Anchors that modify dynamic pharmacokinetics

Abstract

Therapeutic oligonucleotides are provided that include pharmacokinetic (PK)-modifying anchors. Methods for treating a disease or disorder are provided that include administering to a subject therapeutic oligonucleotides that include one or more PK-modifying anchors.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 794,123, filed on January 18, 2019, and incorporates by reference in its entirety the disclosure thereof herein.

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

[0003] This disclosure relates to compounds that include an anchor for modifying pharmacokinetics (PK) (a (PK)-modifying anchor) based on novel oligonucleotides and having useful applications for RNA interference (RNAi) and other gene therapy techniques. The PK - modifying anchors described herein are patterned to enable efficient regulation of the absorption, distribution, and clearance kinetics of therapeutic oligonucleotides and enhance their tissue distribution. The efficient adjustment of their absorption, distribution, and clearance kinetics can be achieved in blood / plasma, cerebrospinal fluid (CSF), and in other relevant body fluids / biological fluids and tissues.

Background Art

[0004] Oligonucleotides are removed extremely rapidly after cerebrospinal fluid (CSF) injection, with less than 1 - 2% of the injected dose remaining in the brain and spinal cord. One well - understood problem in the use of oligonucleotide therapeutics for central nervous system applications is rapid CSF clearance. In rodents, bolus injection is sufficient to support extensive oligonucleotide distribution in the brain, and bulk CSF flow is the major mechanism underlying distribution. Rapid CSF clearance limits the 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 removed systemically, either through filtration in the kidney or by clearance via the reticuloendothelial system. Retention in secondary tissues other than the liver, kidney, bone marrow, and spleen is the real challenge in the art.

[0006] The need remains 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 finding of a novel platform, a compound comprising an anchor that modifies the dynamic pharmacokinetics ("PK"), where the anchor enables efficient modulation of the absorption, distribution, and clearance kinetics of the component therapeutic oligonucleotides in blood / plasma, CSF, and other body fluids / biological fluids and tissues. A group of block copolymers (e.g., poloxamer 188, etc.) that function as non-immunogenic PK modifiers and are compatible with oligonucleotide compounds are provided herein.

[0008] In one aspect, the present disclosure provides a compound comprising: a first oligonucleotide (where the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5' end, and a 3' end); and an anchor that modifies the pharmacokinetics (PK) comprising an anchor oligonucleotide, any linker, and at least one polymer (where the anchor oligonucleotide comprises or consists of from 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 from about 5 to about 15 nucleotides in length or from 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 a particular embodiment, the compound is of formula (I): [ka] [In formula: O is the first axial cuticle; L is a linker, and is either present or absent; X c These are selected from the group consisting of hydrophobic moieties, sugars, peptides, aptamers, and nucleic acids, and are either present or absent; Z is an anchor that modifies PK. This includes those indicated by [the symbol].

[0012] In certain embodiments, the first oligonucleotide is selected from the group consisting of antisense oligonucleotides (ASOs), synthetic miRNAs, synthetic mRNAs, single-stranded siRNAs, and modified CRISPR guide strands.

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

[0014] In certain embodiments, the first oligonucleotide includes complementarity with the target.

[0015] In certain embodiments, the first oligonucleotide includes complete complementarity with the target.

[0016] In certain embodiments, L includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphodiesters, phosphorothioates, phosphoramidates, amides, carbamates, or combinations thereof.

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

[0018] In certain embodiments, 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, polycaprolactone, 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, the polysaccharide is selected from the group consisting of soluble polyglucose, insoluble polyglucose, cellulose, glycogen, and amylopectin. In certain embodiments, the polypeptide is polylysine, polyarginine, polyalanine, polyisoleucine, polymethionine, polyphenylalanine, polyvaline, polyproline, and polyglycine, as well as any combination thereof. In certain embodiments, 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.

[0019] In certain embodiments, the anchor modifying the PK contains one or more polymers. In certain embodiments, the anchor modifying the PK contains two, three, or four polymers.

[0020] In a particular embodiment, 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 chemically fully modified or chemically partially modified. In certain embodiments, the first oligonucleotide comprises one or more locked nucleic acids (LNAs) or one or more peptide nucleic acids (PNAs). In certain embodiments, the first oligonucleotide comprises one or more S-restricted ethyl (cET) molecules. In a particular embodiment, the first oligonucleotide comprises approximately 50% 2'-methoxy-ribonucleotide, approximately 55% 2'-methoxy-ribonucleotide, approximately 60% 2'-methoxy-ribonucleotide, approximately 65% ​​2'-methoxy-ribonucleotide, approximately 70% 2'-methoxy-ribonucleotide, approximately 75% 2'-methoxy-ribonucleotide, approximately 80% 2'-methoxy-ribonucleotide, approximately 85% 2'-methoxy-ribonucleotide, approximately 90% 2'-methoxy-ribonucleotide, approximately 95% 2'-methoxy-ribonucleotide, approximately 96% 2'-methoxy-ribonucleotide, approximately 97% 2'-methoxy-ribonucleotide, approximately 98% 2'-methoxy-ribonucleotide, approximately 99% 2'-methoxy-ribonucleotide, or approximately 100% 2'-methoxy-ribonucleotide. 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 linked 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, where each nucleotide is either 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 linkages, where the nucleotides at positions 1-6 from the 3' end, or positions 1-7 from the 3' end, are connected to adjacent nucleotides via phosphorothioate linkages.

[0023] In certain embodiments, the compound comprises a second oligonucleotide comprising at least 12 consecutive nucleotides, a 5' end, and a 3' end; where 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 is attached to X c using a linker, L. 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. In certain embodiments, X c is attached at the 5' end, the 3' end, an internal position, or a mixed position of the second oligonucleotide. In certain embodiments, X c is attached at the 3' end of the second oligonucleotide. In certain embodiments, X c is selected from the group consisting of a fatty acid, a steroid, a secosteroid, a lipid, a ganglioside, a nucleoside analog, and an endocannabinoid. In certain embodiments, X c comprises an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof. In certain embodiments, Xc X has affinity for one or both low-density lipoproteins and medium-density lipoproteins. In certain embodiments, X c X is a saturated or unsaturated moiety having fewer than three double bonds. In certain embodiments, X c X has affinity for high-density lipoproteins. In a particular embodiment, X c This 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 one, two, three, or four 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 (LNAs) or one or more peptide nucleic acids (PNAs). In certain embodiments, the anchor oligonucleotide comprises one or more S-restricted ethyl (cET) molecules. In certain embodiments, the anchor oligonucleotide comprises alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides. In certain embodiments, the nucleotides of the anchor oligonucleotide consist of alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, and include at least two adjacent phosphorothioate nucleotide interlinks at the 5' and 3' ends. In certain embodiments, the nucleotides of the anchor oligonucleotide consist of alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, and include phosphorothioate nucleotide interlinks at all nucleotide positions. In certain embodiments, the anchor oligonucleotide comprises nucleic acids with at least two adjacent 2',4'-constrained 2'O-ethyl crosslinks at the 5' and 3' ends. In certain embodiments, the anchor oligonucleotide comprises 2',4'-constrained 2'O-ethyl crosslinked nucleic acids at all nucleotide positions, with phosphorothioate internucleotide bonds between each adjacent nucleotide. In certain embodiments, the anchor oligonucleotide comprises alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, with at least two 2',4'-constrained 2'O-ethyl crosslinked nucleic acids at the 5' and 3' ends. In certain embodiments, the anchor oligonucleotide comprises peptide nucleic acids at all nucleotide positions.

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

[0028] In a particular embodiment, at least two oligonucleotides are crosslinked.

[0029] In certain embodiments, the compounds further include nanoparticles, inserts, polycations, or mixtures thereof.

[0030] In a particular embodiment, a) the first oligonucleotide has a length between 21 and 25 nucleotides; b) the second oligonucleotide has a length between 13 and 17 nucleotides; and c) the anchor oligonucleotide has a length between 5 and 8 nucleotides.

[0031] In a particular embodiment, a) the first oligonucleotide has a length of 21 nucleotides; b) the second oligonucleotide has a length of 13 nucleotides; and c) the anchor oligonucleotide has a length of 8 nucleotides.

[0032] In a particular embodiment, a) the first oligonucleotide has a length of 23 nucleotides; b) the second oligonucleotide has a length of 15 nucleotides; and c) the anchor oligonucleotide has a length of 8 nucleotides.

[0033] In a particular embodiment, a) the first oligonucleotide is 25 nucleotides long; b) the second oligonucleotide is 17 nucleotides long; and c) the anchor oligonucleotide is 8 nucleotides long.

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

[0035] In certain embodiments, PK-modifying anchors affect the stability of oligonucleotide therapeutic agents over time in parts of the body including the heart, kidneys, liver, spleen, adrenal glands, pancreas, lungs, blood, plasma, brain, or mixtures thereof, where the effect includes changes in the volume of distribution, area under the curve, clearance, maximum half-life concentration, bioavailability, or mixtures thereof.

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

[0037] In one embodiment, the Disclosure provides a compound comprising a first oligonucleotide (wherein the first oligonucleotide is 21 nucleotides in length, having a 5' end and a 3' end, and is complementary to a target); a second oligonucleotide (wherein the second oligonucleotide is 13 nucleotides in length, having a 5' end and 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, having a 5' end and 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, wherein the first oligonucleotide constitutes complementarity with both the second oligonucleotide and the anchor oligonucleotide, forming an asymmetric double helix.

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

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

[0040] In yet another embodiment, the present disclosure provides a method for treating a patient who is in need of treatment for a disease or disorder, the method comprising administering the above-mentioned compound to the patient.

[0041] In one embodiment, the present disclosure provides a method for treating a patient with a disease or disorder, comprising administering an asymmetric oligonucleotide double helix comprising a first oligonucleotide, a second oligonucleotide, and an anchor oligonucleotide, wherein the anchor oligonucleotide comprises a pharmacokinetic modifying portion, and wherein the first oligonucleotide can pair with both the second oligonucleotide and the anchor oligonucleotide to form an asymmetric oligonucleotide double helix.

[0042] In one embodiment, the Disclosure provides a method for delivering a compound to the liver of a subject, comprising administering to the subject a compound comprising: a first oligonucleotide (wherein the first oligonucleotide comprises at least 16 consecutive nucleotides, a 5' end, a 3' end, and is complementary to the target); a second oligonucleotide (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 (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).

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

[0044] In a particular embodiment, the first oligonucleotide chain comprises 10-50 nucleotides, the second oligonucleotide chain comprises 10-50 nucleotides, and the anchor oligonucleotide comprises 5-15 nucleotides.

[0045] In certain embodiments, the first oligonucleotide chain and the second oligonucleotide chain form an siRNA or dsRNA having a double-stranded region. In certain embodiments, the double-stranded region is about 10 to about 50 base pairs long. In certain embodiments, the 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 long. In certain embodiments, the double-stranded region is 13, 14, 15, 16, 17, or 18 base pairs long.

[0046] In certain embodiments, the first oligonucleotide chain has a length of at least 16 nucleotides, the second oligonucleotide chain has a length of at least 11 nucleotides, and the anchor oligonucleotide has a length of about 5–15 nucleotides. In certain embodiments, the first oligonucleotide chain has a length of about 21–23 nucleotides, the second oligonucleotide chain has a length of about 13–16 nucleotides, and the anchor oligonucleotide has a length of about 5–10 nucleotides. In certain embodiments, the first oligonucleotide chain has a length of 21 nucleotides, the second oligonucleotide chain has a length of 13 nucleotides, and the anchor oligonucleotide has a length of 8 nucleotides.

[0047] In certain embodiments, at least one chemically modified nucleotide includes 2'-O-methyl-ribonucleotide, 2'-fluoro-ribonucleotide, phosphorothioate nucleotide interlinking, locked nucleic acid, 2',4'-restricted 2'O-ethyl crosslinked nucleic acid, peptide nucleic acid, or a mixture thereof.

[0048] In certain embodiments, the second oligonucleotide chain includes a ligand bound at its 5' end, its 3' end, an internal position, or a combination thereof.

[0049] In certain embodiments, the second-chain ligands include lipids, lipophilic substances, terpenes, sugars, peptides, proteins, alkyl chains, lectins, glycoproteins, hormones, drugs, carbohydrates, antibodies, aptamers, vitamins, cationic dyes, bioactive conjugates, porphyrins, polycyclic aromatic hydrocarbons, synthetic polymers, or mixtures thereof. In certain embodiments, the second-chain ligands include fatty acids, steroids, secosteroids, polyamines, gangliosides, nucleoside analogs, endocannabinoids, omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, conjugated linolenic acid, saturated fatty acids, or mixtures thereof. In certain embodiments, the ligands of the second chain include cholesterol, docosahexaenoic acid, conjugated phosphatidylcholine, N-acetylgalactosamine, dichloroacetic acid, epithelial cell adhesion molecule aptamers, 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-(oleolyl)lithocholic acid, O3-(oleolyl)cholenic acid, dimethoxytrityl, phenoxazine, or mixtures thereof.

[0050] In certain embodiments, the second chain further includes a linker that binds a ligand to the second chain.

