NAZEF NAIM (US)
WO2019079781A2 | 2019-04-25 | |||
WO2019075419A1 | 2019-04-18 | |||
WO2010033225A2 | 2010-03-25 | |||
WO2011133871A2 | 2011-10-27 | |||
WO2015188197A2 | 2015-12-10 | |||
WO2014088920A1 | 2014-06-12 | |||
WO2016100401A1 | 2016-06-23 |
US8372968B2 | 2013-02-12 | |||
US8883996B2 | 2014-11-11 | |||
US8513207B2 | 2013-08-20 | |||
US8927705B2 | 2015-01-06 | |||
US20170049909W | 2017-09-01 | |||
US201662383207P | 2016-09-02 | |||
US201662393401P | 2016-09-12 | |||
US8927513B2 | 2015-01-06 | |||
US201662393401P | 2016-09-12 | |||
US20080274462A1 | 2008-11-06 | |||
US20070254362A1 | 2007-11-01 | |||
US20110294869A1 | 2011-12-01 | |||
US201662378635P | 2016-08-23 |
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CLAIMS What is claimed is: 1. An oligonucleotide comprising: a sense strand comprising 17-36 nucleotides, wherein the sense strand has a first region (R1) and a second region (R2), wherein the second region (R2) of the sense strand comprises a first subregion (S1), a second subregion (S2) and a tetraloop (L) or triloop (triL) that joins the first and second regions, wherein the first and second subregions form a second duplex (D2); an antisense strand comprising 20-22 nucleotides, wherein the antisense strand includes at least 1 single-stranded nucleotide at its 3′-terminus, wherein the sugar moiety of the nucleotide at position 5 of the antisense strand is modified with a 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′- fluoro (2′-F), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-[2-(methylamino)-2- oxoethyl] (2′-O-NMA), and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA), and wherein the sense strand and antisense strand are separate strands; and a first duplex (D1) formed by the first region of the sense strand and the antisense strand, wherein the first duplex has a length of 12-20 base pairs and has 7-10 nucleotides that are modified at the 2′-position of the sugar moiety with 2′-F. 2. The oligonucleotide of claim 1, wherein the sugar moiety at positions 2 and 14 of the antisense strand is modified with 2’-F. 3. The oligonucleotide of claim 2, wherein the sugar moiety at each of up to 3 nucleotides at positions 1, 3, 7, and 10 of the antisense strand is additionally modified with 2’-F. 4. The oligonucleotide of any one of claims 1-3, wherein the sugar moiety of each of the nucleotides at positions 8-11 of the sense strand is additionally modified with 2′-F. 5. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 1-7 and 12-17 or 12-20 of the sense strand are modified with a 72 modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2’-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-[2- (methylamino)-2-oxoethyl] (2′-O-NMA),, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′- FANA). 6. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 2, 5, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2’-O-methyl (2′-OMe), 2’-O-methoxyethyl (2′-MOE), and 2’- deoxy-2’-fluoro-β-d-arabinonucleic acid (2′-FANA). 7. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 1, 2, 5, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′- deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). 8. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′- deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). 9. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′- deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). 73 10. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 10, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′- deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). 11. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 2, 3, 5, 10, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′- deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). 12. The oligonucleotide of claim 1, wherein the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′- deoxy-2′-fluoro-β-d-arabinonucleic acid (2′-FANA). 13. The oligonucleotide of claim 1 or 12, wherein the antisense strand has 3 nucleotides that are modified at the 2′-position of the sugar moiety with 2′-F. 14. The oligonucleotide of any of the preceding claims, wherein the second duplex has a length of 1-6 base pairs. 