TECHNICAL FIELD

This disclosure relates to compounds comprising novel oligonucleotide-based pharmacokinetic (PK)-modifying anchors with useful applications for RNA interference (RNAi) and other gene therapy technologies. The PK-modifying anchors described herein are patterned to enable efficient modulation of absorption, distribution and clearance kinetics of therapeutic oligonucleotides to enhance their tissue distribution. Efficient modulation of the absorption, distribution and clearance kinetics can be achieved in blood/plasma, cerebrospinal fluid (CSF) and other relevant bodily/biological fluids and tissues.

BACKGROUND

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

Similarly, when administered IV or SC, oligonucleotides are rapidly removed systemically by elimination through the kidney filtration or via the reticuloendothelial system. Retention in secondary tissues beyond liver, kidney, bone marrow and spleen is a real challenge in the field.

There remains a need for self-delivering siRNA that is characterized by efficient RISC entry, minimum 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

The present invention is based on the discovery of compounds comprising novel platform, dynamic pharmacokinetic (“PK”)-modifying anchors, where the anchors enable efficient modulation of the absorption, distribution, and clearance kinetics of component therapeutic oligonucleotides in blood/plasma, CSF, and other bodily/biological fluids and tissues. A panel of block co-polymers (e.g., poloxamer 188 and the like) are provided herein that function as non-immunogenic PK modifiers and which are compatible with oligonucleotide chemistry.

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

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

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

In certain embodiments, the compound comprises formula (I):
Xc-L-O-Z  (I)
wherein:O is the first oligonucleotide;L is a linker, and is present or absent;Xcis selected from the group consisting of a hydrophobic moiety, a sugar, a peptide, an aptamer and a nucleic acid, and is present or absent; andZ is the PK-modifying anchor.

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

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

In certain embodiments, the first oligonucleotide comprises complementarity to a target.

In certain embodiments, the first oligonucleotide comprises perfect complementarity to a target.

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, Xcis 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, Xccomprises an N-Acetylgalactosamine (GalNAc) moiety or a derivative thereof. In certain embodiments, Xchas an affinity for one or both of a low-density lipoprotein and an intermediate density lipoprotein. In certain embodiments, Xcis a saturated or unsaturated moiety having fewer than three double bonds. In certain embodiments, Xchas an affinity for high density lipoprotein. In certain embodiments, Xcis a polyunsaturated moiety having three or more double bonds.

In certain embodiments, the at least one polymer is selected from the group consisting of a hydrophobic polycarbonate, a polyester, an amphiphilic block copolymer, a hydrophobic block polymer, a polysaccharide and a polypeptide. In certain embodiments, the hydrophobic polycarbonate is poly(DTR carbonate). In certain embodiments, the polyester is selected from the group consisting of a polyhydroxyalkanoate, a polycaprolactone, a poly(hyroxybuterate-hydroxyvalerate), a polyglycolic acid and a 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(diphenylamine) and poly(tetrahydrofurfuryl methacrylate). In certain embodiments, the polysaccharide is selected from the group consisting of a soluble polyglucose, a non-soluble polyglucose, cellulose, glycogen and amylopectin. In certain embodiments, the polypeptide is a polylysine, a polyarginine, a polyalanine, a polyisoleucine, a polymethionine, a polyphenylalanine, a polyvaline, a polyproline and a polyglycine and any combinations 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.

In certain embodiments, the PK-modifying anchor comprises more than one polymer.

In certain embodiments, the PK-modifying anchor comprises 2, 3 or 4 polymers.

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

In certain embodiments, the first oligonucleotide comprises one or more chemically-modified nucleotides. In certain embodiments, the first oligonucleotide is fully chemically-modified or partially chemically-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-constrained-ethyls (cETs). In certain embodiments, the first oligonucleotide comprises about 50% 2′-methoxy-ribonucleotides, about 55% 2′-methoxy-ribonucleotides, about 60% 2′-methoxy-ribonucleotides, about 65% 2′-methoxy-ribonucleotides, about 70% 2′-methoxy-ribonucleotides, about 75% 2′-methoxy-ribonucleotides, about 80% 2′-methoxy-ribonucleotides, about 85% 2′-methoxy-ribonucleotides, about 90% 2′-methoxy-ribonucleotides, about 95% 2′-methoxy-ribonucleotides, about 96% 2′-methoxy-ribonucleotides, about 97% 2′-methoxy-ribonucleotides, about 98% 2′-methoxy-ribonucleotides, about 99% 2′-methoxy-ribonucleotides or about 100% 2′-methoxy-ribonucleotides. In certain embodiments, the first oligonucleotide comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides. In certain embodiments, the nucleotides of the first oligonucleotide are connected via phosphodiester linkages, phosphorothioate linkages or a combination of phosphodiester and phosphorothioate linkages.

