Compositions and methods for modulating HBV and TTR expression

Abstract

To provide oligomeric compounds with conjugate groups.SOLUTION: The present invention provides a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 8 to 80 linked nucleosides and has a nucleobase sequence at least 85%, 90%, 95%, or 100% complementary to a specific sequence encoding transthyretin (TTR).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application has been filed in electronic format along with a Sequence Listing, which is provided as a 16 Kb file named BIOL0248WOSEQ_ST25.txt created on May 1, 2014. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0002] The principle behind antisense technology is that antisense compounds hybridize to target nucleic acids and modulate the amount, activity, and / or function of the target nucleic acid. For example, in certain cases, antisense compounds result in alterations in the transcription or translation of the target. Such modulation of expression can be achieved, for example, by target mRNA degradation or occupancy-based inhibition. One example of modulation of RNA target function by degradation is RNase H-based degradation of target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing via a mechanism that utilizes the RNA-induced silencing complex (RISC). Another example of modulation of RNA target function is by occupancy-based mechanisms, such as those naturally used by microRNAs. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. Binding of an antisense compound to a microRNA prevents the microRNA from binding to its messenger RNA target, thus disrupting microRNA function. MicroRNA mimics can enhance native microRNA function. Certain antisense compounds alter the splicing of pre-mRNA. Regardless of the specific mechanism, sequence specificity makes antisense compounds attractive as a means of target validation and gene functioning, as well as therapeutic agents that selectively modulate the expression of genes involved in disease pathogenesis.

[0003] Antisense technology is an effective means of regulating the expression of one or more specific gene products and may therefore prove uniquely useful in many therapeutic, diagnostic, and research applications. Chemically modified nucleosides can be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for the target nucleic acid. In 1998, the antisense compound Vitravene® (fomilirsen, developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to receive marketing approval from the U.S. Food and Drug Administration (FDA) and is currently used to treat cytomegalovirus (CMV)-induced retinitis in AIDS patients.

[0004] New chemical modifications have improved the strength and efficacy of antisense compounds, enabling oral delivery, enhancing subcutaneous administration, and reducing the potential for side effects, leading to improved patient convenience. Chemical modifications that increase the strength of antisense compounds allow for lower doses, reducing the potential for toxicity and overall treatment costs. Modifications that increase resistance to degradation result in slower elimination from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined within a single compound to further optimize the compound's efficacy. Summary of the Invention

[0005] In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides antisense oligonucleotides complementary to nucleic acid transcripts. In certain embodiments, the present disclosure provides a method comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides a method comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of the nucleic acid transcript in the cell.

[0006] The asialoglycoprotein receptor (ASGP-R) has been previously described. See, for example, Park et al., PNAS vol. 102, No. 47, pp. 17125-17129 (2005). Such receptors are expressed on liver cells, specifically hepatocytes. Furthermore, it has been shown that compounds containing clusters of three N-acetylgalactosamine (GalNAc) ligands can bind to ASGP-R, resulting in the uptake of the compounds into cells. See, for example, Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp. 5216-5231 (May 2008). Thus, conjugates containing such GalNAc clusters have been used to promote the uptake of certain compounds into liver cells, specifically hepatocytes. For example, it has been shown that certain GalNAc-containing conjugates increase the activity of double-stranded siRNA compounds in liver cells in vivo. In such cases, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex. Because sense strand is disposed of before antisense strand finally hybridizes with target nucleic acid, there is little concern that conjugate will hinder activity.Typically, conjugate is attached to the 3' end of sense strand of siRNA.See, for example, U.S. Patent No. 8,106,022.Certain conjugate groups described herein are more active and / or easier to synthesize than conjugate groups previously described.

[0007] In certain embodiments of the present invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to, RNase H-based antisense compounds and antisense compounds that alter the splicing of pre-mRNA target nucleic acids. In such embodiments, the conjugate should remain attached to the antisense compound for a period of time sufficient to provide a benefit (improved cellular uptake), but should not be cleaved or otherwise interfere with subsequent steps required for activity, such as hybridization to the target nucleic acid and interaction with RNase H or enzymes involved in splicing or splicing regulation. This balance of properties is more important in the context of single-stranded antisense compounds than in the context of siRNA compounds, and the conjugate may be attached solely to the sense strand. Disclosed herein are conjugated single-stranded antisense compounds that exhibit improved potency in liver cells in vivo compared to the same antisense compound lacking the conjugate. Given the required balance of properties of these compounds, such improved potency is surprising.