15. The oligonucleotide of any of the preceding claims, wherein the second duplex comprises at least one bicyclic nucleotide. 16. The oligonucleotide of claim 15, wherein the second duplex has a length of 1-3 base pairs. 74 17. The oligonucleotide of any of the preceding claims, wherein the triloop has a nucleotide sequence of GAA or AAA or wherein the tetraloop is an RNA tetraloop selected from the group consisting of GAAA, UNCG, GNRA, or CUUG or a DNA tetraloop selected from the group consisting of d(GNAB), d(CNNG), or d(TNCG), wherein N is any one of U, A, C, G and R is G or A. 18. The oligonucleotide for reducing RNA expression of claim 1, wherein the sugar moiety of each nucleotide in the second duplex is modified with 2′-O-methyl (2′-OMe). 19. The oligonucleotide for reducing RNA expression of any of the preceding claims, wherein at least one of the nucleotides in the tetraloop or the triloop is conjugated to a ligand. 20. The oligonucleotide for reducing RNA expression of claim 19, wherein 1-3 nucleotides in the triloop or 1-4 nucleotides in the tetraloop are conjugated to a ligand. 21. The oligonucleotide for reducing RNA expression of claim 19 or 20, wherein the ligand comprises N-acetylgalactosamine. 22. The oligonucleotide for reducing RNA expression of the preceding claims, wherein the nucleotide at position 1 of the antisense strand comprises a phosphate mimic. 23. The oligonucleotide for reducing RNA expression of any of the preceding claims, wherein the sense strand comprises 36 nucleotides and the antisense strand comprises 22 nucleotides. 24. A single-stranded oligonucleotide comprising 20-22 nucleotides, wherein the sugar moiety of each of the nucleotides at positions 2, 5, and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7, and 10 of the antisense strand is modified with 2′-F and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from the group consisting of 2′-O-propargyl, 2′-O-propylamin, 2′-amino, 2′-ethyl, 2′-aminoethyl (EA), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-[2- (methylamino)-2-oxoethyl] (2′-O-NMA), and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (2′- FANA). 75 25. The single-stranded oligonucleotide of claim 24, wherein the single-stranded oligonucleotide comprises 20 nucleotides. 26. The single-stranded oligonucleotide of claim 24, wherein the single-stranded oligonucleotide comprises 21 nucleotides. 27. The single-stranded oligonucleotide of claim 24, wherein the single-stranded oligonucleotide comprises from 20 to 23 nucleotides. 28. A pharmaceutical composition comprising any one of the preceding claims and a pharmaceutically acceptable carrier. 29. A method for reducing expression of a target gene in a subject, comprising administering the oligonucleotide of any one of claims 1-23, the single stranded oligonucleotide of claims 24-27, or the composition of claim 28 to the subject in an amount sufficient to reduce expression of a target gene in the subject. 30. A method of treating or preventing a disease or disorder in a subject comprising administering to the subject the oligonucleotide of any one of claims 1-23, the single stranded oligonucleotide of claims 24-27, or the composition of claim 28 in an amount sufficient to inhibit expression of a gene causing disease in the subject. 76 |
Table A: Sequence information for the oligonucleotides in Tables 1-8.
In the modification patterns of Table A:
“M” refers to a 2'-OMe modified nucleotide;
“F” refers to a 2'-F modified nucleotide;
“S” refers to a nucleotide with a 3’-phosphorothioate linkage;
“{MS}” refers to a 2'-OMe modified nucleotide with a 3’-phosphorothioate linkage; “{FS}” refers to a 2'-F modified nucleotide with a 3’-phosphorothioate linkage; “[prg-peg-GalNAc]” refers to a nucleotide having a 2’-GalNAc conjugate:
“{Px-FS}” refers to a 2'-F modified nucleotide with a 3’-phosphorothioate linkage, and 5’ phosphonate or vinylphosphonate; “{Px-MS}” refers to a 2 -OMe modified nucleotide with a 3’-phosphorothioate linkage, and 5’ phosphonate or vinylphosphonate.