[0051] In one embodiment, the disclosure provides an asymmetric double helix comprising a first oligonucleotide of 21 nucleotides in length, a second oligonucleotide chain of 13 nucleotides in length, and an anchor oligonucleotide chain of 8 nucleotides in length, wherein each oligonucleotide chain alternately comprises 2'-fluororibonucleotides and 2'-methoxyribonucleotides, the first chain comprising two adjacent phosphorothioate nucleotide bonds at its 5' end and eight adjacent phosphorothioate nucleotide bonds at its 3' end, the second oligonucleotide chain comprising two adjacent phosphorothioate nucleotide bonds at its 5' end and two adjacent The present invention provides an asymmetric double helix, comprising a phosphorothioate nucleotide interbonding and a linker that binds at the 3' end to a molecule containing cholesterol, dichloroacetate, docosahexaenoic acid, or N-acetylgalactosamine, wherein the anchor oligonucleotide chain binds at the 5' end to seven adjacent phosphorothioate nucleotide interbonding and a linker that binds at the 3' end to a pharmacokinetic modifying portion containing a polyethylene glycol polymer with a molecular weight between 10,000 and 40,000 daltons, wherein the first oligonucleotide chain can pair with both the second oligonucleotide chain and the anchor oligonucleotide chain to form an asymmetric double helix.

[0052] In one embodiment, the present disclosure provides a method for treating a patient with a disease or disorder, comprising administering an asymmetric oligonucleotide duplex comprising a first oligonucleotide chain, a second oligonucleotide chain, and an anchor oligonucleotide chain, wherein the anchor oligonucleotide chain is a pharmacokinetic modifying portion, and the first oligonucleotide chain may pair with both the second oligonucleotide chain and the anchor oligonucleotide chain to form an asymmetric oligonucleotide duplex.

[0053] In one embodiment, the disclosure provides an asymmetric hairpin double helix comprising a hairpin oligonucleotide chain and an anchor oligonucleotide chain, wherein the hairpin oligonucleotide chain includes an overhang and can be paired with the anchor oligonucleotide chain to form an asymmetric hairpin double helix, and the anchor oligonucleotide chain includes a portion that modifies pharmacokinetics.

[0054] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an asymmetric oligonucleotide duplex, comprising a first oligonucleotide chain, a second oligonucleotide chain, and an anchor oligonucleotide chain, wherein the anchor oligonucleotide chain comprises a pharmacokinetic modifying portion, and the first oligonucleotide chain may pair with both the second oligonucleotide chain and the anchor oligonucleotide chain to form an asymmetric oligonucleotide duplex.

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

[0056] In certain embodiments, the first chain and anchor chain contain approximately 35% to approximately 100% GC content. [Brief explanation of the drawing]

[0057] The above and other features and advantages of the present invention will be more fully understood from the following detailed description of exemplary embodiments, which will be considered in conjunction with the attached drawings. The patent or application file will include at least one drawing drawn in color. A copy of the publication of this patent or patent application, along with the color drawing, will be provided by the relevant authority upon request and payment of the necessary fees.

[0058] [Figure 1] The chemical structure of an asymmetric siRNA according to a specific exemplary embodiment is schematically shown. In non-limiting examples, the hydrophobically modified siRNA (hsiRNA) shown herein consists of an asymmetric double helix formed by a 21-oligonucleotide (21-mer) antisense chain and a 13-mer sense chain containing a hydrophobic cholesterol moiety. The asymmetric hsiRNA further comprises a complementary oligonucleotide anchor (e.g., an octamer) to which a polymer that modifies the pharmacokinetics (PK) is attached. The complementary oligonucleotide anchor (e.g., an octamer) hybridizes with the complementary antisense chain. 2'-O-methyl is shown in black, 2'-fluoro is shown in gray, and phosphorothioate bonds are shown with red dashes.

[0059] [Figure 2] An exemplary configuration of an asymmetric siRNA with a complementary oligonucleotide-containing anchor is schematically shown. The antisense strand includes an overhang that can pair with the oligonucleotide anchor. In non-limiting examples, shown herein are a 21-mer antisense strand that can hybridize with the following: a 13-mer sense strand and an octamer oligonucleotide anchor; a 14-mer sense strand and a heptamer anchor; a 15-mer sense strand and a hexamer anchor; or a 16-mer sense strand and a pentamer anchor. In certain embodiments, the hybridized oligomer may contain one, two, three or more mismatches. See Figure 24.

[0060] [Figure 3]Schematic embodiments of the PK-modifying moiety, comprising hydrophilic polycarbonate, block copolymer (e.g., amphiphilic block copolymer, hydrophilic block copolymer, or poloxamer), polyethylene glycol, and polysaccharide (e.g., dextrin or chitosan), are schematically shown.

[0061] [Figure 4] Two asymmetric siRNA duplexes linked together according to certain exemplary embodiments further described herein are schematically shown. As shown, the PK-modifying portions are attached to each oligonucleotide anchor such that the siRNA construct contains two PK-modifying portions when the oligonucleotide anchors bind to the siRNA duplex. In this embodiment, the upper scaffold represents the use of dynamically PK-modifying anchors, while the lower scaffold consists of stably attached PK modifiers. Without intending to be limited by scientific theory, given the dynamic properties of the upper scaffold, this is expected to enable improved distribution and retention in vivo.

[0062] [Figure 5] A schematic diagram illustrates an exemplary configuration for attaching PK-modifying portions to oligonucleotides. The branching pattern allows for the attachment of multiple PK-modifying polymers to each oligonucleotide anchor. siRNA and polymers modifying one, two, three, or four PKs are shown here.

[0063] [Figure 6] A schematic representation of an asymmetric siRNA is shown, which is either unconjugated to a lipid or conjugated to one. Exemplary lipids include, but are not limited to, cholesterol, docosahexaenoic acid-conjugated phosphatidylcholine (PC-DHA), dichloroacetic acid (DCA), or epithelial cell adhesion molecule (EpCAM) aptamers.

[0064] [Figure 7] A schematic diagram of cholesterol-conjugated siRNA and its delivery system is shown. Suitable delivery systems include, but are not limited to, lipid nanoparticles, exosomes, and microvesicles.

[0065] [Figure 8A-8B] Figure 8B and Figure 8A show the blood / plasma circulation time and area under the curve for unconjugated (unconjugated) and cholesterol-conjugated (conjugated) hsiRNAs after intravenous injection. 20 mg / kg was administered via tail vein injection to female FVB / N mice (approximately 9-12 weeks old).

[0066] [Figures 9A-9G] This study demonstrates the effects of PK-modifying anchors on in vivo biodistribution. Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric double helix contained an antisense strand of a 21-mer oligonucleotide and a sense strand of a 13-mer oligonucleotide. A fully phosphorothioate-treated octameric oligonucleotide anchor was used. 20 mg / kg was administered by tail vein injection to female FVB / N mice (approximately 9-12 weeks old). After 48 hours, the antisense strand was quantified using a peptide nucleic acid hybridization assay. The biodistribution of hsiRNA 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) is shown.

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

[0068] [Figure 11A-11C]The effects of PK-modifying anchors on the in vivo biodistribution of hsiRNA compounds in the central nervous system after intracerebroventricular injection (Figures 11A and 11B) and intrathecal injection (Figure 11C) are shown. In Figure 11A, 4 nanomoles (or approximately 250 μg) of hsiRNA were injected into the lateral ventricle, resulting in a concentration of approximately 2 nanomoles / ventricle. In Figure 11B, 20 nanomoles of hsiRNA were 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 were injected intrathecally between L5 and L6. The distribution of hsiRNA in the mouse spine is shown in Figure 11C. 48 hours after injection, mouse brain and spinal tissue were collected and stained with DAPI (nucleus, blue). Brain and tissue were imaged using a Leica DMi8 fluorescence microscope.

[0069] [Figure 12] A hydrophobic polycarbonate polymer according to a specific exemplary embodiment is shown.

[0070] [Figure 13] A polyester polymer according to a specific exemplary embodiment is shown.

[0071] [Figure 14] This shows an amphiphilic block copolymer according to a specific exemplary embodiment.

[0072] [Figure 15] A hydrophilic block copolymer according to a specific exemplary embodiment is shown.

[0073] [Figure 16] This shows a polysaccharide polymer according to a specific exemplary embodiment.

[0074] [Figure 17] This shows the distribution within the kidney after IV administration.

[0075] [Figure 18]This shows the intrahepatic distribution after IV administration.

[0076] [Figure 19] This shows the distribution within the spleen after IV administration.

[0077] [Figure 20] This shows the distribution within the kidney after subcutaneous (SC) administration.

[0078] [Figure 21] This shows the intrahepatic distribution after SC administration.

[0079] [Figure 22] This shows the intrasplenic distribution after SC administration.

[0080] [Figure 23] This shows the distribution of SC in the skin after administration.

[0081] [Figure 24] For Tm optimization, a schematic representation of an exemplary configuration containing an asymmetric siRNA and a mismatched complementary oligonucleotide anchor is shown.

[0082] [Figure 25] A schematic representation of an exemplary asymmetric siRNA containing various chemical modifications is shown.

[0083] [Figure 26] A schematic diagram illustrates dynamic oligonucleotide anchors for use in the delivery of other classes of nucleotides, such as ASOs (e.g., those shown to be compatible with RNase H or splice switching), microRNAs, mRNAs, and CRISPR guide strands.

[0084] [Figure 27]A schematic diagram illustrates exemplary configurations of an asymmetric siRNA and an anchor containing a complementary oligonucleotide. The circles represent fixed sequences of multiple dynamic oligonucleotide anchors that may be used in siRNA constructs targeting various different mRNAs. In certain embodiments, the antisense strand increases in length up to 23 nucleotides in total. In certain embodiments, nucleotides from positions 18 to 23 do not hybridize with the mRNA target. In certain embodiments, fixed / conserved oligonucleotide anchor regions are provided that may 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, pentameric or decameral anchors are used.

[0085] [Figures 28A-28B]The graph shows that PK-modifying anchors dynamically improved the blood / plasma circulation time of the parent hsiRNA compound. After subcutaneous injection, PK-modifying anchors broadened the area under the curve for both unconjugated (Figure 28A) and cholesterol-conjugated (Figure 28B) hsiRNAs. PK-modifying anchors delayed the time to peak and slowed the clearance rate of the parent hsiRNA compound. 20 mg / kg was administered via tail vein injection to female FVB / N mice (approximately 9-12 weeks old). The antisense strand was quantified by a peptide nucleic acid (PNA) hybridization assay, as previously described by Godinho et al. (2017) in Nucleic Acids Therapeutics. Briefly, this assay uses a cy3-labeled PNA probe that hybridizes with the antisense strand, which is then quantified by HPLC. AUC was calculated using the model-independent trapezoidal method in GastroPlus and Simulations Plus. Polyethylene glycol (PEG) was used as the PK modifying moiety in the model, and a fully phosphorothioated octamer was used as the anchor in the model, with the circulation time of each parent as21-s13 compound being adjusted. Both the length and chemical properties of the PK modifying moiety and anchor may be adjusted according to the purpose / target of delivery.

[0086] [Figure 29]The graph shows that PK-modifying anchors regulated the systemic in vivo distribution of parental hsiRNA compounds. PK-modifying anchors significantly affected the in vivo distribution of unconjugated (red) and cholesterol-conjugated (black) hsiRNAs after subcutaneous injection. Thus, PK-modifying anchors improved the delivery of unconjugated oligonucleotides to most organs. 20 mg / kg tail vein injections were administered between the scapulae of female FVB / N mice (approximately 9-12 weeks old). Antisense strands were quantified after 48 hours by PNA hybridization assay. Polyethylene glycol (PEG) was used as the PK-modifying moiety in the model, and fully phosphorothioated octamers were used as the anchors in the model, to regulate the circulation time of individual parental as21-s13 compounds. Both the length and chemical properties of the PK-modifying moiety and anchors may be adjusted according to 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. Binding of the anchors to the asymmetric siRNA duplex was performed with and without cholesterol conjugates. The antisense strand of the asymmetric siRNA duplex was 21 nucleotides long, the sense strand was 13 nucleotides long, and the anchor complementary to the tail of the antisense strand was 8 nucleotides long. The asymmetric siRNA duplex was Cy3-labeled at the 5' end of the sense strand.

[0088] [Figure 31] This shows typical siRNA structures used to measure blood concentration profiles and tissue distribution profiles when administered systemically via intravenous and subcutaneous injection.

[0089] [Figures 32A-32F]Figure 31 shows the blood concentration profiles of the PK-modifying anchors that pair with the asymmetric siRNA double-stranded structures of the panel. Polyethylene glycol (PEG) was used as the polymer to modify the PK. The siRNA asymmetric double-stranded structures contained an antisense strand of a 21-mer oligonucleotide and a sense strand of a 13-mer oligonucleotide. Fully phosphorothioated octameric oligonucleotide anchors were used. 20 mg / kg was administered by tail vein injection to female FVB / N mice (approximately 9-12 weeks old). After 48 hours, the antisense strand was quantified using a peptide nucleic acid hybridization assay. Blood concentration levels of siRNA are shown for unconjugated siRNA (Figure 32A), GalNAc-conjugated siRNA (Figure 32B), DHA-conjugated siRNA (Figure 32C), branched siRNA (Figure 32D), cholesterol-conjugated siRNA (Figure 32E), and DCA-conjugated siRNA (Figure 32F).