[0008] In certain embodiments, the conjugate group herein comprises a cleavable moiety. As mentioned above, without wishing to be bound by a mechanism, it is logical that the conjugate should remain on the compound long enough to provide enhanced uptake, after which a portion of the conjugate, or ideally all of the conjugate, is cleaved to release the parent compound (e.g., antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments take advantage of endogenous nucleases in cells by linking the remainder of the conjugate (cluster) to the antisense oligonucleotide via a nucleoside through one or more cleavable bonds, such as a phosphodiester bond. In certain embodiments, the cluster is linked to the cleavable nucleoside via a phosphodiester bond. In certain embodiments, the cleavable nucleoside is linked to the antisense oligonucleotide (antisense compound) via a phosphodiester bond. In certain embodiments, In some embodiments, the conjugate group may contain two or three cleavable nucleosides. In these embodiments, these cleavable nucleosides are linked to each other, to the antisense compound, and / or to the cluster by a cleavable bond (such as a phosphodiester bond). Certain conjugates herein do not contain a cleavable nucleoside, but instead contain a cleavable bond. It is shown that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond (cleavable bond) that is vulnerable to cleavage in cells.

[0009] In certain embodiments, the conjugated antisense compound is a prodrug. Such a prodrug is administered to an animal and ultimately metabolized to a more active form. For example, the conjugated antisense compound is cleaved to remove all or part of the conjugate, resulting in an active (or more active) form of the antisense compound lacking all or part of the conjugate.

[0010] In certain embodiments, the conjugate is attached to the 5'-terminus of the oligonucleotide. Certain such 5'-conjugates are cleaved more efficiently than counterparts with the same conjugate attached to the 3'-terminus. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, the efficacy of oligonucleotides containing a conjugate at the 5'-terminus is higher than that of oligonucleotides containing a conjugate at the 3'-terminus (see, e.g., Examples 56, 81, 83, and 84). Furthermore, 5'-linkages allow for simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3'-to-5' direction. To create a 3'-conjugated oligonucleotide, a pre-conjugated 3' nucleoside is typically attached to the solid support, and the oligonucleotide is then assembled as usual. However, attaching the conjugated nucleoside to the solid support complicates synthesis. Furthermore, by using this approach, the conjugate then remains present throughout the synthesis of the oligonucleotide and may become degraded during subsequent steps or may limit the types of reactants and reagents that can be used. Using the 5'-conjugated oligonucleotide structures and techniques described herein, the oligonucleotide can be synthesized using standard automated techniques to incorporate the conjugate with the final (5'-most) nucleoside, or the oligonucleotide can be synthesized after it has been cleaved from the solid support.

[0011] Given the state of the art and this disclosure, one skilled in the art can easily prepare any of the conjugates and conjugated oligonucleotides described herein. Furthermore, the synthesis of certain such conjugates and conjugated oligonucleotides disclosed herein is easier and / or requires fewer steps than the synthesis of previously disclosed conjugates, thus being less expensive and providing manufacturing advantages. For example, the synthesis of certain conjugates involves fewer synthetic steps, resulting in increased yields, compared to previously described conjugates. Conjugates such as GalNAc3-10 in Example 46 and GalNAc3-7 in Example 48 are much simpler than previously described conjugates, such as those described in U.S. Patent No. 8,106,022 or U.S. Patent No. 7,262,177, which require the construction of many chemical intermediates. Therefore, these and other conjugates described herein are advantageous over previously described compounds when used in conjunction with any oligonucleotide, including single-stranded oligonucleotides and either strand of a double-stranded oligonucleotide (e.g., siRNA).

[0012] Similarly, conjugate groups having only one or two GalNAc ligands are disclosed herein. As shown, such conjugate groups improve the activity of antisense compounds. Such compounds are easier to prepare than conjugates containing three GalNAc ligands. Conjugate groups containing one or two GalNAc ligands are suitable for use in single-stranded oligonucleotides and and any antisense compound containing either strand of a double-stranded oligonucleotide (e.g., siRNA).