In the modified sequences of Table A:
“[mN]” refers to a 2'-OMe modified nucleotide; “[fN]” refers to a 2'-F modified nucleotide;
“[Ns]” refers to a nucleotide with a 3’-phosphorothioate linkage;
“[mNs]” refers to a 2'-OMe modified nucleotide with a 3’-phosphorothioate linkage; “[fNs]” refers to a 2'-F modified nucleotide with a 3’ -phosphor othioate linkage; “[prgG-peg-GalNAc]” refers to a G nucleotide having a 2’-GalNAc conjugate:
“[prgA-peg-GalNAc]” refers to an A nucleotide having a 2’-GalNAc conjugate:
[5VPfUs]” refers to a 5'-vinylphosphonate 2'-F uridine with a 3’-phosphorothioate linkage:
“[5VPmUs]” refers to a 5’-vinylphosphonate 2'-OMe uridine with a 3’-phosphorothioate linkage:
“ [Phosphonate-40-mUs]” refers to a 5'-phosphonate-4’-Oxy-2'-OMe uridine with a 3 ’ -phosphorothioate linkage:
[0086] In some embodiments, the antisense strand has 3 nucleotides that are modified at the 2’-position of the sugar moiety with a 2′-F. In some embodiments, the sugar moiety at positions 2, 5, and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7, and 10 of the antisense strand are modified with a 2’-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 5, and 14 of the antisense strand is modified with the 2’-F. In other embodiments, the sugar moiety at each of the positions at positions 1, 2, 5, and 14 of the antisense strand is modified with the 2’-F. In still other embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 7, and 14 of the antisense strand is modified with the 2’-F. In yet another embodiment, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 10, and 14 of the antisense strand is modified with the 2’-F. In another embodiment, the sugar moiety at each of the positions at positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with the 2'-F. (b) 5′ Terminal Phosphates [0087] In some embodiments, 5’-terminal phosphate groups of oligonucleotides enhance the interaction with Argonaute 2. However, oligonucleotides comprising a 5’-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5’ phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 1′ end of an oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5′-phosphate group (“phosphate mimic”). [0088] In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”). See, for example, International Patent Application PCT/US2017/049909, filed on September 1, 2017, U.S. Provisional Application numbers 62/383,207, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, filed on September 2, 2016, and 62/393,401, filed on September 12, 2016, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, the contents of each of which relating to phosphate analogs are incorporated herein by reference. In some embodiments, an oligonucleotide provided herein comprises a 4′-phosphate analog at a 5′-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. In other embodiments, a 4′-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4′-carbon of the sugar moiety or analog thereof. In certain embodiments, a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula –O–CH 2 –PO(OH) 2 or –O–CH 2 –PO(OR) 2 , in which R is independently selected from H, CH 3 , an alkyl group, CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , CH 2 OCH 2 CH 2 Si (CH 3 ) 3 , or a protecting group. In certain embodiments, the alkyl group is CH 2 CH 3 . More typically, R is independently selected from H, CH 3 , or CH 2 CH 3 . (c). Modified Intranucleoside Linkages [0089] In some embodiments, an oligonucleotide may comprise a modified internucleoside linkage. In some embodiments, phosphate modifications or substitutions may result in an oligonucleotide that comprises at least one (e.g., at least 1, at least 2, at least 3 or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages. [0090] A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage. [0091] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. (d) Base modifications [0092] In some embodiments, oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1′ position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain nitrogen atom. See e.g., U.S. Published Patent Application No. 20080274462. In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic). [0093] In some embodiments a universal base is a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference single- stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid comprising the mismatched base. [0094] Non-limiting examples of universal-binding nucleotides include inosine, 1-β-D- ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. NUCLEIC ACIDS RES. 1995 Nov 11;23(21):4363-70; Loakes et al., 3- Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. NUCLEIC ACIDS RES. 1995 Jul 11;23(13):2361-6; Loakes and Brown, 5-Nitroindole as a universal base analogue, NUCLEIC ACIDS RES. 1994 Oct 11;22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications). (e) Reversible Modifications [0095] While certain modifications to protect an oligonucleotide from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione). [0096] In some embodiments, a reversibly modified nucleotide comprises a glutathione- sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See U.S. Published Application No. 2011/0294869 originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015/188197 to Solstice Biologics, Ltd. (“Solstice”), Meade et al., NATURE BIOTECHNOLOGY, 2014,32:1256-1263 (“Meade”), PCT Publication No. WO 2014/088920 to Merck Sharp & Dohme Corp, each of which are incorporated by reference for their disclosures of such modifications. This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g. glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al. J. A M . C HEM . S OC .2003,125:940-950). [0097] In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and/or lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed, and the result is a cleaved oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release. [0098] In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2’-carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5′-carbon of a sugar, particularly when the modified nucleotide is the 5′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3′-carbon of sugar, particularly when the modified nucleotide is the 3′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., U.S. Prov. Appl. No. 62/378,635, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof, which was filed on August 23, 2016, and the contents of which are incorporated by reference herein for its relevant disclosures. (iv) Targeting Ligands [0099] In some embodiments, it may be desirable to target the oligonucleotides of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs or may avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit for the oligonucleotide. Accordingly, in some embodiments, oligonucleotides disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, oligonucleotides disclosed herein may be modified to facilitate delivery of the oligonucleotide to the hepatocytes of the liver. In some embodiments, an oligonucleotide comprises a nucleotide that is conjugated to one or more targeting ligand. [0100] A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer. For example, a targeting ligand may be an RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferring, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties. [0101] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligand are conjugated to a 2 to 4 nucleotide overhang or extension on the 5’ or 3’ end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5’ or 3’ end of the sense strand and 1, 2, 3 or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand. [0102] GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotides of the instant disclosure may be used to target these oligonucleotides to the ASGPR expressed on cells. [0103] In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide of the instant disclosure is conjugated to a one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties. [0104] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of tetraloop are each conjugated to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of triloop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5’ or 3’ end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand where each GalNAc moiety is conjugated to one nucleotide. [0105] In some embodiments, an oligonucleotide herein comprises a monovalent GalNAc attached to a guanidine nucleotide, referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol- Guanidine-GalNAc, as depicted below: [0106] In some embodiments, an oligonucleotide herein comprises a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol- Adenine-GalNAc, as depicted below.