[0090] [Figures 33A-33F] Figure 31 shows the tissue distribution profiles of the PK-modifying anchors that pair with the asymmetric siRNA double-stranded structures of the panel. Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric double-stranded structures contained an antisense strand of a 21-mer oligonucleotide and a sense strand of a 13-mer oligonucleotide. Fully phosphorothioated octameric oligonucleotide anchors were used. 20 mg / kg was administered by tail vein injection to female FVB / N mice (approximately 9-12 weeks old). After 48 hours, the antisense strand was quantified using a peptide nucleic acid hybridization assay. The blood concentration levels of siRNA are shown for unconjugated siRNA (Figure 33A), GalNAc-conjugated siRNA (Figure 33B), DHA-conjugated siRNA (Figure 33C), bifurcated siRNA (Figure 33D), cholesterol-conjugated siRNA (Figure 33E), and DCA-conjugated siRNA (Figure 33F).

[0091] [Figures 34A-34B]Figure 31 shows the blood concentration profiles of PK-modifying anchors that pair with unconjugated siRNA (Figure 34A) or bifurcated siRNA (Figure 34B). Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric double helix contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. Fully phosphorothioated octameric oligonucleotide anchors were used. 20 mg / kg was administered by tail vein injection to female FVB / N mice (approximately 9-12 weeks old). The antisense strand was quantified after 48 hours using a peptide nucleic acid hybridization assay.

[0092] [Figures 35A-35B] Figure 31 shows the tissue distribution profiles of PK-modifying anchors that pair with unconjugated siRNA (Figure 35A) or bifurcated siRNA (Figure 35B). Polyethylene glycol (PEG) was used as the PK-modifying polymer. The siRNA asymmetric double helix contained a 21-mer oligonucleotide antisense strand and a 13-mer oligonucleotide sense strand. Fully phosphorothioated octameric oligonucleotide anchors were used. Subcutaneous injection of 20 mg / kg was administered to female FVB / N mice (approximately 9-12 weeks old). The antisense strand was quantified after 48 hours using a peptide nucleic acid hybridization assay.

[0093] [Figure 36] The delivery scheme for aptamer-siRNA chimeras with PK-modifying anchors is described. Subcutaneous injection of 20 mg / kg was administered to Balb-c mice with tumors. These mice had 4T1E breast cancer cell line tumors and P815 mast cell tumors.

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

[0095] [Figures 38A-38C] This shows the tissue distribution profiles of PK-modifying anchors that pair with unconjugated or bifurcated siRNAs. The siRNAs were delivered via intravenous or subcutaneous administration. Polyethylene glycol (PEG) was used as the PK-modifying polymer. Figure 38A shows the liver distribution, Figure 38B shows the spleen distribution, and Figure 38C shows the kidney distribution.

[0096] [Figure 39] This shows the tissue distribution profile to the mouse placenta with unconjugated siRNA and PK-modifying anchors that pair with it. siRNA was delivered via subcutaneous administration. Two doses of 20 mg / kg were delivered, as shown 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]The efficacy of sFlT-1 mRNA silencing in selected tissues was demonstrated using a PK-modifying anchor paired with unconjugated siRNA. siRNA was delivered subcutaneously. Two doses of 20 mg / kg were delivered, as shown in the timeline. Pregnant female FVB / N mice (approximately 9-12 weeks old, 6-8 mice / group) were used. Branched DNA (bDNA) was used for mRNA quantification. Target mRNA and sFlt-1 levels were measured in placental, liver, and kidney tissues. Mouse body weight profiles were measured to demonstrate that the use of the PK-modifying anchor did not induce acute systemic toxicity. A panel of blood chemistry and complete blood count values ​​was also measured to demonstrate that the use of the PK-modifying anchor did not induce acute systemic toxicity.

[0098] [Figure 41] This report describes the delivery of GalNAc-conjugated siRNA to the liver via intravenous or subcutaneous administration. The distribution of siRNA-PK-modified anchors 21-13-8 and 25-17-8 was compared. The siRNA-PK-modifying anchors 25-17-8 contained a conserved sequence from nucleotide positions 18–25 of the antisense tail of the asymmetric siRNA, comprising 25 nucleotides. These eight nucleotide conserved sequences were complementary to the eight nucleotide anchors.

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

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

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

[0102] [Figure 45]The design of universal sequences for PK-modifying anchors is schematically shown. Option 1 manipulates a universal sequence of 6 nucleotides starting at nucleotide position 18 of a 23-nucleotide antisense strand 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, with the other 2 nucleotides (which will vary depending on the target sequence selected for the antisense strand) being complementary to the nucleotides at positions 16 and 17 of the antisense strand. Option 2 manipulates an 8-nucleotide universal sequence starting at nucleotide position 18 of a 25-nucleotide antisense strand 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] This study demonstrates the mRNA silencing effect of targeted Htt mRNA. Antisense sequences of 23 nucleotides (Figure 46A) and 25 nucleotides (Figure 46B) were used. Dose-response was performed by incubating HeLa cells for 72 hours. bDNA assays were used for mRNA evaluation. Results were normalized to HPRT or PPIB. [Modes for carrying out the invention]

[0104] This disclosure relates to therapeutic oligonucleotides (e.g., therapeutic siRNAs) comprising pharmacokinetic (PK) modifying anchors. The therapeutic oligonucleotides comprising PK modifying anchors provided herein efficiently modulate the absorption, distribution, and clearance kinetics in relevant body fluids / 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, kidneys, liver, spleen, adrenal glands, pancreas, lungs, blood (e.g., plasma), and brain tissue.

[0105] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In cases of potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless contextually required otherwise, singular forms include plurals, and plural forms include singulars. The use of “or” means “and / or” unless otherwise specified. The use of the word “containing,” as well as other terms such as “contains” and “contained,” is not restrictive. Where used herein, unless otherwise specified, singular “a,” “an,” and “the” include plural references. Thus, for example, “protein” includes multiple protein molecules.

[0106] In general, the nomenclature used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids is well known and commonly used in the art. The methods and techniques provided herein are generally carried out in accordance with conventional methods well known in the art and, unless otherwise noted, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out as commonly achieved in the art or as described herein, according to the manufacturer's specifications. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry described herein, as well as their laboratory operations and techniques, are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, preparation, formulation and delivery of pharmaceuticals, and patient treatment.

[0107] To make the disclosure easier to understand, we have selected and defined the following terms.

[0108] As used herein, the terms “pharmacokinetic modifier” or “PK modifier” refer 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, the PK modifier affects the stability of the therapeutic agent at one or more sites in a target (e.g., in the heart, kidneys, liver, spleen, adrenal gland, pancreas, lungs, blood (e.g., plasma), and / or brain tissue). The PK parameter to be modified is the volume of distribution (V d ), area under the curve (AUC), clearance (CL), half-life (t 1 / 2 ), maximum concentration (C max Examples include, but are not limited to, bioavailability (F) and bioavailability (F).

[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 may be complementary to the oligonucleotide, for example, 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 the oligonucleotide, for example, via hybridization of the oligonucleotide anchor, to an overhang of a double-stranded nucleic acid sequence or a portion of a single-stranded oligonucleotide. The polymer portion of Z may include a PK-modifying portion.

[0110] In certain embodiments, the polymers described herein (e.g., PK-modifying polymers) are attached directly to oligonucleotide anchors (e.g., without a separate linker).

[0111] In certain embodiments, the oligonucleotide anchor is linked to the polymer via a linker that provides a functional group that bonds the polymer to the oligonucleotide anchor. In certain embodiments, the linker may be, for example, an alkyl chain from about 1 carbon to about 25 carbons, or, for example, a well-defined propylene or ethylene glycol chain of about 1 to about 25 units. An exemplary linker is [ka] That is the case.

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

[0113] In certain embodiments, the target oligonucleotide includes 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 contains more than one polymer. In certain exemplary embodiments, Z contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polymers. In certain exemplary embodiments, Z contains 2, 3, 4, or more polymers.

[0115] In a particular exemplary embodiment, Z is a polymer portion whose molecular weight varies from about 2,000 Da to about 100,000 Da, encompassing all values ​​in between. In a particular exemplary embodiment, 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, encompassing all values ​​in between. In certain exemplary embodiments, the molecular weight of the polymer is approximately 10,000 Da, approximately 15,000 Da, approximately 20,000 Da, approximately 25,000 Da, approximately 30,000 Da, approximately 35,000 Da, approximately 40,000 Da, approximately 45,000 Da, approximately 50,000 Da, approximately 55,000 Da, approximately 60,000 Da, approximately 65,000 Da, approximately 70,000 Da, approximately 75,000 Da, approximately 80,000 Da, approximately 85,000 Da, approximately 90,000 Da, approximately 95,000 Da, or approximately 100,000 Da, including all values ​​in between. In certain exemplary embodiments, the molecular weight of the polymer is approximately 2,000 Da, approximately 4,500 Da, approximately 10,000 Da, approximately 20,000 Da, approximately 40,000 Da, or approximately 100,000 Da.

[0116] In certain exemplary embodiments, suitable polymers may include one or a combination of hydrophilic polycarbonates, polyethylene glycol (PEG), block copolymers (e.g., including amphiphilic or hydrophilic block copolymers), poloxamers, polysaccharides (e.g., including dextrin or chitosan), and poly(lactic acid-coglycolic acid) (PLGA). Exemplary embodiments of suitable PK-modifying portions are shown in Figure 3.

[0117] In certain exemplary embodiments, the PK-modified 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 a particular exemplary embodiment, the polymer used in Z is PEG, for example, 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-220, PE Includes one or any combination of G-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, etc.

[0119] In certain exemplary embodiments, the polymer used in Z includes poloxamers. 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 such as Synperonics (Croda Healthcare), Pluronics (BASF), and Kolliphor (BASF) are also suitable.

[0120] In a particular exemplary embodiment, the polymer used in Z includes, for example, hydrophobic polycarbonates such as tyrosine-induced polycarbonates (Figure 12).

[0121] In certain exemplary embodiments, the polymer used in Z includes, for example, polyesters such as polyhydroxyalkanoate (PHA), polycaprolactone (PCL), poly(hydroxybuterate-hydroxyvalerate), polyglycolic acid (PGA), and polylactic acid (PLA) (Figure 13).

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

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

[0124] In certain exemplary embodiments, the polymer used in Z comprises a polypeptide, for example, 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 point (Tm) of a nucleotide anchor is optimized to reduce the clearance rate of the associated oligonucleotide. In certain exemplary embodiments, the Tm of the anchor is between approximately 37°C and approximately 70°C, and includes all values ​​in between. In certain exemplary embodiments, the Tm is approximately 37°C, approximately 38°C, approximately 39°C, approximately 40°C, approximately 41°C, approximately 42°C, approximately 43°C, approximately 44°C, approximately 45°C, approximately 46°C, approximately 47°C, approximately 48°C, approximately 49°C, approximately 50°C, approximately 51°C, approximately 52°C, approximately 53°C, approximately 54°C, approximately 55°C, approximately 56°C, approximately 57°C, approximately 58°C, approximately 59°C, approximately 60°C, approximately 61°C, approximately 62°C, approximately 63°C, approximately 64°C, approximately 65°C, approximately 66°C, approximately 67°C, approximately 68°C, approximately 69°C, or approximately 70°C, and includes all values ​​in between. In a particular exemplary embodiment, Tm is between approximately 37°C and approximately 40°C, and includes all values ​​in that range. In a particular exemplary embodiment, Tm is between approximately 40°C and approximately 45°C, and includes all values ​​in that range. In a particular exemplary embodiment, Tm is between approximately 45°C and approximately 50°C, and includes all values ​​in that range. In a particular exemplary embodiment, Tm is between approximately 50°C and approximately 55°C, and includes all values ​​in that range. In a particular exemplary embodiment, Tm is between approximately 55°C and approximately 60°C, and includes all values ​​in that range.

[0126] In certain exemplary embodiments, a polymer modifying multiple PKs may be attached to single-stranded oligonucleotides, partially double-stranded oligonucleotides, or fully double-stranded nucleic acid double helixes. Exemplary embodiments are shown in Figure 5, which illustrates various configurations useful for attaching PK-modifying polymers to oligonucleotide anchors. In certain exemplary embodiments, the PK-modifying polymer may 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 the polymer modifying multiple PKs. Certain exemplary embodiments include one, two, or three PK-modifying polymers attached to the 3' end, 5' end, or both the 3' and 5' ends of a polynucleotide anchor.

[0127] In certain exemplary embodiments, Z modulates the delivery of branched oligonucleotides, in which two or more double-stranded oligonucleotides are linked together. In certain embodiments, a 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 may be selected from the group consisting of ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphodiesters, phosphorothioates, phosphoramidates, amides, carbamates, and any combination thereof. In certain embodiments, L is bonded 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 yet another specific embodiment, L is a divalent linker L2: [ka]

[0131] In yet another specific embodiment, L is [ka] A trivalent or divalent linker selected from the group consisting of the following.

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

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

[0134] In a specific exemplary embodiment, X c The substance is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, and endocannabinoids.

[0135] In a specific exemplary embodiment, X cThese are neuromodulatory lipids, such as endocannabinoids. Non-exclusive examples of endocannabinoids include, but are not limited to, anandamide, arachidonoylethanolamine, 2-arachidonylglyceryl ether (norazine ether), 2-arachidonylglycerol, and N-arachidonoyldopamine.

[0136] In a specific exemplary embodiment, X c These are omega-3 fatty acids. Non-exclusive examples of omega-3 fatty acids include, but are not limited to, hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), ceridic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA, thymnodonic acid), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA, clupanodonic acid), docosahexaenoic acid (DHA, ceruvianic acid), tetracosapentaenoic acid, and tetracosahexaenoic acid (herringic acid).