[0013] In certain embodiments, the conjugates herein do not substantially change tolerance to a certain extent. For example, it is shown herein that the immunogenicity of conjugated antisense compounds is lower than that of unconjugated parent compounds. Embodiments in which tolerance remains the same (or even remains the same even if tolerance is only slightly reduced compared to increased potency) due to improved potency have improved therapeutic properties.

[0014] In certain embodiments, conjugation allows antisense compounds to be altered in ways that would otherwise have less attractive results.For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with a phosphodiester linkage results in some degree of improved tolerability.For example, in certain cases, the immunogenicity of such antisense compounds with one or more phosphodiesters is lower than that of the same compound in which each linkage is a phosphorothioate linkage.However, in certain cases, as shown in Example 26, similarly replacing one or more phosphorothioate linkages with a phosphodiester linkage also results in reduced cellular uptake and / or loss of potency.In certain embodiments, the conjugated antisense compounds described herein tolerate such linkage changes with little or no loss of uptake and potency compared to their fully phosphorothioate conjugated counterparts. Indeed, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides containing conjugates and at least one phosphodiester internucleoside linkage actually exhibit increased potency in vivo, even when compared to fully phosphorothioate counterparts containing the same conjugate. Moreover, because conjugation results in a substantial increase in uptake / potency, a slight loss of that substantial increase may be acceptable to achieve improved tolerability. Thus, in certain embodiments, the conjugated antisense compound contains at least one phosphodiester linkage.

[0015] In certain embodiments, the conjugation of antisense compounds herein leads to increased delivery, uptake and activity in hepatocytes.Therefore, more compound is delivered to liver tissue.However, in certain embodiments, this increased delivery alone does not clearly increase overall activity.In certain such embodiments, more compound enters hepatocytes.In certain embodiments, even this increased hepatocyte uptake does not clearly increase overall activity.In such embodiments, the productive uptake of conjugated compounds increases.For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase the concentration of antisense oligonucleotides in hepatocytes compared with non-parenchymal cells.This concentration is beneficial for the oligonucleotides that target genes expressed in hepatocytes.

[0016] In certain embodiments, the conjugated antisense compound herein reduces renal exposure.For example, as shown in Example 20, the concentration of antisense oligonucleotides comprising certain embodiments of GalNAc-containing conjugates is lower in kidney than the concentration of antisense oligonucleotides that lack GalNAc-containing conjugates.This has several beneficial therapeutic implications.In therapeutic indications that do not require activity in the kidney, exposure to the kidney carries the risk of nephrotoxicity and does not merit any corresponding benefit.In addition, high concentration in the kidney typically leads to compound loss in urine, resulting in faster clearance.Therefore, in the case of non-renal target, accumulation in the kidney is undesirable.

[0017] In certain embodiments, the present disclosure provides an antisense conjugate represented by the formula: providing a compound, [ka] During the ceremony, A is an antisense oligonucleotide, B is a cleavable moiety, C is a conjugated linker; D is a branched group; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0018] In the figures above and similar figures herein, the branching group "D" repeats branching as necessary to accommodate the number of (E-F) groups, as indicated by "q." Thus, when q=1, the formula is: [ka] For q=2, the formula is: [ka] For q=3, the formula is: [ka] For q=4, the formula is: [ka] For q=5, the formula is: [ka]

[0019] In certain embodiments, conjugated antisense compounds are provided having the following structure: [ka]

[0020] In certain embodiments, conjugated antisense compounds are provided having the following structure: [ka]

[0021] In certain embodiments, conjugated antisense compounds are provided having the following structure: [ka]

[0022] In certain embodiments, conjugated antisense compounds are provided having the following structure: [ka]

[0023] In embodiments having more than one of a specified variable (e.g., more than one "m" or "n"), unless otherwise indicated, each such specified variable is independently selected. Thus, for structures having more than one n, each n is independently selected and may or may not be identical to one another. DETAILED DESCRIPTION OF THE INVENTION

[0024] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the present disclosure. As used herein, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both multiple elements and components comprising one unit and multiple elements and components comprising two or more subunits, unless expressly stated otherwise.