[0107] An example of such conjugation is shown below for a loop comprising from 5′ to 3′ the nucleotide sequence GAAA (L = linker, X = heteroatom) stem attachment points are shown. Such a loop may be present, for example, at positions 27-30 of the molecule shown in FIG.1A. In the chemical formula, is used to describe an attachment point to the oligonucleotide strand.
[0108] Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication Number WO2016100401 A1, which published on June 23, 2016, and the contents of which is incorporated herein by reference in its entirety. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. [0109] An example is shown below for a loop comprising from 5′ to 3′ the nucleotides GAAA, in which GalNac moieties are attached to nucleotides of the loop using an acetal linker. Such a loop may be present, for example, at positions 27-30 of the molecule shown in FIG.10. In the chemical formula, is an attachment point to the oligonucleotide strand. [0110] Any appropriate method or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal- based linkers are disclosed, for example, in International Patent Application Publication Number WO2016100401 A1, which published on June 23, 2016, and the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. A “labile linker” refers to a linker that can be cleaved, e.g., by acidic pH. A “fairly stable linker” refers to a linker that cannot be cleaved. [0111] In some embodiments, a duplex extension (e.g., of up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide. In some embodiments, the oligonucleotides of the present disclosure do not have a GalNAc conjugated. III. Formulations [0112] Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. [0113] Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotides into cells. For example, cationic lipids, such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine, can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer’s instructions. [0114] Accordingly, in some embodiments, a formulation comprises a lipid nanoparticle. In some embodiments, an excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013). [0115] In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin). [0116] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. [0117] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. [0118] In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent or more, although the percentage of the active ingredient(s) may be between about 1% 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. [0119] Even though a number of embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated. IV. Methods of Use (a) Reducing RNA Expression in Cells [0120] In some embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of RNA in the cell. Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses RNA (e.g., hepatocytes, macrophages, monocyte-derived cells, prostate cancer cells, cells of the brain, endocrine tissue, bone marrow, lymph nodes, lung, gall bladder, liver, duodenum, small intestine, pancreas, kidney, gastrointestinal tract, bladder, adipose and soft tissue and skin). In some embodiments, the cell is a primary cell that has been obtained from a subject and that may have undergone a limited number of a passages, such that the cell substantially maintains is natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides. [0121] In some embodiments, oligonucleotides disclosed herein can be introduced using appropriate nucleic acid delivery methods including injection of a solution containing the oligonucleotides, bombardment by particles covered by the oligonucleotides, exposing the cell or organism to a solution containing the oligonucleotides, or electroporation of cell membranes in the presence of the oligonucleotides. Other appropriate methods for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others. [0122] The consequences of inhibition can be confirmed by an appropriate assay to evaluate one or more properties of a cell or subject, or by biochemical techniques that evaluate molecules indicative of RNA expression (e.g., RNA, protein). In some embodiments, the extent to which an oligonucleotide provided herein reduces levels of expression of RNA is evaluated by comparing expression levels (e.g., mRNA or protein levels to an appropriate control (e.g., a level of RNA expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of RNAi expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean. [0123] In some embodiments, administration of an oligonucleotide as described herein results in a reduction in the level of RNA expression in a cell. In some embodiments, the reduction in levels of RNA expression may be a reduction to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower compared with an appropriate control level of RNA. The appropriate control level may be a level of RNAi expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell according to a method disclosed herein is assessed after a finite period of time. For example, levels of RNA may be analyzed in a cell at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introduction of the oligonucleotide into the cell. [0124] In some embodiments, an oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotides (e.g., its sense and antisense strands). In some embodiments, an oligonucleotide is delivered using a transgene that is engineered to express any oligonucleotide disclosed herein. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject. (b) Treatment Methods [0125] Aspects of the disclosure relate to methods for reducing RNA expression in for attenuating the onset or progression of various diseases. In some embodiments, the disclosure provides methods for using RNAi oligonucleotides of the invention for treating subjects having or suspected of having liver conditions such as, for example, cholestatic liver disease, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). In some embodiments, the disclosure provides RNAi oligonucleotides described herein for use in treating subjects having or suspected of having liver conditions such as, for example, cholestatic liver disease, NAFLD and NASH. In some embodiments, the disclosure provides RNAi for the preparation of a medicament for treatment of subjects having or suspected of having liver conditions such as, for example, cholestatic liver disease, NAFLD and nonalcoholic steatohepatitis NASH. [0126] In a further aspect, the present invention relates to a method for treating a subject having a disease or at risk of developing a disease caused by the expression of a target gene. In this embodiment, the oligonucleotides can act as novel therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, hematopoietic disorders, cardiovascular disorders, liver disorders, viral diseases, or metabolic disorders. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that expression of the target gene is silenced. Because of their high specificity, the oligonucleotides of the present invention specifically target mRNAs of target genes of diseased cells and tissues. [0127] In the prevention of disease, the target gene may be one which is required for initiation or maintenance of the disease, or which has been identified as being associated with a higher risk of contracting the disease. In the treatment of disease, the oligonucleotide can be brought into contact with the cells or tissue exhibiting the disease. For example, oligonucleotide substantially identical to all or part of a mutated gene associated with cancer, or one expressed at high levels in tumor cells, e.g., aurora kinase, may be brought into contact with or introduced into a cancerous cell or tumor gene. [0128] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin. As used herein, the terms “cancer,” “hyperproliferative,” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state of condition characterized by rapidly proliferating cell growth. These terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Proliferative disorders also include hematopoietic neoplastic disorders, including diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. [0129] The present invention can also be used to treat a variety of immune disorders, in particular those associated with overexpression of a gene or expression of a mutant gene. Examples of hematopoietic disorders or diseases include, without limitation, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, kerato-conjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing, loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy. [0130] In another embodiment, the invention relates to a method for treating viral diseases, including but not limited to human papilloma virus, hepatitis C, hepatitis B, herpes simplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus. Oligonucleotides of the invention are prepared as described herein to target expressed sequences of a virus, thus ameliorating viral activity and replication. The molecules can be used in the treatment and/or diagnosis of viral infected tissue, both animal and plant. Also, such molecules can be used in the treatment of virus-associated carcinoma, such as hepatocellular cancer. [0131] The oligonucleotide of the present invention can also be used to inhibit the expression of the multi-drug resistance 1 gene ("MDR1"). "Multi-drug resistance" (MDR) broadly refers to a pattern of resistance to a variety of chemotherapeutic drugs with unrelated chemical structures and different mechanisms of action. Although the etiology of MDR is multifactorial, the overexpression of P-glycoprotein (Pgp), a membrane protein that mediates the transport of MDR drugs, remains the most common alteration underlying MDR in laboratory models (Childs and Ling, 1994). Moreover, expression of Pgp has been linked to the development of MDR in human cancer, particularly in the leukemias, lymphomas, multiple myeloma, neuroblastoma, and soft tissue sarcoma (Fan et al.). Recent studies showed that tumor cells expressing MDR-associated protein (MRP) (Cole et al., 1992), lung resistance protein (LRP) (Scheffer et al., 1995) and mutation of DNA topoisomerase II (Beck, 1989) also may render MDR. [0132] In some embodiments, the target