[0137] In another embodiment, X c These are omega-6 fatty acids. Non-exclusive 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), docosadenoic acid, adrenaline, docosapentaenoic acid (osbondic acid), tetracosatetraenoic acid, and tetracosapentaenoic acid.

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

[0139] In another embodiment, X cThis is a conjugated linoleic acid. Non-exclusive examples of conjugated linoleic acids include, but are not limited to, α-calendic acid, β-calendic acid, jacalic acid, α-eleostearic acid, β-eleostearic acid, catalpic acid, and punisic acid.

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

[0141] In another embodiment, X c This 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 X is selected from the group consisting of docosanoic acid (DCA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). In a particular embodiment, X c This is docosanic acid (DCA). In another specific embodiment, X c This is DHA. In another specific embodiment, X c It is EPA.

[0143] In another embodiment, X c It is a secosteroid. In a specific embodiment, X c This is calciferol. In another embodiment, X c It is a steroid other than cholesterol.

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

[0145] In yet another embodiment of the oligonucleotide, X c It is characterized by a cLogP value that falls within 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" means an oligonucleotide consisting of at least 16 consecutive nucleotides, wherein the oligonucleotide has complementarity with the 5' end, 3' end, and target. In one embodiment, the oligonucleotide has sufficient complementarity with the target to form a hybrid. In certain embodiments, the complementarity is >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50%. In one embodiment, the oligonucleotide has perfect complementarity with the target. In yet 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 yet another particular embodiment, the nucleotides at positions 1 and 2 from the 3' end of the oligonucleotide are linked 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 linked to adjacent nucleotides via phosphorothioate bonds. In yet another particular embodiment, the oligonucleotide comprises 2'-fluoro modifications at each of the nucleotides at positions 2 and 14 from the 5' end, and 2'-methoxy modifications at each other's nucleotide positions.

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

[0149] In one embodiment, O comprises an asymmetric double helix. The lengths of the sense and antisense strands of the asymmetric double helix may vary. In certain exemplary embodiments, the asymmetric double helix 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 double helix 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 double helix 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 double helix 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 may vary with respect to the length of the oligonucleotide sense strand. In some embodiments, the sense chain is a 13-mer, 14-mer, 15-mer, or 16-mer oligonucleotide, and the oligonucleotide anchor is an octa-mer, 7-mer, 6-mer, or 5-mer oligonucleotide (see Figure 2). In certain embodiments, the oligomer formed by the hybridization may contain one, two, three or more mismatches (see Figure 24).

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

[0151] In a particular embodiment, O is a therapeutic RNA, such as ASO, ssRNA, etc., and the oligonucleotide anchor is a 15-mer, 14-mer, 13-mer, 12-mer, 11-mer, 10-mer, 10-mer, 9-mer, 8-mer, 7-mer, 6-mer, or 5-mer oligonucleotide.

[0152] When used herein in relation to 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 terms “nucleotide analog,” “modified nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, including ribonucleotides or deoxynucleotides that do not exist in nature. Exemplary nucleotide analogs are modified at any position that alters the specific chemical properties of the nucleotide, but still retains the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and for adenosine and / or guanosine, the 8-position, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Nucleotide analogs also include deazanucleotides, e.g., 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 involve modifications to the sugar moiety 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. Patents 5,858,988 and 6,291,438.

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

[0156] When used herein in relation to 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 at the 3' carbon of its ribose ring.

[0158] As used herein, the term "5' end" refers to the end of a nucleic acid that contains a phosphate group bonded to the 5' carbon of its 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" means a nucleoside having a phosphate group at its 3' or 5' sugar hydroxyl group.

[0161] An RNAi agent, such as siRNA, having a strand that is "sufficiently complementary to the target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to induce RNAi-mediated disruption of the target mRNA.

[0162] As used herein, “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) means an RNA molecule that, when produced by recombinant techniques, substantially contains no other cellular material or culture, or, when chemically synthesized, substantially contains no chemical precursors or other compounds.

[0163] The term "discriminative 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 regulatory region of the target gene (e.g., a DNA sequence encoding a promoter or enhancer factor). In other embodiments, the target polynucleotide sequence is the target mRNA encoded by the target gene.

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

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

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

[0167] As used herein, the terms “overhang” or “tail” mean a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides located at the 3' end of one or both of the sense and antisense strands of an siRNA duplex that is single-stranded, i.e., does not form a duplex with the other strand.

[0168] As used herein, the terms “antisense oligonucleotide” or “ASO” mean 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) in order to effectively block a region of the target RNA, for example, in an effective manner to inhibit the translation of the target mRNA and / or the splicing of the target pre-mRNA. An antisense oligonucleotide having a “sequence sufficiently complementary to the target RNA” means that the antisense agent has a sequence sufficient to mask the binding site of a protein that would otherwise regulate splicing, and / or the antisense agent has a sequence sufficient to mask the binding site of a ribosome, and / or the antisense agent has a sequence sufficient to modify the three-dimensional structure of the targeted RNA in order to prevent splicing and / or translation.

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

[0170] In certain exemplary embodiments, the siRNA of the present invention comprises a double-stranded region of a nucleotide or nucleotide analog of about 8 to 20 base pairs in length, a nucleotide or nucleotide analog of about 10 to 18 base pairs in length, a nucleotide or nucleotide analog of about 12 to 16 base pairs in length, or a nucleotide or nucleotide analog of about 13 to 15 base pairs in length (e.g., a double-stranded 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 includes one or two overhangs. In certain embodiments, each overhang of the siRNA contains 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 overhang of the siRNA of the present invention has a length of about 4, about 5, about 6, or about 7 nucleotides. In certain embodiments, the sense strand overhang has the same number of nucleotides as the antisense strand overhang. In other embodiments, the sense strand overhang has fewer nucleotides than the antisense strand overhang. In other embodiments, the antisense strand overhang has fewer nucleotides than the sense strand overhang.

[0172] In certain exemplary embodiments, the siRNA of the present invention comprises a sense strand and / or antisense strand, each having a length of about 10, about 15, about 20, about 25, or about 30 nucleotides. In certain embodiments, the siRNA of the present invention comprises a sense strand and / or antisense strand, each having a length between about 15 and about 25 nucleotides. In certain embodiments, the siRNA of the present invention comprises a sense strand and an antisense strand, each having a length of about 20 nucleotides. In certain embodiments, the sense strand and antisense strand of the siRNA are of the same length. In other embodiments, the sense strand and antisense strand of the siRNA are of different lengths.

[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 and about 35 nucleotides. In other exemplary embodiments, the siRNA of the present invention has a total length of about 20 and about 30 nucleotides. In other exemplary embodiments, the siRNA of the present invention has a total length of about 22 and 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 terms “chemically modified nucleotide,” “nucleotide analog,” “modified nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, including ribonucleotides or deoxyribonucleotides, which do not exist in nature. Exemplary nucleotide analogs are modified at any position that alters the specific chemical properties of the nucleotide, but still retains the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and for adenosine and / or guanosine, the 8-position, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified nucleotides (e.g., alkylated, e.g., N6-methyladenosine, or as known in the art); 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 involve modifications to the sugar moiety 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. Patents 5,858,988 and 6,291,438.

[0176] As used herein, the term “metabolically stabilized” means an RNA molecule containing 2'-ribose modifications to replace a native 2'-hydroxyl group with a 2'-O-methyl group or a 2'-fluoro group. In certain embodiments, the double-stranded region of the siRNA contains one or two 2'-fluoro modifications and / or 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% of 2'-methoxy modifications. In certain exemplary embodiments, the antisense strand contains 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% of 2'-methoxy modifications. In certain exemplary embodiments, the sense strand contains 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% of 2'-methoxy modifications. In certain exemplary embodiments, the sense strand contains no 2'-fluoro modifications and contains 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% of 2'-methoxy modifications. In certain exemplary embodiments, a single-stranded RNA is provided that contains two 2'-fluoro modifications and contains at least about 90%, at least about 91%, at least about 92%, at least about 93%, or at least about 94% of 2'-methoxy modifications. In certain exemplary embodiments, single-stranded RNA is provided that is free of 2'-fluoro modifications and contains 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% of 2'-methoxy modifications.

[0177] As used herein, the term "phosphorothioate" refers to a phosphate group of a nucleotide modified by substituting one or more oxygens of the phosphate group with sulfur. A phosphorothioate further contains a cationic counterion (e.g., sodium, potassium, calcium, magnesium, etc.). The term "phosphorothioate-modified nucleotide" refers to a nucleotide bonded to another nucleotide by one or two phosphorothioate bonds. In certain embodiments, the single-stranded tail of the siRNA of the present invention comprises or constitutes a phosphorothioate-modified nucleotide.

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

[0179] Certain internucleotide bonds provided herein, for example, including phosphodiesters and phosphorothioates, are understood to contain a formal charge of -1 at physiological pH, which will be equilibrated by a cationic moiety, for example, 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” means a liposome formulation, where liposomes may be used, for example, to form nanoparticles with nucleic acids to facilitate the internalization of nucleic acids into cells. Without being constrained by theory, liposomes suitable for use are those that readily fuse with the phospholipid bilayer of the cell membrane, thereby enabling the penetration of nucleic acids into cells. In one embodiment, the compound comprises nanoparticles, inserts, polycations, or mixtures thereof.

[0181] In a particular embodiment, the compounds of this disclosure are of formula (I): [ka] [In the formula, the solid line "-" represents "X c This is indicated by [representing the means of interaction between "L", "O", and "Z"]. In certain embodiments, the interaction is via base pair complementarity, such as base pair complementarity between the oligonucleotide O and the anchor oligonucleotide Z that modifies PK. In certain embodiments, the interaction is X c This is a covalent bond, such as a bond between L and O, or between L and O.

[0182] Pharmaceutical composition and administration method In one embodiment, a pharmaceutical composition is provided herein 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 containing pharmacokinetic-modifying anchors described herein, and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition comprises one double-stranded chemically modified nucleic acid containing a pharmacokinetic-modifying anchor described herein, and a pharmaceutically acceptable carrier. In yet another particular embodiment, the pharmaceutical composition comprises two double-stranded chemically modified nucleic acids containing pharmacokinetic-modifying anchors described herein, and a pharmaceutically acceptable carrier.

[0183] The present invention relates to the use of the above-described agents for the therapeutic treatment described above. Accordingly, the modulators of the present invention (e.g., RNAi agents) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically include nucleic acid molecules, proteins, antibodies, or modulating compounds, and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” encompasses all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of conventional media or agents in a composition should be considered unless they are incompatible with the active compound. Complementary active compounds may also be incorporated into the composition.

[0184] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous (IV), intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), intravitreous, intra-articular, intranasal, vaginal, rectal, sublingual, and transmucosal administration. In certain exemplary embodiments, the pharmaceutical compositions of the present invention are delivered to the cerebrospinal fluid (CSF) by routes of administration including, but not limited to, intrastriatal (IS), intraventricular (ICV), and intrathecal (IT) administration (e.g., administration via pump, infusion, etc.). Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antimicrobial agents such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate, or phosphate; and agents to adjust isotonicity such as sodium chloride or dextrose. pH may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be placed in ampoules, disposable syringes, or glass or plastic vials for multiple doses.

[0185] Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate 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 a certain extent for easy injection. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersants, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it would be appropriate to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Long-term absorption of the injectable composition can be achieved by incorporating absorption-delaying agents, such as aluminum monostearate and gelatin, into the composition.

[0186] A sterile injection solution can be prepared by incorporating the active compound in the required amount, along with the components or combinations of components described above as necessary, into a suitable solvent, followed by sterile filtration. Generally, dispersions are prepared by compounding the active compound into a sterile vehicle containing a dispersion medium as a base and other necessary components derived from the components described above. In the case of sterile powders for producing sterile injection solutions, exemplary methods of preparation are vacuum drying and freeze-drying, thereby obtaining the active ingredient powder and any additional desired components from the previously sterile-filtered solution.

[0187] The toxicity and therapeutic effects of such compounds can be determined, for example, by standard pharmacokinetic procedures in cell cultures or experimental animals to measure the LD50 (the dose at which 50% of the population is lethal) and ED50 (the dose at which 50% of the population is therapeutically effective). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a large therapeutic index are particularly suitable. While compounds exhibiting toxic side effects can also be used, care must be taken to design a delivery system that delivers such compounds to the targeted infected tissue site in order to minimize the possibility of damaging uninfected cells and thereby reduce 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, including an ED50 that is little to no toxicity. The dosage 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 first be estimated from a cell culture assay. The dose can be formulated in animal studies to achieve a range of circulating plasma concentrations, including the EC50 (i.e., the concentration of the test compound that achieves half of the maximum response) determined in cell culture. The dose may also be formulated in animal studies by confirming the gene-silencing effect of the oligonucleotide on tissue concentrations. Such information can be used to more accurately determine a useful dose in humans. Plasma concentrations may be measured, for example, by high-performance liquid chromatography.

[0189] Treatment method As used herein, “treatment” or “to treat” is defined as an application or administration of a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding such agent) for the purpose of curing, healing, reducing, mitigating, altering, modifying, improving, enhancing, or influencing a patient with a disease or disorder, symptoms of a disease or disorder, or a predisposition to the disease or disorder, or the predisposition to the disease, or as an application or administration of the therapeutic agent to tissues or cell lines isolated from the patient.