[0025] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are expressly incorporated herein by reference in their entirety for all purposes. A. Definition

[0026] Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis and Certain such techniques and procedures can be used in chemical analysis. Research” Edited by Sangvi and Cook, American Chemical Society, Washington DC, 1994, “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21 st edition, 2005, and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca. Raton, Florida, and Sambrook et al., “Molecular Cloning, A laboratory Manual,”2 ndEdition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. Where permitted, all patents, applications, published applications, and other publications, and other materials referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0027] Unless otherwise indicated, the following terms have the following meanings.

[0028] As used herein, "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (those found in DNA and RNA) and modified nucleosides. Nucleosides can be linked to a phosphate moiety.

[0029] As used herein, " chemical modification " refers to the chemical difference of a compound when compared with its counterpart that exists in nature.Oligonucleotide chemical modification includes nucleoside modification (including sugar moiety modification and nucleic acid base modification) and internucleoside bond modification.With respect to oligonucleotide, chemical modification does not include only difference in nucleic acid base sequence.

[0030] As used herein, "furanosyl" means a structure containing a five-membered ring containing four carbon atoms and one oxygen atom.

[0031] As used herein, "naturally occurring sugar moiety" means ribofuranosyl found in naturally occurring RNA or deoxyribofuranosyl found in naturally occurring DNA.

[0032] As used herein, "sugar moiety" means the naturally occurring or modified sugar moiety of a nucleoside.

[0033] As used herein, "modified sugar moiety" means a substituted sugar moiety or sugar surrogate.

[0034] As used herein, "substituted sugar moiety" refers to a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to, furanosyl containing substituents at the 2', 3', 5', and / or 4' positions. Certain substituted sugar moieties are bicyclic sugar moieties.

[0035] As used herein, "2'-substituted sugar moiety" means a furanosyl containing a substituent at the 2' position other than H or OH. Unless otherwise indicated, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of the 2'-substituted sugar moiety is not a bicyclic sugar moiety). does not form a bridge to the child).

[0036] As used herein, "MOE" means -OCH2CH2OCH3.

[0037] As used herein, "2'-F nucleoside" refers to a nucleoside containing a sugar containing a fluorine at the 2' position. Unless otherwise specified, the fluorine in a 2'-F nucleoside is at the ribo position (replacing the OH of natural ribose).

[0038] As used herein, the term "sugar surrogate" refers to structures that do not contain furanosyl and can replace the naturally occurring sugar moiety of a nucleoside so that the resulting nucleoside subunits can be linked together and / or to other nucleosides to form oligomeric compounds that can hybridize to complementary oligomeric compounds. Such structures include rings containing a different number of atoms than furanosyl (e.g., 4-, 6-, or 7-membered rings), substitution of furanosyl oxygen with non-oxygen atoms (e.g., carbon, sulfur, or nitrogen), or both a change in the number of atoms and a substitution of oxygen. Such structures can also contain substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally containing additional substituents). Sugar surrogates also include more complex sugar substitutes (e.g., the acyclic systems of peptide nucleic acids). Sugar surrogates include, but are not limited to, morpholino, cyclohexenyl, and cyclohexitol.

[0039] As used herein, "bicyclic sugar moiety" means a modified sugar moiety comprising a 4- to 7-membered ring (including, but not limited to, furanosyl) containing a bridge connecting two atoms of the 4- to 7-membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4- to 7-membered ring is a sugar ring. In certain embodiments, the 4- to 7-membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.

[0040] As used herein, "nucleotide" refers to a nucleoside that further comprises a phosphate linking group. As used herein, "linked nucleosides" may or may not be linked by a phosphate bond, and therefore include, but are not limited to, "linked nucleotides." As used herein, "linked nucleosides" are nucleosides that are linked in a contiguous sequence (i.e., no additional nucleosides are present between the linked sequences).

[0041] As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to create a nucleoside that can be incorporated into an oligonucleotide, and which can bind to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. The nucleobase can be naturally occurring or modified.

[0042] As used herein, the term "unmodified nucleobase" or "naturally occurring nucleobase" means a naturally occurring heterocyclic nucleobase in RNA or DNA, where the purines are based on adenine (A) and guanine (G), and the pyrimidines are based on thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

[0043] As used herein, "modified nucleobase" means any nucleobase that is not a naturally occurring nucleobase.