gene may be a target gene from any mammal, such as a human target. Any gene may be silenced according to the method described herein. Exemplary target genes include, but are not limited to, Factor VII, Eg5, PCSK9, TPX2, apoB, LDHA, SAA, TTR, HBV, HCV, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, HMGB1 gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21(WAFl/CIPl) gene, p27(KIPl) gene, PPM1D gene, HAO1 gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumor suppressor genes, p53 tumor suppressor gene, LDHA, HMGB1, HAO1, and combinations thereof. [0133] Methods described herein are typically involved administering to a subject in an effective amount of an oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that is capable of treating a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject’s size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently. [0134] In some embodiments, a subject is administered any one of the compositions disclosed herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides disclosed herein are administered intravenously or subcutaneously. [0135] As a non-limiting set of examples, the oligonucleotides of the instant disclosure would typically be administered quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly. For example, the oligonucleotides may be administered every week or at intervals of two, or three weeks. The oligonucleotides may be administered daily. [0136] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters. EXAMPLES [0137] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope. Example 1: Sense Strand Analyzed by Replacing 2′-F with 2′-OMe at Positions 17 and 19. [0138] A double stranded RNA (dsRNA) that targets HAO1was selected for structure activity relationship (SAR) analysis. The dsRNA comprises a tetraloop, where each base is conjugated to a simple sugar, N-acetylgalactosamine (GalNAc). The sense and antisense strands of the dsRNA are modified with 2′-F at positions 8-11 and at positions 2 and 14, respectively. These modifications increased RNAi potency as compared to the dsRNA modified with 2′-OMe at the same positions. Accordingly, the just-noted 2′-F modifications were held constant during SAR described herein. [0139] To test the effects of replacing 2′-F with 2′-OMe, a series of dsRNA were constructed as shown in Table 1. To analyze potency of the dsRNA, HAO1 mRNA knockdown was measured at 48 hours after transfection of different concentrations of dsRNA in a HAO1 stable cell line. Potency was then calculated as half maximal inhibitory concentration (IC 50 ). Similar potency was determined for each of the tested dsRNA as shown in Figures 1A-1C. Taken together, these results demonstrate that 2′-OMe modifications are well tolerated on the sense strand of the dsRNA. Table 1. Sense Strand Structure Activity Relationship (SAR).
Example 2: Antisense Strand Analyzed by Replacing 2' -F with 2'-0Me at Positions 15, 17, and 19.
[0140] As shown in Table 2, the antisense strand was investigated by replacing 2'-F with 2'- OMe at positions 15, 17, and 19 on the antisense strand. Modifications of the sense strand of the dsRNA were kept constant in this analysis (Table 2). Similar potency was determined for each of the tested dsRNA as shown in Figures 2A-2D. Taken together, these results demonstrate that 2'- OMe modifications are well tolerated at positions 15, 17, and 19 of the antisense strand of the dsRNA.
Table 2. Antisense Strand SAR (#1).
Example 3: Antisense Strand Analyzed by Replacing 2'-F with 2'-OMe at Positions 1-10.
[0141] As shown in Table 3, the antisense strand was investigated by replacing 2'-F with 2'- OMe at positions 1-10 on the antisense strand, also referred to as the seed region. As shown in Figures 3A-3H, 2'-OMe modifications at positions 7 and 9 were well tolerated. However, as 2'-F modifications are replaced with 2-OMe at positions 2 and 5, and at other positions in the seed region, the RNAi potency as determined by IC 50 value decreased (Figures 3A-3G). Taken together, the results demonstrate that 2'-OMe is poorly tolerated at the seed region of the antisense strand, and that position 5 prefers modification with 2'-F over 2'-OMe.
Table 3. Antisense Strand SAR (#2).
Example 4: Antisense Strand Analyzed by Replacing 2'-F with 2'-OMe at Positions 1, 6, 8, 10, and 15. [0142] As shown in Table 4, the antisense strand was investigated by replacing 2'-F with 2'- OMe at positions 1, 6, 8, 10, and 15 on the antisense strand. As shown in Figures 4A-4E, 2'-OMe modification at position 15 was well tolerated, which was consistent with results obtained in Example 2. The effect of 2' modification on position 1 of the antisense strand, which contains a phosphate mimic on the 5'-end, was examined. Similar potency between 2'-OMe and 2'-F on position 1 were observed (FIGs. 4C-4D). Next, the effect 2' modification on only positions 2 and 14 of the antisense strand was examined, and similar IC50 values were obtained as compared to others tested (FIGs. 4A-4E). Taken together, the results demonstrate that 2'-OMe is tolerated on the antisense strand.