[0190] In one embodiment, a method is provided for preventing the disease or disorder described above in a subject, 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 may be identified, for example, by any diagnostic or prognostic assay described herein, or a combination thereof. Administration of the prophylactic agent may be made before the onset of characteristic symptoms of the disease or disorder, so that the disease or disorder is prevented or its progression is delayed.

[0191] Design of Ava molecules In some embodiments, the siRNA molecule of the present invention is a double helix comprising a sense strand and a complementary antisense strand, wherein the antisense strand is sufficiently complementary to the target mRNA for RNAi mediation. In particularly exemplary embodiments, the siRNA molecule has a length of about 10 to 50 or more nucleotides, i.e., each strand contains 10 to 50 nucleotides (or nucleotide analogs). In particularly exemplary embodiments, the siRNA molecule has a length of about 16 to 30 nucleotides, for example, each strand having a length of 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, where one of the strands is sufficiently complementary to the target region. The chain can be aligned such that, when the chain is annealed, there are at least about one, two, or three unaligned bases at the ends of the chain (i.e., so that no bases complementary to the opposite chain are produced) resulting in an overhang of about one, two, or three residues at one or both ends of the double helix. The chain can be aligned such that, at the ends of the chain, there are about five, six, seven, or eight unaligned bases that form an overhang. The siRNA molecule can have a length of about 10 to 50 or more nucleotides, i.e., each chain can contain about 10 to about 50 nucleotides (or nucleotide analogs). In a particularly exemplary embodiment, the siRNA molecule has a chain length of about 16 to about 30, for example, 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, where one of the chains is substantially complementary to the target sequence and the other chain is identical or substantially identical to the first chain.

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

[0193] 1. siRNA should be specific to the target sequence. The first strand should be complementary to the target sequence, and the other strands should be 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, siRNA with a low G / C content (35-55%) may be more active than siRNA 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 a selected target site. In particularly exemplary embodiments, the sense strand contains 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 particularly exemplary embodiments, the sense strand contains about 13, about 14, about 15, or about 16 nucleotides. However, those skilled in the art will understand that siRNAs with a length of less than about 10 nucleotides or more than about 20 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of the invention, provided that they retain their 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 PKR responses (i.e., are sufficiently short in length). However, longer RNA silencing agents may be useful, for example, in cell types that cannot 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 the target sequence so that the siRNA can mediate RNAi. Generally, a nucleotide sequence-containing siRNA is provided that is sufficiently identical to the target sequence portion of the target gene for RISC-mediated cleavage of the target gene. Thus, in certain exemplary embodiments, the sense strand of the siRNA is designed to have a sequence that is sufficiently identical to the target portion. For example, the sense strand may have 100% identity with the target site. However, 100% identity is not required. Identity greater than about 80% between the sense strand and the target RNA sequence, e.g., 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%, and even 100% identity can be achieved. The present invention has the advantage of being able to tolerate specific 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 in the target region, for example, the target region differing by at least one base pair between the wild-type and mutant alleles, for example, the target region containing a gain-of-function mutation, and the other strand being the same as or substantially identical to the first strand. Furthermore, siRNA sequences with slight insertions or deletions of one or two nucleotides may also be effective in mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions may be effective in inhibition.

[0196] Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percentage identity of two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into the first or second sequence for optimal alignment). Next, the nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are compared. If the position in the first sequence is occupied by the same residue at the corresponding position in the second sequence, then the molecules at that position are identical. The percentage identity between the 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 a penalty may be imposed on the score for the number and / or length of any gaps introduced as desired.

[0197] Comparing sequences and measuring the percentage identity between two sequences can be achieved using mathematical algorithms. In one embodiment, sorting was performed over specific portions of sorted sequences that had sufficient identity, but not over portions with a lower degree of identity (i.e., local sorting). A non-restrictive example of a local sorting algorithm used to compare sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68, which is modified as described 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 yet another embodiment, alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the length of the aligned sequences (i.e., gapped alignment). To obtain gapped alignment for comparison, gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. In yet another embodiment, alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the entire length of the aligned sequences (i.e., global alignment). A non-restrictive 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, the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties can be used.

[0199] 3. The antisense or guide strand of an siRNA is typically of a different length from the sense strand and contains complementary nucleotides. In one embodiment, the siRNA strands may be paired so as to have a 3' overhang of about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides. The overhang may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang may contain (or consist of) deoxyribonucleotides, e.g., dTs, or nucleotide analogs, or other suitable non-nucleotide material. The nucleotides in the overhang may 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. Using any method known in the field, compare potential targets with appropriate genome databases (human, mouse, rat, etc.) and exclude any target sequences that have significant homology with other coding sequences. One such method for searching for such sequence homology is known as BLAST, which is available on the National Center for Biotechnology Information website.

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

[0202] Further general information on the design and use of siRNA can be found in "The siRNA User Guide," available on 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 with a target sequence (e.g., hybridizing with 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 hybridizing with 1xSSC at 70°C or 1xSSC at 50°C, 50% formamide, followed by washing with 0.3xSSC at 70°C, or hybridizing with 4xSSC at 70°C or 4xSSC at 50°C, 50% formamide, followed by washing with 1xSSC at 67°C. The hybridization temperature of hybrids expected to be less than 50 base pairs in length should be 5–10°C lower than the hybrid's melting temperature (Tm), where Tm is determined by the following formula. For hybrids with a length of less than 18 base pairs, Tm(°C) is 2(A+T base #) + 4(G+C base #). For hybrids with lengths of 18 and 49 base pairs, 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 1xSSC is 0.165M). Further examples of strict conditions for polynucleotide hybridization are provided in Sambrook, J., EF 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, edited by FM Ausubel et al., John Wiley & Sons, Inc., sections 2.10 and 6.3–6.4, which are incorporated herein by attribution.

[0204] A negative control siRNA will have the same nucleotide composition as the selected siRNA but will not have significant sequence homology to the appropriate genome. Such a negative control may 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 gene in the appropriate genome. In addition, a negative control siRNA may 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 A newly synthesized target mRNA, radiolabeled with 3P, is detected by autoradiography on an agarose gel. The presence of target mRNA cleavage indicates mRNA nuclease activity. A suitable control includes the omission of siRNA and the use of non-target cDNA. Alternatively, the control siRNA is selected to have the same nucleotide composition as the selected siRNA but without 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 gene in the appropriate genome. In addition, the negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0206] The siRNA may be designed to target any of the target sequences described above. The siRNA comprises an antisense strand that is sufficiently complementary to the target sequence in order to mediate the silencing of the target sequence. In certain embodiments, the RNA silencing agent is siRNA.

[0207] The siRNA-mRNA complementation site that provides optimal mRNA specificity and maximum mRNA cleavage is selected.

[0208] siRNA-like molecules The siRNA-like molecule of the present invention has a sequence that is "sufficiently complementary" to the target sequence of the target mRNA in order to direct gene silencing by either RNAi or translational repression (i.e., it has a sequence strand). The siRNA-like molecule is designed in the same way as an siRNA molecule, but the degree of sequence identity between the sense strand and the target RNA is approximately the same as that observed between a miRNA and its target. In general, as the degree of sequence identity between a 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 alternative embodiments, if post-transcriptional gene silencing by translational repression of the 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 within the target mRNA (e.g., within the 3'-UTR of the 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 translational repression mechanism is cooperative, in certain embodiments, multiple (e.g., two, three, four, five, or six) complementary sites may be targeted.

[0209] The ability of an siRNA-like double helix 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 in the complementary site. In one embodiment, if gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central part of the complementary site, and as a result, the double helix formed by the miRNA guide strand and target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003). In another embodiment, 2, 3, 4, 5, or 6 consecutive or discontinuous non-identical nucleotides are introduced. The non-identical nucleotides may be selected so that they form fluctuating 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., the antisense strand).

[0210] Modified RNA silencing agent In certain embodiments of the present invention, the RNA silencing agent (or any part thereof) described above may be modified to further improve the activity of the agent. For example, the RNA silencing agent described above may be modified by any of the following modifications. The modifications may, in part, further enhance target recognition, enhance the stability of the agent (e.g., prevent degradation), promote cellular uptake, enhance targeting efficiency, improve binding efficacy (e.g., with targets), improve patient tolerance to the agent, and / or reduce toxicity.

[0211] 1) Modifications that enhance target identification In certain embodiments, the RNA silencing agent of the present invention may be replaced with an unstable nucleotide to enhance single nucleotide target recognition (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 attribution). Such modifications may be sufficient to neutralize the specificity of the RNA silencing agent to non-target mRNA (e.g., wild-type mRNA) without significantly affecting the specificity of the RNA silencing agent to target mRNA (e.g., gain-of-function mutant mRNA).

[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. The universal nucleotide comprises a base portion that is capable of base-pairing indiscriminately with any of the four conventional nucleotide bases (e.g., A, G, C, U). The universal nucleotide is usable because it has relatively little effect on the stability of the RNA double helix, or the double helix formed by the guide strand and target mRNA of the RNA silencing agent. Exemplary universal nucleotides include those having an inosine base portion or an inosine analog base portion, 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 analog thereof.

[0213] In certain embodiments, the RNA silencing agent of the present invention is modified by introducing at least one destabilizing nucleotide to up to five nucleotides from the specificity-determining nucleotide (i.e., the nucleotide that recognizes disease-related polymorphisms). For example, the destabilizing nucleotide may be introduced at a position up to five, four, three, two, or one nucleotide from the specificity-determining nucleotide. In an exemplary embodiment, the destabilizing nucleotide may be introduced at a position three nucleotides from the specificity-determining nucleotide (i.e., so that there are two stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide). In an RNA silencing agent having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide may be introduced into the strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide may be introduced into the same strand or strand portion that contains the specificity-determining nucleotide.

[0214] 2) Modifications to enhance efficacy and specificity In certain embodiments, the RNA silencing agents of the present invention may be modified to promote enhanced efficacy and specificity through the intervention of RNAi, in accordance with asymmetric design rules (see U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). Such modifications select the sense strand to facilitate the entry of the antisense strand of siRNA (e.g., siRNA designed using the method of the present invention, or siRNA produced from shRNA) into RISC, thereby preferentially inducing cleavage or repression of translation of the 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 with respect to the binding strength or base pair strength between the antisense strand 5' end (AS5') and the sense strand 5' end (S3') of the RNA silencing agent.

[0215] In one embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced such that there are fewer G:C base pairs between the 5' end of the antisense strand and the 3' end of the sense strand than between the 3' end of the antisense strand and the 5' end of the sense strand. In yet another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced such that there is at least one mismatched base pair between the 5' end of the initial or antisense strand and the 3' end of the sense strand. In a particular embodiment, 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 yet another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced such that there is at least one fluctuation base pair, for example, G:U, between the 5' end of the initial or antisense strand and the 3' end of the sense strand. In yet another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced to include at least one base pair comprising a rare nucleotide, such as inosine (I). In a particular embodiment, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another particular embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced to include at least one base pair comprising a modified nucleotide.

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

[0217] In certain embodiments, the present invention features an RNA silencing agent that may comprise first, second, and third strands, any of which may be modified by substituting an internal nucleotide with a modified nucleotide to enhance in vivo stability compared to the corresponding unmodified RNA silencing agent. As defined herein, “internal nucleotide” is any nucleotide located at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. Internal nucleotides may be located 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 the substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand are modified by the substitution of 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 yet another embodiment, the sense strand and / or antisense strand are modified by substitutions of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand are modified by substitutions of all internal nucleotides.

[0218] In certain embodiments, the RNA silencing agent (e.g., any combination of a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide) may optionally contain at least one modified nucleotide analog. The nucleotide analog may be positioned such that target-specific silencing activity, e.g., RNAi-mediated activity or translational repression activity, is substantially unaffected in the 5' and / or 3' terminal regions of the siRNA molecule. In particular, these terminals may be stabilized by incorporating the modified nucleotide analog.

[0219] Exemplary nucleotide analogs include sugar- and / or skeletal-modified ribonucleotides (i.e., modifications to the phosphate-sugar backbone in any of the first, second, and / or third oligonucleotides). For example, the phosphodiester bond in native RNA may be modified to include at least one nitrogen or sulfur heteroatom. In the exemplary skeletal-modified ribonucleotide, the phosphodiester group connected to an adjacent ribonucleotide is replaced by a modifying group, such as a phosphorothioate group. In the exemplary sugar-modified ribonucleotide, the 2'OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is a C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0220] In certain embodiments, the modifications are 2'-fluoro, 2'-amino, and / or 2'-thio modifications. Certain exemplary modifications include 2'-fluorocytidine, 2'-fluorouridine, 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-aminocytidine, 2'-aminouridine, 2'-aminoadenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, and / or 5-aminoallyluridine. In certain embodiments, the 2'-fluororibonucleotides are all uridines and cytidines. Further exemplary modifications include 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2'-aminobutyrylpyreneuridine, 5-fluorocytidine, and 5-fluorouridine. 2'-deoxynucleotides and 2'-ohm nucleotides may also be used within portions of the modified RNA-silencing agents of the present invention. Further modification residues include deoxy-abasic, inosine, N3-methyluridine, N6,N6-dimethyladenosine, pseudouridine, purine ribonucleosides, and ribavirin. In certain exemplary embodiments, the 2' portion is a methyl group such that the linking portion is a 2'-O-methyl oligonucleotide.