In certain embodiments, (1) the first oligonucleotide comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides, wherein each nucleotide is a 2′-methoxy-ribonucleotide or a 2′-fluoro-ribonucleotide, and the nucleotides at positions 2 and 14 from the 5′ end of the first oligonucleotide are not 2′-methoxy-ribonucleotides; and (2) the nucleotides of the first oligonucleotide are connected to adjacent nucleotides via phosphodiester or phosphorothioate linkages, wherein 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.

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

In certain embodiments, the second oligonucleotide comprises a conjugate moiety, Xc. In certain embodiments, the second oligonucleotide is attached to Xcwith 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, Xcis attached at the 5′ end, at the 3′ end, at an internal position, or a mixture thereof of the second oligonucleotide. In certain embodiments, Xcis attached at the 3′ end of the second oligonucleotide. In certain embodiments, Xcis 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, Xccomprises an N-Acetylgalactosamine (GalNAc) moiety or a derivative thereof. In certain embodiments, Xchas an affinity for one or both of a low-density lipoprotein and an intermediate density lipoprotein. In certain embodiments, Xcis a saturated or unsaturated moiety having fewer than three double bonds. In certain embodiments, Xchas an affinity for high density lipoprotein. In certain embodiments, Xcis a polyunsaturated moiety having three or more double bonds.

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 relative to the first oligonucleotide.

In certain embodiments, 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-constrained-ethyls (cETs). In certain embodiments, the anchor oligonucleotide comprises alternating 2′-O-methyl ribonucleotides and 2′-fluoro ribonucleotides. In certain embodiments, the anchor oligonucleotide comprises alternating 2′-O-methyl ribonucleotides and 2′-fluoro ribonucleotides and at least two adjacent phosphorothioate internucleotide linkages at a 5′ end and a 3′ end. In certain embodiments, the anchor oligonucleotide comprises alternating 2′-O-methyl ribonucleotides and 2′-fluoro ribonucleotides and phosphorothioate internucleotide linkages at every nucleotide position. In certain embodiments, the anchor oligonucleotide comprises at least two adjacent 2′,4′-constrained 2′O-ethyl bridged nucleic acids at a 5′ end and a 3′ end. In certain embodiments, the anchor oligonucleotide comprises a 2′,4′-constrained 2′O-ethyl bridged nucleic acids at every nucleotide position and phosphorothioate internucleotide linkages between each adjacent nucleotide. In certain embodiments, the anchor oligonucleotide comprises alternating 2′-O-methyl ribonucleotides and 2′-fluoro ribonucleotides and at least two 2′,4′-constrained 2′O-ethyl bridged nucleic acids at a 5′ end and a 3′ end. In certain embodiments, the anchor oligonucleotide comprises a peptide nucleic acid at every nucleotide position.

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

In certain embodiments, at least two of the oligonucleotides are cross-linked.

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

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

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

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

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

In certain embodiments, the polymer comprises PEG.

In certain embodiments, the PK-modifying anchor influences stability of an oligonucleotide therapeutic agent over time in a body part comprising, a heart, a kidney, a liver, a spleen, an adrenal gland, a pancreas, a lung, blood, plasma, a brain, or a mixture thereof, wherein the effect comprises a change in volume of distribution, area under curve, clearance, half-life maximum concentration, bioavailability, or a mixture thereof.

In certain embodiments, the number of nucleotides in the first oligonucleotide comprises a same number of nucleotides in the second oligonucleotide and anchor oligonucleotide combined. In certain embodiments, the number of nucleotides in the first oligonucleotide comprises a greater number of nucleotides than in the second oligonucleotide and anchor oligonucleotide combined. In certain embodiments, the number of nucleotides in the first oligonucleotide comprises a lesser number of nucleotides than in the second oligonucleotide and anchor oligonucleotide combined.