Table 4. Antisense Strand SAR (#3).
Example 5: Antisense Strand Analyzed by Addition of 2′-F at Positions 3-6. [0143] Next, a low 2′-F pattern (2′-F at positions 2 and 14 only of the antisense strand) was chosen as the starting point, and 2′-F was gradually added in the seed region at positions 3-6 to probe the sensitivity in that region. As shown in Table 5, the starting molecule had the same modification pattern as the last molecule shown in Table 4 except that the molecules contain different phosphate mimics on antisense position 1. Based on the IC 50 results, 2′-F modification at position 5 showed an increase in potency compared to 2′-F modification at positions 3, 4, and 6 (FIGs. 5A-5H). These result further confirmed that position 5 may prefer 2′-F over 2′-OMe in some low 2′-F patterns. Furthermore, increased potency was observed when 2′-F on position 5 was tested in combination with 2′-F on other positions, such as 2′-F at position 3 or position 6 (FIGs.5A-5H). Table 5. Antisense Strand SAR Seed Region (Round 2 – Positions 3-6). 62
Example 6: Antisense Strand Analyzed by Replacing 2'-F with 2'-OMe at Positions 7 to 10, and Maintaining 2'-F at positions 3 and 5. [0144] Next, positions 7 to 10 on the antisense strand were investigated (Table 6). In this analysis, 2'-F modification was maintained at positions 5 and 3, and a phosphate mimic with 2'-F modification was maintained on position 1. As shown in FIG. 6A, control 1 showed an excellent IC50 (3.5 pM) after 66 hrs of transfection in the HAOl stable cell line. In order to probe the impact of 2'-F on positions 7 to 10, 2'-OMe was added on position 9 of the sense strand. This modification will provide a wider dynamic range for examination of the changes in IC50s. As shown in Figure 6, the IC50 of control 2 is >10 fold higher than control 1 (FIGs. 6A-6B). As 2'-F was substituted on positions 7 through 10, an increase in potency was observed (FIGs. 6A-6F). The results showed that the potency was improved with 2'-F modification on position 7 or position 10, but not with 2'- F on position 8 or position 9.
Table 6. Antisense Strand SAR (Round 2 - Positions 7-10)
Example 7: Minimal 2'-F Set forHAOl In Vivo Study
[0145] Taken together, the potency experimental results proved herein demonstrated that the antisense strand is more sensitive to 2'-OMe modifications than the sense strand. Positions on the antisense strand that preferred 2'-F over 2'-OMe were identified, which included positions 2, 3, 5, 7, 10, and 14. Among positions 3, 5, 7, and 10, position 5 was more pronounced in its preference for 2'-F over 2'-OMe. Modification patterns on the just noted positions may provide opportunities to balance potency, duration, and tolerability. The experimental results also showed that the sense strand can tolerate more 2'-OMe modifications than the antisense strand. Further, positions 8-11 on the sense strand preferred 2'-F over 2'-OMe, yet 2'-OMe insertion in this region was tolerated, especially when combined with optimal modifications on the antisense strand.
[0146] To test the in vivo activity of HAOl conjugates comprising minimal 2'-F and heavy 2'- OMe modification patterns, mice were administered the HAOl conjugates, and target knockdown was evaluated. HAOl conjugates tested in mice are shown in Table 7. A HAOl conjugate comprising heavy 2'-F was used as a control.
Table 7. HAOl Conjugates for In Vivo Studies.