[0221] In exemplary embodiments, the RNA silencing agent of the present invention (e.g., any combination of a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide) comprises locked nucleic acid (LNA). The LNA contains glycosylated nucleotides that are resistant to nuclease activity (extremely stable) and have single nucleotide recognition for 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 2'-O, 4'-C-ethylene-bridged nucleic acids that can be modified, such as 2'-deoxy-2''-fluorouridine. Furthermore, LNA enhances the specificity of oligonucleotides by constraining the sugar moiety to a 3'-end structure, thereby pre-organizing the nucleotides for base pairing and increasing the melting temperature of oligonucleotides by up to 10°C per base.

[0222] In yet 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 peptide nucleic acid (PNA). The 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 skeleton that is highly resistant to nuclease digestion and confers improved binding specificity to the molecule (Nielsen et al., Science, (2001), 254: 1497-1500).

[0223] In yet another exemplary embodiment, the RNA silencing agent of the present invention (e.g., any combination of the first, second, and third oligonucleotides) comprises a phosphorodiamidate morpholino oligomer (PMO). The PMO comprises a modified nucleotide having a standard nucleic acid base bound to a methylenemorpholine ring linked via a phosphorodiamidate group instead of a phosphate group (Summerton et al., (1997) antisense & Nucleic Acid Drug Development. 7 (3): 187-95).

[0224] Ribonucleotides with modified nucleic acid bases, i.e., ribonucleotides containing at least one non-naturally occurring nucleic acid base instead of a naturally occurring nucleic acid base, are also provided. The bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleic acid bases include, but are not limited to, uridine and / or cytidine modified at position 5, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at position 8, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenosine; and O- and N-alkylated nucleotides, e.g., N6-methyladenosine, which are suitable. Note that the above modifications may be combined.

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

[0226] As other exemplary modifications, (a) 2'-modifications, such as those in the sense or antisense strand, but especially the provision of 2'-OMe moieties at U in the sense strand, or in the 3'-overhang, such as the provision of a 2'-OMe moiety at the 3'-end (the 3'-end means the 3'-atom of the molecule or the maximal 3'-portion, such as maximal 3'-P or 2'-position, as indicated in context); (b) modifications of the backbone, such as replacing O with S in the phosphate backbone, such as the provision of phosphorothioate modifications replacing P with S, especially in the antisense strand at U or A or both; (c) replacement of U with a C5 amino linker; (d) replacement of A with G (the sequence change is preferably located in the sense strand, rather than the antisense strand); and (e) modifications at the 2', 6', 7', or 8'-positions. 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 there are few such modifications in the antisense strand. Further exemplary modifications include the use of methylated P at the 3'-end in the 3'-overhang; combinations of 2'-modifications, such as the provision of 2'-OMe moieties and modifications of the backbone, such as replacing P with S, such as the provision of phosphorothioate modifications, or the use of methylated P at the 3'-end in the 3'-overhang; modifications using 3'-alkyls; modifications with pyrrolidones lacking a base at the 3'-end in the 3'-overhang; modifications with naproxen, ibuprofen or other moieties that inhibit degradation at the 3'-end.

[0227] 4) Modifications for enhancing cellular uptake In other embodiments, the RNA silencing agent (e.g., any combination of the first, second, and third oligonucleotides) may be chemically modified to enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the present invention encompasses RNA silencing agents that are conjugated or unconjugated (e.g., at the 3' end of the sense strand) to yet another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), etc. Conjugation can be achieved by methods known in the art, for example, using the methods described in Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to inserts, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes 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, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl) glycerol, geranyloxyhexyl group, hexadecyl glycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) chenodeoxycholic acid, dimethoxytrityl, or phenoxazine.

[0229] 5) Linking ligand Other entities can be linked to the RNA silencing agent of the present invention. For example, ligands can be linked to the RNA silencing agent to improve stability, thermodynamics of hybridization with target nucleic acids targeting specific tissues or cell types, or cell permeability by mechanisms such as endocytosis-dependent or independent mechanisms. Ligands and associated modifications can also increase sequence specificity and consequently reduce off-site labeling. Linked ligands may contain 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 double helix. Intercalators may be aromatic compounds, e.g., polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking ability and may include systems with two, three, or four fused rings. Universal bases described herein may be included in ligands. In one embodiment, cleavage groups may be included that contribute to the inhibition of a target gene by cleaving the target nucleic acid. The cleavage group may 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 may include, for example, Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the bulge site by free metal ions such as Lu(III). In some embodiments, the peptide ligand may be linked to an RNA silencing agent to promote the cleavage of target RNA at the bulge region, for example. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (Cycram) can be conjugated with a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage.The linking 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 acridine analogs can enhance sequence specificity. For example, neomycin B has high affinity for RNA compared to DNA, but low sequence specificity. The acridine analog, neo-5-acridine, has high affinity for HIV Rev-response factors (RREs). In some embodiments, guanidine analogs (guanidinoglycosides) of aminoglycoside ligands are linked to the RNA silencing agent. In guanidinoglycosides, the amine group of an amino acid is exchanged for a guanidine group. The attachment of a guanidine analog can enhance the cellular permeability of RNA silencing agents. The linking ligand may be a polyarginine peptide, peptoid, or peptide mimetic that can enhance the cellular uptake of oligonucleotide agents.

[0230] An exemplary ligand binds to a conjugated carrier either directly or indirectly through an intervening tether, e.g., covalently. In an exemplary embodiment, the ligand binds to the carrier through an intervening tether. In an exemplary embodiment, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent it incorporates. In an exemplary embodiment, the ligand provides enhanced affinity to selected targets, e.g., molecules, cells or cell types, compartments, e.g., cellular or organ compartments, body tissues, organs, or regions, compared to the absence of such a 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 agents or polymer molecules containing any combination of monomers and / or natural or modified ribonucleotides described herein. Generally, ligands may include, for example, therapeutic modifiers to enhance uptake; for example, diagnostic compounds or reporter groups to monitor distribution; crosslinking agents; moieties that confer nuclease resistance; and natural or non-natural nucleic acid bases. Common examples include lipophilic substances, lipids, steroids (e.g., ubaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin-targeting molecules, polycationic agents, peptides, polyamines, and peptide mimetic compounds. Ligands may include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids, or lipids. Ligands may also be synthetic polymers, such as recombinant or synthetic molecules including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolated) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0232] Ligands may also include target groups, such as agents that target cells or tissues, such as lectins, glycoproteins, lipids or proteins, or antibodies that bind to specific cell types, such as kidney cells. Target groups may include tyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, transferrin-mimicking peptides, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, or RGD peptides or RGD peptide mimics. Other examples of ligands include dyes, inserts (e.g., acridine and substituted acridines), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidium aminoglycosides, artificial endonucleases, lipophilic molecules (e.g., cholesterol (and its thio analogues)), cholic acid, cholanic acid, lithocholic acid, adamantane acetate, 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, for example, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 Alkyl groups (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)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide) This includes alkylating agents, phosphates, amino acids, mercaptosaccharides, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0233] Ligands can be proteins, such as glycoproteins or peptides, molecules that have a specific affinity for a colligand, or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteocytes. Ligands may also include hormones and hormone receptors. They may also encompass lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species such as polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. The ligand may be, for example, a lipopolysaccharide, a p38MAP kinase activator, or an NF-κB activator.

[0234] A ligand can be a substance, such as a drug, that can enhance the uptake of RNA silencing agents into cells by, for example, disrupting the cytoskeleton of a cell, such as by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. Drugs may include, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlaquinolide, latruncrine A, phalloidin, swinholide A, indanosine, or myoserbine. Ligands can increase the uptake of RNA silencing agents into cells by, for example, activating an inflammatory response. Exemplary ligands having 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 a lipid or lipid-based molecule binds to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand distributes the conjugate to a target tissue, such as a non-renal target tissue of the body. For example, the target tissue may be the liver, including hepatic parenchymal cells. Other molecules capable of binding to HSA may also be used as ligands. For example, naproxen or aspirin may be used. Lipids or lipid-based ligands can be used to (a) increase the conjugate's resistance to degradation, (b) enhance targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Lipid-based ligands can be used to modulate, for example, control the binding of the conjugate to target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to target the kidney and therefore less likely to be removed from the body. A lipid or lipid-based ligand that does not bind as strongly to HSA can be used to make the conjugate target the kidney. In certain embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand may bind to HSA with sufficient affinity so that the conjugate is distributed to tissues other than the kidney.However, in certain embodiments, the affinity is not strong enough to prevent the HSA-ligand from being reversed. In yet another specific embodiment, the lipid-based ligand binds weakly to HSA or not at all, so that the conjugate is distributed to the kidney. Other parts that target renal cells may also be used instead of, or in addition to, the lipid-based ligand.

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

[0236] In another embodiment, the ligand is a cell permeation agent, for example, a helical cell permeation agent. The agent may be amphiphilic. Exemplary agents are peptides such as Tat or Antennapedia. If the agent is a peptide, it may be modified, including the binding of peptidyl mimes, invertma, non-peptide or pseudopeptide compounds, and the use of D-amino acids. In certain embodiments, the helical agent is an alpha-helical agent, which may optionally have an oily phase and an oil-phobic phase.

[0237] The ligand may be a peptide or a peptide mime. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules that, like natural peptides, can be folded into a defined three-dimensional structure. The attachment of peptides and peptide mimes to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, for example, by enhancing cellular recognition and absorption. The peptide or peptide mime portion may be about 5 to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mime may be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., mainly consisting of Tyr, Trp, or Phe). The peptide portion may be a dendrimeric peptide, a restricting peptide, or a cross-linked peptide. The peptide portion may be an L-peptide or a D-peptide. In another way, the peptide portion may contain a hydrophobic membrane transition sequence (MTS). Peptides or peptide mimes can be encoded by random sequences of DNA, such as peptides identified from phage-represented libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptide mime linked to the RNA silencing agent via incorporated monomer units is a cell-targeting peptide such as an arginine-glycine-aspartate (RGD) peptide or RGD mime. The peptide portion can range in length from approximately 5 to approximately 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 may be utilized.

[0238] All references that may be cited throughout this application (including references, patents, patent applications, and websites) are expressly incorporated herein by attribution for all purposes, just as the references therein. Unless otherwise noted, the disclosures will utilize prior art in immunology, molecular biology, and cell biology that is well known in the art.

[0239] This disclosure also incorporates all of its well-known techniques in the fields of molecular biology and drug delivery, provided that the sources are clearly indicated.

[0240] [Examples] Example 1. Oligonucleotide containing an anchor that modifies the dynamic pharmacokinetics (PK) for cerebrospinal fluid and systemic delivery. 1.1 Anchors modifying PK A major challenge in the field of therapeutic oligonucleotides is that non-serum-bound oligonucleotides are cleared from cerebrospinal fluid (CSF) and blood / plasma within minutes of injection. This rapid clearance is the primary factor limiting the delivery of oligonucleotides to tissues beyond the liver and kidneys. In the central nervous system, the primary mechanism behind oligonucleotide distribution across the brain is bulk CSF flow. Oligonucleotides are rapidly removed from the central nervous system, which limits their distribution in organisms with large and complex brains (including humans). The PK-modifying molecular anchors disclosed herein are patterned to enable efficient modulation of the absorption, distribution, and clearance kinetics of therapeutic oligonucleotides, thereby enhancing their tissue distribution. Efficient modulation of absorption, distribution, and clearance kinetics can be achieved in blood / plasma, cerebrospinal fluid (CSF), and other relevant body fluids / 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 PK-modifying anchors described herein dynamically modulate the size of therapeutic oligonucleotides, resulting in the modification of clearance kinetics for tissue uptake and distribution. The dynamic properties of this concept are achieved through the optimization of the anchor size and chemical composition. PK-modifying anchors of optimal size for modulating CSF clearance rates and systemic clearance are described herein. In addition, a panel of non-immunogenic polymers (including poloxamer 188) and block polymers that function as pharmacokinetic-modifying portions are described herein.

[0242] 1.2 PK-modifying anchors dynamically improved the blood / plasma circulating time of hsiRNA compounds. As shown in Figure 2, the effect of hydrophobically modified siRNA (hsiRNA) with PK-modifying anchors on blood / plasma circulation time was tested. Anchors of PK-modifying molecules were measured to enhance 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 polymer for PK modification. Octamer oligonucleotides with a phosphorothioate backbone were used as model oligonucleotide anchors. The PK-modifying anchors were hybridized to asymmetric hsiRNA double helixes containing a 21-mer oligonucleotide antisense chain and a 13-mer oligonucleotide sense chain. Increasing the length of the PEG portion significantly improved the circulation time of the hsiRNA compound.

[0243] Female FVB / N mice (approximately 9-12 weeks old) were administered a 20 mg / kg tail vein injection. The antisense strand was quantified using a peptide nucleic acid (PNA) hybridization assay, as previously described by Godinho et al., 2017 (Nucleic Acids Therapeutics). In short, this assay uses a cy3-labeled PNA probe that hybridizes with the antisense strand, followed by quantification by HPLC. The area under the curve (AUC) was calculated using the model-independent trapezoidal method with GastroPlus and Simulations Plus.

[0244] 1.3 PK-modifying anchors regulated the systemic in vivo distribution of hsiRNA compounds. As shown in Figure 9, the effect of PK-modifying anchors on the biodistribution of hsiRNA was tested. 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 polymer for PK modification. Octamer oligonucleotides with a phosphorothioate backbone were used as model oligonucleotide anchors. The PK-modifying anchors were hybridized to asymmetric hsiRNA double helixes containing a 21-mer oligonucleotide antisense chain and a 13-mer oligonucleotide sense chain.

