucleotides and 2'-methoxyribonucleotides; the first strand comprises two adjacent phosphorothioate internucleotide linkages at the 5'-terminus and eight adjacent phosphorothioate internucleotide linkages at the 3'-terminus; the second oligonucleotide strand comprises two adjacent phosphorothioate internucleotide linkages at the 5'-terminus and two adjacent phosphorothioate internucleotide linkages at the 3'-terminus, and a linker attached at the 3'-terminus to a molecule comprising cholesterol, dichloroacetate, docosahexaenoic acid, or N-acetylgalactosamine; the anchor oligonucleotide strand comprises a linker at its 5'-end having seven adjacent phosphorothioate internucleotide linkages and at its 3'-end attached to a pharmacokinetic-modifying moiety comprising a polyethylene glycol polymer having a molecular weight between 10,000 and 40,000 daltons; and the first oligonucleotide strand pairs with both the second oligonucleotide strand and the anchor oligonucleotide strand to form an asymmetric duplex; Asymmetric duplexes can be formed.

94. 1. A method for treating a patient with a disease or disorder, comprising administering an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand, wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety, and the first oligonucleotide strand is capable of pairing with both the second oligonucleotide strand and the anchor oligonucleotide strand to form an asymmetric oligonucleotide duplex.

95. An asymmetric hairpin duplex comprising a hairpin oligonucleotide strand and an anchor oligonucleotide strand, wherein the hairpin oligonucleotide strand comprises an overhang and is capable of pairing with the anchor oligonucleotide strand to form an asymmetric hairpin duplex, and the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety.

96. A pharmaceutical composition comprising an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand, wherein the anchor oligonucleotide strand comprises a moiety that modifies pharmacokinetics, and the first oligonucleotide strand is capable of pairing with both the second oligonucleotide strand and the anchor oligonucleotide strand to form an asymmetric oligonucleotide duplex.

97. A universal anchor oligonucleotide having a length of about 5 to 8 nucleotides and comprising a pharmacokinetic-modifying moiety at the 5'-end, wherein at least one nucleotide comprises a chemical modification, wherein the anchor oligonucleotide is capable of binding to an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand and a second oligonucleotide strand, and wherein the sequence of the oligonucleotide anchor is complementary to a region at the 3'-end of the first oligonucleotide strand.

98. 99. The universal anchor oligonucleotide of claim 98, wherein the first strand and the anchor strand comprise from about 35 to about 100% GC content.

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