[0147] As shown in FIGs. 7A-7H, HAOl conjugates comprising minimal 2'-F and heavy 2'- OMe modification patterns showed excellent potency (IC50s) in vitro in the HAOl stable cell line,
and their IC50s were comparable to the heavy 2′-F control. The HAO1 conjugates shown in Table 7 were also administered to mice by subcutaneous injection of a single dose of 1 mpk. Liver HAO1 mRNA expression relative to the PBS control group was measured 3 days post dose. As shown in FIG.7I, the HAO1 conjugates comprising minimal 2′-F and heavy 2′-OMe modification patterns showed comparable KD activities in vivo compared to those of the heavy 2′-F control. No difference was detected between either 2′-F or 2′-OMe modifications in combination with a phosphate mimic on position 1 of the antisense strand. No difference was observed at day 3 for the comparison of 2′-OMe vs 2′-F on antisense position 1 in combination with a phosphate mimic. These results demonstrated a correlation between the in vitro and in vivo activities of the HAO1 conjugates comprising minimal 2′-F and heavy 2′-OMe modification patterns described herein. Example 8: HAO1 Duration Study [0148] Modification with 2′-OMe typically provides better metabolic stability toward nuclease degradation than modification with 2′-F. Therefore, minimal 2′-F and heavy 2′-OMe modified nucleic acids should last longer in the cell. To test whether nucleic acids modified with 2′-OMe persist longer in the cell, duration studies were conducted using selected HAO1 conjugates test in the previous in vivo study (Table 8). As shown in FIG.8, minimal 2′-F and heavy 2′-OMe modified nucleic acids showed better mRNA knockdown at longer time points, and therefore, better duration of RNAi activity in vivo, as compared to the heavy 2′-F control. Table 8. Selected HAO1 Conjugates for HAO1 Duration Studies. 67
Example 9: APOC3 Conjugates Having Minimal 2 '-F and Heavy 2 '-OMe Modifications [0149] To confirm that nucleic acids having minimal 2'-F and heavy 2'-OMe modification patterns can be applied to other target sequences, modification patterns of the HAOl conjugates shown in Table 7 were transferred onto an APOC3 sequence. The resulting APOC3 conjugates shown in Table 9 were tested in vitro and in vivo.
Table 9. APOC3 Conjugates.
[0150] For in vitro experiments, HEK-293 cells were co-transfected with 100 ng of pcDNA3- mAPOC3 plasmid (containing cDNA for mouse APOC3) and siRNAs at the indicated
concentration using Dharmafect Duo reagent (Dharmacon) according to the manufacturer’s protocol. The next day the cells were lysed and RNA was purified using the SV96 kit (Promega). The purified RNA was reverse transcribed using High-capacity RT kit (Life Technologies) and APOC3 cDNA was quantified at RT-qPCR using gene assays for mouse APOC3, normalized against human SFRS9. As shown in FIG. 9, APOC3 conjugates having minimal 2′-F and heavy 2′-OMe modification patterns were well tolerated and showed similar in vitro activity as compared to the heavy 2′-F control. [0151] For in vivo experiments, CD-1 mice were divided into study groups and were dosed subcutaneously with 1 mg/kg of the assigned APOC3 conjugate. Animals were bled on day 7 post dose via lateral tail vein puncture with a collection volume of 10 µL. Collected whole blood was diluted immediately 1:5000 in cold PBS, and subsequently frozen at -20 °C. Whole blood at a final dilution of 1:10,000 was used for determining plasma APOC3 levels using the Cloud Clone Corporation ELISA (SEB890Mu). As seen in FIG. 9, APOC3 conjugates having minimal 2′-F and heavy 2′-OMe modification patterns showed good activity while the heavy 2′-F control did not show activity on day 7 post dose. Example 10: GYS2 Conjugates Having Minimal 2′-F and Heavy 2′-OMe Modifications [0152] To confirm that nucleic acids having minimal 2′-F and heavy 2′-OMe modification patterns can be applied to other target sequences, modification patterns of the HAO1 conjugates shown in Table 7 were transferred onto different GYS2 sequences. The resulting GYS2 conjugates are shown in Table 10. Two minimal 2′-F patterns were chosen and compared to a heavy 2′-F pattern (Table 10). For each of the three patterns, either 3 phosphorothioates (3PS) or 2 phosphorothioates (2PS) were included on the 5′-end of the antisense strand. GYS2 conjugates contained 3 GalNAc conjugated nucleotides in the loop region. Four different GYS2 sequences comprising the patterns in Table 10 were tested. Table 10. Modification Patterns for GYS2 Conjugates. 70
[0153] As shown in FIG. 10, minimal 2'-F and heavy 2'-OMe modification patterns 1 and 2 were well tolerated in vivo compared to the heavy 2'-F control, specifically these patterns were tolerated 4 days after a single subcutaneous dose of 0.5 mg/kg. Similar results were obtained for each of the four GYS2 sequences tested.
[0154] In sum, several advanced tetraloop GalXC designs were developed with reduced 2'-F content and increased 2'-OMe content that can be applied to multiple target genes and sequences with optimal potency and duration.