[0245] Many embodiments of pharmacokinetic modifying anchors and oligonucleotide anchors are possible. The length and chemical properties of the anchors can be adjusted according to the purpose and target delivery. As shown in Figure 9, pharmacokinetic modifying anchors are, This significantly affected the biodistribution of unconjugated and cholesterol-conjugated siRNAs after intravenous injection. Figure 9 shows a positive correlation between elongation of the PEG portion length and improved delivery of most organs to unconjugated oligonucleotides.

[0246] Female FVB / N mice (aged approximately 9 - 12 weeks) were given a 20 mg / kg tail vein injection. After 48 hours, the antisense strand was quantified using a PNA hybridization assay.

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

[0248] Female FVB / N mice (aged approximately 9 - 12 weeks) were given a 20 mg / kg tail vein injection. Tissues were collected 48 hours after injection and mRNA was quantified using the QuantiGene b-DNA assay as described by Coles et al., 2015.

[0249] The anchor that modifies PK also delivered the hsiRNA compound to the kidney (Figure 20), liver (Figure 21), spleen (Figure 22), and skin (Figure 23) after subcutaneous administration.

[0250] 1.5 The anchor that modifies PK regulated the in vivo biodistribution of the hsiRNA compound within the central nervous system after intraventricular and intrathecal injection. The effects of various hsiRNA constructs on in vivo biodistribution were tested 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 were injected into the lateral ventricle, resulting in a concentration of approximately 2 nanomoles / ventricle. In Figure 11B, 20 nanomoles of hsiRNA were 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 were injected intrathecally between L5 and L6. The distribution of hsiRNA in the mouse spine is shown in Figure 11C.

[0251] hsiRNA constructs were fabricated starting with a 21-mer oligonucleotide antisense chain and a 13-mer oligonucleotide sense chain. Various attachments tested included cholesterol alone, cholesterol-anchor alone, cholesterol-2000Da PK-modifying anchor, cholesterol-4500Da PK-modifying anchor, cholesterol-free anchor alone, cholesterol-free 2000Da PK-modifying anchor, cholesterol-free 4500Da PK-modifying anchor, and cholesterol-free hsiRNA alone. Mouse brain and spinal tissues were collected 48 hours after injection and stained with DAPI (nucleus, blue). Brain and tissues were imaged using a Leica DMi8 fluorescence microscope.

[0252] PK-modified anchors enabled unique diffusion and retention of the highly lipophilic conjugate in the mouse brain after intracerebroventricular injection (Figure 11A). Immobilization of a larger PEG portion onto the hsiRNA cholesterol-conjugated compound improved penetration into the brain parenchyma. PK-modified anchors enabled unique diffusion and retention of the highly lipophilic conjugate in the mouse spine after intrathecal administration (Figure 11B). Immobilization of a larger PEG portion onto the hsiRNA cholesterol-conjugated compound improved penetration into the spinal cord parenchyma, as observed in brain tissue.

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

[0254] Example 2. Conjugated oligonucleotide containing an anchor that modifies dynamic pharmacokinetics (PK). 2.1 Anchors modifying PK The PK-modifying anchor was paired with different conjugated asymmetric siRNAs, as shown in Figure 31. Specifically, siRNAs having a 21-nucleotide antisense strand and a 13-nucleotide sense strand were conjugated with one of cholesterol, DCA, DHA, or GalNAc. Dibranched siRNA compounds, in which a linker connects the two siRNAs at the 3' end of the sense strand, were also tested. Each conjugated asymmetric siRNA was paired with a PK-modifying anchor containing a 40 kDa PEG moiety, where all internucleotide links are phosphorothioates.

[0255] 2.2 PK-modifying anchors dynamically improved blood / plasma circulation time of intravenously injected siRNA compounds. As shown in Figure 32, the effect of PK-modifying anchors on the blood / plasma circulation time of conjugated siRNAs was tested. Anchors of PK-modifying molecules 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 old). The antisense strand was quantified using a peptide nucleic acid (PNA) hybridization assay, as previously described by Godinho et al. in 2017 (Nucleic Acids Therapeutics). In short, this assay uses a cy3-labeled PNA probe that hybridizes with the antisense strand, followed by quantification by HPLC.

[0257] 2.3 PK-modifying anchors regulated the systemic in vivo distribution of intravenously administered siRNA compounds. As shown in Figure 33, the effect of PK-modifying anchors on the biodistribution of conjugated siRNA in Example 2.2 was tested. siRNA localization was tested with respect to the pancreas, lungs, heart, adrenal glands, spleen, kidneys, muscles, and liver. Similar to 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 indicate 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 kidneys. Since the kidneys are clearance tissues, avoiding renal evasion may increase the serum half-life and biodistribution of conjugated siRNA.

[0258] These results also demonstrate an unexpected enhancement of hepatic delivery that PK-modifying anchors confer to GalNAc-conjugated siRNA. As shown in Figure 33B, the amount of siRNA with PK-modifying anchors conjugated to GalNAc is more than twice that of siRNA without PK-modifying anchors conjugated to GalNAc. While GalNAc conjugation is known to promote hepatic delivery of siRNA, the addition of PK-modifying anchors may promote higher hepatic delivery beyond GalNAc conjugation, potentially enhancing the therapeutic effect of GalNAc-conjugated siRNA.

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

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

[0261] 2.5 PK-modifying anchors regulated the systemic in vivo distribution of siRNA compounds administered subcutaneously. As shown in Figure 35, the effect of PK-modifying anchors on the biodistribution of the siRNA from Example 2.4 was tested. siRNA localization was tested in the pancreas, lungs, heart, adrenal glands, spleen, kidneys, muscles, and liver. As in Example 2.4, unconjugated siRNA (Figure 35A) and di-siRNA (Figure 35B) were also tested.

[0262] 2.6 PK-modifying anchors regulated the blood / plasma circulation time and systemic in vivo distribution of subcutaneously administered aptamer-siRNA chimeric compounds. As shown in Figures 36 and 37, the effects of PK-modifying anchors on the blood / plasma circulating time (Figure 37A) and biodistribution (Figure 37B) of aptamer-siRNA chimeras were tested. The 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 Balb-c mice with tumors. These mice contained both tumors derived from 4T1E cells and tumors derived from P815 cells. Sense strands were quantified by a peptide nucleic acid (PNA) hybridization assay, as previously described by Godinho et al., 2017 (Nucleic Acids Therapeutics). Tissue was collected for the assay 48 hours after injection. In this assay, the EPCAM-binding aptamer-siRNA conjugate was taken up by 4T1E tumors expressing the EPCAM receptor, while P815 was used as a negative control. As shown in Figures 37A and 37B, the PK-modifying anchor increased circulation time and improved delivery to target tumors by 2- to 4-fold compared to the adapter-siRNA conjugate without the PK-modifying anchor.

[0264] 2.7 PK-modifying anchors regulated the systemic in vivo distribution of dibranch siRNA compounds administered intravenously and subcutaneously. As shown in Figure 38, the effect of PK-modifying anchors on the biodistribution of unconjugated and di-siRNAs was measured compared to intravenous and subcutaneous injection. siRNA localization was examined with respect to the liver (Figure 38A), spleen (Figure 38B), and kidney (Figure 38C). The results show that PK-modifying anchors enhanced the delivery of unconjugated and di-siRNAs to the liver and other secondary distribution organs after SC and IV administration. PK-modifying anchors also reduced renal clearance of both siRNA scaffolds. The experiment was performed in triple duplication, and fluorescence tissue images are shown 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 siRNA unconjugated with a PK-modifying anchor on the biodistribution of the mouse placenta was measured. Two separate subcutaneous injections of 20 mg / kg were administered to pregnant female FVB / N mice (approximately 9-12 weeks old, 4 mice per group). Tissues were collected 48 hours after the second injection. Antisense strands were quantified using a peptide nucleic acid (PNA) hybridization assay, as previously described by Godinho et al., 2017 (Nucleic Acids Therapeutics). The results showed that the PK-modifying anchor enhanced placental distribution tenfold 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 targeted mRNA silencing of unconjugated siRNA in mouse placentas was measured. The experiment was conducted 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 showed that targeted mRNA silencing using PK-modifying anchors was comparable to that of siRNA without PK-modifying anchors. To demonstrate that PK-modifying anchors do not cause acute systemic toxicity, mouse body weight profiles (body weight % increase), blood chemistry characteristics, and total blood counts were measured. The results demonstrated that mouse body weight profiles, blood chemistry characteristics, and total blood counts were comparable between controls injected with saline and mice injected with PK-modifying anchors. These results demonstrate that PK-modifying anchors do not induce 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 hepatic delivery of GalNAc-conjugated siRNA was measured. The assay utilized two different types of asymmetric siRNA. The first was siRNA with a 21-nucleotide antisense strand, a 13-nucleotide sense strand, and an 8-nucleotide anchor (indicated as 21-13-8). The second was siRNA with a 25-nucleotide antisense strand, a 17-nucleotide sense strand, and an 8-nucleotide anchor (indicated as 25-17-8). The results demonstrated that PK-modifying anchors enhanced hepatic delivery in both the 21-13-8 siRNA format and the 25-17-8 siRNA format, both intravenously and subcutaneously, compared to siRNA without PK-modifying anchors. This enhancement was particularly strong in hepatocytes. These results also indicate an unexpected enhancement of hepatic delivery that PK-modifying anchors confer to GalNAc-conjugated siRNA. As shown in Example 2.3 above, it is known that GalNAc conjugates promote hepatic delivery of siRNA, but the addition of PK-modifying anchors promotes advanced hepatic delivery beyond GalNAc conjugates and may be useful in enhancing the therapeutic effect of GalNAc-conjugated siRNA.

[0268] Example 3. Development of a conserved universal oligonucleotide anchor sequence The experiments described in Examples 1 and 2 were performed using asymmetric siRNA targeting sFlt-1 mRNA. This siRNA is in 21-13 (antisense length-sense length) format. Therefore, it has an 8-nucleotide antisense tail to which an 8-nucleotide anchor sequence can be bound. The tail sequence of the asymmetric siRNA targeting sFlt-1 mRNA utilizes a G / C nucleotide-rich tail sequence (with a G / C nucleotide content of 87.5%). siRNAs with G / C nucleotide-poor sequences were tested to measure whether the PK-modifying anchor successfully bound to its target antisense 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, 8-nucleotide with a 40kDa PEG moiety: [ka] A PK-modifying anchor, a 6-nucleotide having a 40kDa PEG moiety: [ka] sFLT1-2283: Antisense strand, 21-nucleotide: [ka] Sense strand, 13-nucleotide: [ka] PK-modifying anchor, 8-nucleotide with a 40kDa PEG moiety: [ka]

[0269] In the above sequence, "V" corresponds to the 5'-vinylphosphonate moiety, "m" to the 2'-O-methyl modification, "f" to the 2'-fluoro modification, "#" to the phosphorothioate nucleotide bond, and "40k" to the 40kDa PEG moiety. The bold / underlined portion of the antisense sequence corresponds to the 8-nucleotide tail to which the PK-modifying anchor binds. 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 HttsiRNA was used with a 6-nucleotide or 8-nucleotide anchor, there was no detectable shift of the siRNA in the gel shift assay. This was true for molar ratios of siRNA to anchor of 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 efforts to develop conserved universal anchor and antisense tail sequences, the effects of varying the lengths of the anchor and adjacent sense strands on anchor tail binding were measured. As shown in Figure 43, 21-13siRNA was used with 8-nucleotide, 7-nucleotide, 6-nucleotide, and 5-nucleotide anchors. The 7-, 6-, and 5-nucleotide anchors each contain a gap in the sequence between the adjacent sense strand and the anchor (1-, 2-, and 3-nucleotide gaps, respectively). When a gap remains within the sense strand and oligonucleotide anchor, binding efficiency decreases because the positive effect of coaxial stacking is lost. The 7-nucleotide anchor required four times the molar amount to enable a complete shift in gel electrophoresis assays. As shown in Figure 44, this decrease in binding efficiency could be mitigated by increasing the sense strand length and closing the gap between the sense strand and the anchor. 7-nucleotide anchors, 6-nucleotide anchors, and 5-nucleotide anchors were used with 14-nucleotide sense strands, 15-nucleotide sense strands, and 16-nucleotide sense strands, respectively. Gel-shift assays demonstrated that using a 14-nucleotide sense strand with a 7-nucleotide anchor reduced the decrease in binding efficiency compared to the 13-nucleotide sense strand / 7-nucleotide anchor combination.

[0271] Several options were tested to design anchor sequences that modify the universal PK. 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 the first example of Option 1, a 17-nucleotide sense strand was used with a 6-nucleotide anchor sequence complementary to the 6-nucleotide universal sequence of the antisense strand. In the second example of Option 1, a 15-nucleotide sense strand was used with an 8-nucleotide anchor sequence complementary to the 6-nucleotide universal sequence of the antisense strand, with the other two nucleotides complementary to the nucleotides at positions 16 and 17 of the antisense strand, and which would change 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 complementary to the 8-nucleotide universal sequence of the antisense strand (Figure 45).

[0272] The three alternative universal PK modification anchor sequence solutions described above were tested against Htt mRNA targets. The mRNA silencing effect of the target Htt mRNA was measured. 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 whose conserved region is located at nucleotide 16 (data points enclosed in the box "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 conserved universal sequences may be available for any siRNA, thus solving any of the challenges associated with tail sequences with poor G / C content (Figures 46A, 46B).