[0044] As used herein, "modified nucleoside" means a nucleoside that contains at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. Modified nucleosides contain a modified sugar moiety and / or a modified nucleobase.

[0045] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside that includes a bicyclic sugar moiety.

[0046] As used herein, "constrained ethyl nucleoside" or "cEt" means a nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge.

[0047] As used herein, "locked acid nucleoside" or "LNA" means a nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge.

[0048] As used herein, "2'-substituted nucleoside" means a nucleoside that includes a substituent at the 2' position other than H or OH. Unless otherwise indicated, a 2'-substituted nucleoside is not a bicyclic nucleoside.

[0049] As used herein, "deoxynucleoside" refers to a nucleoside containing a 2'-H-furanosyl sugar moiety found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, 2'-deoxynucleosides may contain modified nucleobases or may contain RNA nucleobases (e.g., uracil).

[0050] As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.

[0051] As used herein, "oligonucleoside" means an oligonucleotide in which none of the internucleoside linkages contain a phosphorus atom. As used herein, oligonucleotide includes oligonucleosides.

[0052] As used herein, "modified oligonucleotide" means an oligonucleotide that contains at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0053] As used herein, a "bond" or "linking group" means a group of atoms that joins two or more other groups of atoms together.

[0054] As used herein, "internucleoside linkage" means the covalent bond between adjacent nucleosides in an oligonucleotide.

[0055] As used herein, "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0056] As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0057] As used herein, "terminal internucleoside linkage" means the linkage between the last two nucleosides of an oligonucleotide or a defined region thereof.

[0058] As used herein, "phosphorus linking group" means a linking group that includes a phosphorus atom. Phosphorus linking groups include, but are not limited to, groups having the formula: [ka] During the ceremony, R a and R d are each independently O, S, CH2, NH, or NJ1, where J1 is C1-C6 alkyl or substituted C1-C6 alkyl; R b is O or S, R c is OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino, or substituted amino; J1 is R b is O or S.

[0059] Phosphorus linking groups include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, phosphonates, phosphoramidates, phosphorothioamidates, thionoalkylphosphonates, phosphotriesters, thionoalkylphosphotriesters, and boranophosphates.

[0060] As used herein, "phosphorus internucleoside linking group" means a phosphorus linking group that directly links two nucleosides.

[0061] As used herein, "non-internucleoside phosphorus linking group" refers to a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside.

[0062] As used herein, "neutral linking group" refers to a linking group that is not charged. Neutral linking groups include, but are not limited to, phosphotriester, methylphosphonate, MMI (-CH-N(CH)-O-), amide-3 (-CH-C(=O)-N(H)-), amide-4 (-CH-N(H)-C(=O)-), formacetal (-O-CH-O-), and thioformacetal (-S-CH-O-). Additionally, neutral linking groups include nonionic linkages, including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (e.g., Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, "Antisense Modifications in Antisense Research"). Eds.ACS Symposium Series 580;Chapters 3 and 4 (pp. 40-65). Additionally, neutral bonding groups include nonionic bonds containing mixed N, O, S, and CH2 component moieties.

[0063] As used herein, "neutral internucleoside linking group" means a neutral linking group that directly links two nucleosides.

[0064] As used herein, "non-internucleoside neutral linking group" refers to a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral linking group links two groups, neither of which is a nucleoside.

[0065] As used herein, "oligomeric compound" refers to a polymeric structure comprising two or more substructures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugated groups and / or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. Oligomeric compounds also include naturally occurring nucleic acids. In certain embodiments, an oligomeric compound comprises a backbone of one or more linked monomeric subunits, each linked monomeric subunit being directly or indirectly bound to a heterocyclic base moiety. In certain embodiments, an oligomeric compound may also comprise a monomeric subunit not linked to a heterocyclic base moiety, thereby providing an abasic site. In certain embodiments, the linkages connecting the monomeric subunits, sugar moieties or surrogates, and heterocyclic base moieties may be independently modified. In certain embodiments, linked sugar units, which may or may not contain a heterocyclic base, may be replaced with mimetics, such as monomers in peptide nucleic acids.