In one aspect, the disclosure provides a compound comprising: a first oligonucleotide, wherein the first oligonucleotide is 21 nucleotides in length, with a 5′ end, a 3′ end, and is complementary to a target; a second oligonucleotide, wherein the second oligonucleotide is 13 nucleotides in length, with a 5′ end, a 3′ end, and is complementary to nucleotides 1-13 of the first oligonucleotide; and an anchor oligonucleotide, wherein the anchor oligonucleotide is 8 nucleotides in length, with a 5′ end, a 3′ end, and is complementary nucleotides 14-21 of the first oligonucleotide, wherein the second oligonucleotide is attached to a molecule comprising cholesterol, dichloroacetate, docosahexaenoic acid, or N-acetylgalactosamine at a 3′ end, wherein the anchor oligonucleotide is attached to a polyethylene glycol polymer comprising a molecular weight of between 10,000 and 40,000 Daltons, and wherein the first oligonucleotide comprises complementarity to both the second oligonucleotide and the anchor oligonucleotide to form the asymmetric duplex.

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

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

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

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

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

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

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

In certain embodiments, the first oligonucleotide strand and second oligonucleotide strand form an siRNA or dsRNA having a duplex region. In certain embodiments, the duplex region is about 10 to about 50 base pairs in length. In certain embodiments, the duplex region is about 10 to about 15, about 10 to about 20, about 10 to about 25, or about 10 to about 30 base pairs in length. In certain embodiments, the duplex region is 13, 14, 15, 16, 17, or 18 base pairs.

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

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

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

In certain embodiments, the ligand of the second strand comprises a lipid, a lipophile, a terpene, a sugar, a peptide, a protein, an alkyl chain, a lectin, a glycoprotein, a hormone, 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. In certain embodiments, the ligand of the second strand comprises a fatty acid, a steroid, a secosteroid, a polyamine, a ganglioside, a nucleoside analog, an endocannabinoid, an omega-3 fatty acid, an omega-6 fatty acid, an omega-9 fatty acid, a conjugated linolenic acid, a saturated fatty acid, or a mixture thereof. In certain embodiments, the ligand of the second strand comprises cholesterol, docosahexaenoic acid, conjugated phosphatidylcholine, N-acetylgalactosamine, dichloroacetic acid, epithelial cell adhesion molecule aptamer, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneal, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleolyl)lithocholic acid, O3-(oleolyl)cholenic acid, dimethoxytrityl, phenoxazine, or a mixture thereof.

In certain embodiments, the second strand further comprises a linker attaching the ligand to the second strand.

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

In one aspect, the disclosure provides a method of treating a patient having a disease or disorder comprising administering an asymmetric oligonucleotide duplex comprising an first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety and wherein the first oligonucleotide strand can pair with both the second oligonucleotide strand and the anchor oligonucleotide strand to form the asymmetric oligonucleotide duplex.

In one aspect, the disclosure provides an asymmetric hairpin duplex comprising a hairpin oligonucleotide strand and an anchor oligonucleotide strand wherein the hairpin oligonucleotide strand comprises an overhang and can pair with the anchor oligonucleotide strand to form the asymmetric hairpin duplex and wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety.

In one aspect, the disclosure provides a pharmaceutical composition comprising an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and an anchor oligonucleotide strand wherein the anchor oligonucleotide strand comprises a pharmacokinetic-modifying moiety and wherein the first oligonucleotide strand can pair with both the second oligonucleotide strand and the anchor oligonucleotide strand to form the asymmetric oligonucleotide duplex.

In one aspect, the disclosure provides a universal anchor oligonucleotide of about 5-8 nucleotides in length and a pharmacokinetic-modifying moiety at a 5′ end, wherein at least one nucleotide comprises a chemical modification, wherein the anchor oligonucleotide can bind an asymmetric oligonucleotide duplex comprising a first oligonucleotide strand, a second oligonucleotide strand, and wherein the sequence of the oligonucleotide anchor is complementary to a region at a 3′ end of the first oligonucleotide strand.

In certain embodiments, the first strand and the anchor strand comprise a GC content of between about 35 to about 100%.