[0273] In addition to the in vitro silencing experiments described above, the effect of PK-modifying anchors 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 (denoted as 21-17-8) having a 21-nucleotide antisense strand, a 13-nucleotide sense strand, and an 8-nucleotide anchor. The 25-17-8 siRNA utilized the above-mentioned conserved universal tail sequence, starting at position 18 of the antisense strand. Each siRNA had a GalNAc conjugate for hepatic delivery. The results showed that PK-modifying anchors with conserved universal tail sequences also enhanced hepatic distribution compared to siRNAs without the conserved sequence, for both intravenous and subcutaneous delivery (Figure 41).

Claims

1. A first oligonucleotide containing 16 to 25 consecutive nucleotides, a 5' end, and a 3' end, A second oligonucleotide containing 12 to 17 nucleotides, a 5' end, and a 3' end, and The pharmacokinetic (PK) modifying anchors, which include anchor oligonucleotides linked to at least one polymer, Here, the anchor oligonucleotide contains or consists of 5 to 8 nucleotides, a 5' end, and a 3' end. The first oligonucleotide chain is complementary to the target, and the second oligonucleotide chain forms an asymmetric double helix in complement to the 5' end of the first oligonucleotide chain, and The anchor oligonucleotide chain is complementary to the 3' end of the first oligonucleotide chain. The polymer is an siRNA compound linked to the 5' end of the anchor oligonucleotide, located between 2,000 Da and 100,000 Da.

2. The compound according to claim 1, wherein the polymer is operably linked to an anchor oligonucleotide via a linker.

3. At least one polymer is selected from the group consisting of amphiphilic block copolymers, hydrophobic polycarbonates, polyesters, hydrophobic block polymers, polysaccharides, and polypeptides. The hydrophobic polycarbonate is poly(DTR carbonate), The polyester is selected from the group consisting of polyhydroxyalkanoates, polycaprolactone, poly(hydroxybuterate-hydroxyvalerate), polyglycolic acid, and polylactic acid. 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). The polysaccharide is selected from the group consisting of soluble polyglucose, insoluble polyglucose, cellulose, glycogen, and amylopectin. The compound according to claim 1, wherein the polypeptide is polylysine, polyarginine, polyalanine, polyisoleucine, polymethionine, polyphenylalanine, polyvaline, polyproline, and polyglycine, and any combination thereof.

4. The compound according to claim 3, wherein the amphiphilic block copolymer is selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), acrylonitrile styrene acrylate, and N-(2-hydroxypropyl)methacrylamide.

5. The PK modifying anchor comprises two or more polymers, according to any one of claims 1 to 4. The compound described.

6. The compound according to claim 5, wherein the PK-modifying anchor comprises two, three, or four polymers.

7. The first oligonucleotide contains one or more chemically modified nucleotides, The first oligonucleotide comprises one or more locked nucleic acids (LNAs) or one or more peptide nucleic acids (PNAs), The first oligonucleotide contains one or more S-restricted-ethyl (cET) groups, or The compound according to any one of claims 1 to 6, wherein the first oligonucleotide comprises 50% 2'-methoxyribonucleotide, 55% 2'-methoxyribonucleotide, 60% 2'-methoxyribonucleotide, 65% 2'-methoxyribonucleotide, 70% 2'-methoxyribonucleotide, 75% 2'-methoxyribonucleotide, 80% 2'-methoxyribonucleotide, 85% 2'-methoxyribonucleotide, 90% 2'-methoxyribonucleotide, 95% 2'-methoxyribonucleotide, 96% 2'-methoxyribonucleotide, 97% 2'-methoxyribonucleotide, 98% 2'-methoxyribonucleotide, 99% 2'-methoxyribonucleotide, or 100% 2'-methoxyribonucleotide.

8. The compound according to claim 7, wherein the first oligonucleotide is completely or partially chemically modified.

9. The first oligonucleotide comprises alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides, and the nucleotides of the first oligonucleotide are linked via phosphodiester bonds, phosphorothioate bonds, or a combination of phosphodiester bonds and phosphorothioate bonds. (1) The first oligonucleotide comprises alternating 2'-methoxyribonucleotides and 2'-fluororibonucleotides, where each nucleotide is either a 2'-methoxyribonucleotide or a 2'-fluororibonucleotide, and the nucleotides at positions 2 and 14 from the 5' end of the first oligonucleotide are not 2'-methoxyribonucleotides; and (2) The nucleotide of the first oligonucleotide is connected to an adjacent nucleotide via a phosphodiester or phosphorothioate bond, Here, nucleotides located at positions 1-6 from the 3' end, or at positions 1-7 from the 3' end, are connected to adjacent nucleotides via phosphorothioate bonds. Compound according to any one of claims 1 to 8

10. The compound according to any one of claims 1 to 9, wherein the second oligonucleotide comprises one or more chemically modified nucleotides.

11. The second oligonucleotide contains Xc, which is the conjugate portion. The compound according to any one of claims 1 to 10, wherein Xc is bonded to the 3' end of the second oligonucleotide, at an internal position, or at a mixed position thereof.

12. The compound according to any one of claims 1 to 11, wherein the anchor oligonucleotide is completely complementary to the first oligonucleotide, or the anchor oligonucleotide contains one, two, three, or four mismatches in relation to the first oligonucleotide.

13. The anchor oligonucleotide comprises one or more chemically modified nucleotides, wherein the anchor oligonucleotide comprises one or more locked nucleic acids (LNAs), or one or more peptide nucleic acids (PNAs), or The compound according to any one of claims 1 to 12, wherein the anchor oligonucleotide comprises one or more S-restricted ethyl (cET) or 2'-methoxyribonucleotides.

14. The anchor oligonucleotide may consist of alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, The anchor oligonucleotide comprises alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, and contains at least two adjacent phosphorothioate internucleotide bonds at the 5' and 3' ends, or The compound according to any one of claims 1 to 13, wherein the anchor oligonucleotide comprises alternating 2'-O-methylribonucleotides and 2'-fluororibonucleotides, and includes phosphorothioate internucleotide bonds at all nucleotide positions.

15. The anchor oligonucleotide comprises at least two adjacent 2',4'-restricted 2'O-ethyl crosslinked nucleic acids at its 5' and 3' ends. The anchor oligonucleotide is 2',4'-constrained at all nucleotide positions. - Ethyl-crosslinked nucleic acids, containing phosphorothioate internucleotide bonds between adjacent nucleotides, The compound according to any one of claims 1 to 14, wherein the anchor oligonucleotide comprises 2'-O-methylribonucleotide and 2'-fluororibonucleotide alternately, with at least two 2',4'-restricted 2'O-ethyl crosslinked nucleic acids at the 5' and 3' ends.

16. The compound according to any one of claims 1 to 15, wherein at least two oligonucleotides are crosslinked.

17. A compound according to any one of claims 1 to 16, further comprising nanoparticles, an insert, a polycation, or a mixture thereof.

18. a) The length of the first oligonucleotide is between 21 and 25 nucleotides; b) The length of the second oligonucleotide is between 13 and 17 nucleotides; or a) The length of the first oligonucleotide is 21 nucleotides; b) The length of the second oligonucleotide is 13 nucleotides; and c) The anchor oligonucleotide has a length of 8 nucleotides, or a) The length of the first oligonucleotide is 23 nucleotides; b) The length of the second oligonucleotide is 15 nucleotides; and c) The anchor oligonucleotide has a length of 8 nucleotides, or a) The length of the first oligonucleotide is 25 nucleotides; b) The length of the second oligonucleotide is 17 nucleotides; and c) The anchor oligonucleotide has a length of 8 nucleotides. The compound according to any one of claims 1 to 17.

19. The first oligonucleotide has a length of 21 nucleotides and is complementary to the target mRNA; The second oligonucleotide has a length of 13 nucleotides and is complementary to nucleotides 1-13 of the first oligonucleotide, and The anchor oligonucleotide has a length of 8 nucleotides and is complementary to nucleotides 14-21 of the first oligonucleotide. Here, the second oligonucleotide is bound at its 3' end to a molecule containing cholesterol, docosahexaenoic acid, or N-acetylgalactosamine. Here, the anchor oligonucleotide is bonded to a polyethylene glycol polymer having a molecular weight between 10,000 and 40,000 Da. Here, the first oligonucleotide includes complementarity to both the second oligonucleotide and the anchor oligonucleotide in order to form an asymmetric double helix. The compound according to claim 1.

20. A pharmaceutical composition comprising a compound according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition for treating a disease or disorder in a patient requiring treatment, wherein the treatment comprises administering to the patient a therapeutically effective amount of the pharmaceutical composition according to claim 20.

22. A pharmaceutical composition for treating a subject having a disease or disorder related to a target in the liver of the subject, wherein the pharmaceutical composition comprises an siRNA compound, a) A first oligonucleotide comprising 16 to 25 consecutive nucleotides, a 5' end, and a 3' end, and having complementarity with the target; b) A second oligonucleotide comprising 12 to 17 consecutive nucleotides, a 5' end, and a 3' end; (Here, the second oligonucleotide chain forms an asymmetric double helix in complement to the 5' end of the first oligonucleotide chain, and includes an N-acetylgalactosamine (GalNAc) moiety or a derivative thereof to which the second oligonucleotide is bound at the 3' end.) c) A pharmacokinetic (PK) modifying anchor comprising at least one polymer and an anchor oligonucleotide bound at its 5' end, A pharmaceutical composition in which the anchor oligonucleotide comprises 5 to 8 nucleotides complementary to the 3' end of the first oligonucleotide, and the polymer is between 2,000 Da and 100,000 Da.

23. An asymmetrical double helix comprising a first oligonucleotide chain, a second oligonucleotide chain, and an anchor oligonucleotide chain, wherein each oligonucleotide chain comprises at least one chemically modified nucleotide. Here, the anchor oligonucleotide chain includes a portion that modifies pharmacokinetics, The length of the first oligonucleotide chain, including the 5' and 3' ends, consists of 21 to 25 nucleotides and is complementary to the target. The length of the second oligonucleotide chain, including the 5' and 3' ends, consists of 13 to 17 nucleotides, complementing the 5' end of the first oligonucleotide chain and forming an asymmetric double helix. The anchor oligonucleotide chain is complementary to the 3' end of the first oligonucleotide chain, has a length including a 5' end and a 3' end, and consists of 5 to 8 nucleotides. An siRNA compound in which the 5' end is bound to a pharmacokinetic modifying moiety, and the pharmacokinetic modifying moiety forms an asymmetric double helix containing a polyethylene glycol polymer with a molecular weight between 10,000 and 40,000 daltons.

24. The double-stranded region is 13, 14, 15, 16, or 17 base pairs, The first oligonucleotide chain has a length of 21 to 23 nucleotides, and the second oligonucleotide chain has a length of 13 to 16 nucleotides. The first oligonucleotide chain has a length of 21 nucleotides, the second oligonucleotide chain has a length of 13 nucleotides, and the anchor oligonucleotide chain has a length of 8 nucleotides. At least one chemically modified nucleotide comprises a 2'-O-methyl-ribonucleotide, a 2'-fluoro-ribonucleotide, a phosphorothioate nucleotide interbond, a locked nucleic acid, a 2',4'-restricted 2'O-ethyl crosslinked nucleic acid, a peptide nucleic acid, or a mixture thereof. The siRNA compound according to claim 23, wherein the second oligonucleotide chain comprises a ligand to which it is bound at the 5' end, the 3' end, an internal position, or a combination thereof.

25. The siRNA compound according to claim 24, wherein the ligand of the second oligonucleotide comprises a lipid, a lipophilic substance, 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.

26. An asymmetric double helixen comprising a first oligonucleotide chain of 21 nucleotides in length, a second oligonucleotide chain of 13 nucleotides in length, and an anchor oligonucleotide chain of 8 nucleotides in length, The first oligonucleotide chain is complementary to the target, and the second oligonucleotide chain forms an asymmetric double helix in complement to the 5' end of the first oligonucleotide chain, and The anchor oligonucleotide chain is complementary to the 3' end of the first oligonucleotide chain and includes a 5' end and a 3' end. Here, each oligonucleotide chain alternately contains 2'-fluororibonucleotide and 2'-methoxyribonucleotide. The first oligonucleotide chain includes two adjacent phosphorothioate nucleotide bonds at its 5' end and eight adjacent phosphorothioate nucleotide bonds at its 3' end. The second oligonucleotide chain includes an internucleotide bond between two adjacent phosphorothioates at its 5' end, an internucleotide bond between two adjacent phosphorothioates at its 3' end, and a linker at its 3' end that is bound to a molecule containing cholesterol, docosanate (DCA), docosahexaenoic acid, or N-acetylgalactosamine. An siRNA compound comprising an anchor oligonucleotide chain containing seven adjacent nucleotide-nucleotide bonds of phosphorothioates at its 5' end and a linker at its 3' end that is bound to a pharmacokinetic modifying portion containing a polyethylene glycol polymer with a molecular weight between 10,000 and 40,000 Da.

27. A pharmaceutical composition for treating a patient having a disease or disorder, wherein a therapeutically effective amount of the pharmaceutical composition is administered to the patient, comprising the siRNA compound described in claim 23.

28. A pharmaceutical composition comprising the siRNA compound described in claim 23.

29. The compound according to claim 1, wherein the first oligonucleotide and the anchor oligonucleotide contain 35 to 100% GC content.

Images