[0066] As used herein, "terminal group" refers to one or more atoms attached to either the 3'-end or the 5'-end, or both, of an oligonucleotide. In certain embodiments, the terminal group is a conjugate group. In certain embodiments, the terminal group comprises one or more terminal nucleosides.

[0067] As used herein, "conjugate" or "conjugate group" refers to an atom or group of atoms attached to an oligonucleotide or oligomeric compound. Generally, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to, pharmacodynamic properties, pharmacokinetic properties, binding properties, absorption properties, cellular distribution properties, cellular uptake properties, charge properties, and / or clearance properties.

[0068] As used herein, "conjugated linker" or "linker" in reference to a conjugate group means a portion of the conjugate group, including any atom or group of atoms, that (1) covalently links an oligonucleotide to another portion of the conjugate group, or (2) covalently links two or more portions of the conjugate group.

[0069] Conjugate groups, referred to herein as radicals, provide a bond for forming a covalent bond to an oligomeric compound, such as an antisense oligonucleotide. In certain embodiments, the point of attachment to an oligomeric compound is the 3'-oxygen atom of the 3'-hydroxyl group of the 3'-terminal nucleoside of the oligomeric compound. In certain embodiments, the point of attachment to an oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl group of the 5'-terminal nucleoside of the oligomeric compound. In certain embodiments, the bond for forming the bond to an oligomeric compound is a cleavable bond. In certain such embodiments, such a cleavable bond constitutes all or part of the cleavable moiety.

[0070] In certain embodiments, a conjugate group comprises a cleavable moiety (e.g., a cleavable bond or a cleavable nucleoside) and a carbohydrate cluster moiety, such as a GalNAc cluster moiety. Such carbohydrate cluster moieties comprise a targeting moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster moiety is identified by the number and identity of the ligand. For example, in certain embodiments, a carbohydrate cluster moiety comprises three GalNAc groups and is designated "GalNAc3." In certain embodiments, a carbohydrate cluster moiety comprises four GalNAc groups and is designated "GalNAc4." Specific carbohydrate cluster moieties (with specific tether, branch, and conjugate linker groups) are described herein and are designated by a Roman numeral followed by the subscript "a" or "b." " Therefore, "GalNac3-1 a " refers to a specific carbohydrate cluster portion of a conjugate group having three GalNac groups and specifically specified tether, branching, and linking groups. Such carbohydrate cluster fragments are attached to an oligomeric compound via a cleavable bond or a cleavable moiety such as a cleavable nucleoside.

[0071] As used herein, "cleavable moiety" refers to a bond or group that can be split under physiological conditions. In certain embodiments, the cleavable moiety is cleaved within a cell or intracellular compartment, such as a lysosome. In certain embodiments, the cleavable moiety is cleaved by an endogenous enzyme, such as a nuclease. In certain embodiments, the cleavable moiety comprises an atomic group having one, two, three, four, or more than four cleavable bonds.

[0072] As used herein, "cleavable bond" means any chemical bond that can be split. In certain embodiments, the cleavable bond is selected from among an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a disulfide, or a peptide.

[0073] As used herein, "carbohydrate cluster" means a compound having one or more carbohydrate residues attached to a scaffold or linker group (e.g., for examples of carbohydrate-conjugated clusters, see Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting," Bioconjugate Chemistry, 2003, (14):18-29, or Rensen et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor," J. Med. Chem. 2004, (47):5798-5808, which are incorporated herein by reference in their entireties).

[0074] As used herein, "carbohydrate derivative" means any compound that can be synthesized using a carbohydrate as a starting material or intermediate.

[0075] As used herein, "carbohydrate" means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

[0076] As used herein, "protecting group" means any compound or protecting group known to those skilled in the art. Non-limiting examples of protecting groups are "Protective Groups in Organic Chemistry”, T.W. Greene, P.G.M. Butts, ISBN 0-471-62301-6, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety.

[0077] As used herein, "single-stranded" means an oligomeric compound that is not hybridized to its complement and lacks sufficient self-complementarity to form a stable self-duplex.

[0078] As used herein, "double-stranded" refers to a pair of oligomeric compounds that are hybridized to each other or a single self-complementary oligomeric compound that forms a hairpin structure. In certain embodiments, a double-stranded oligomeric compound comprises a first and a second oligomer. Includes mer compounds.

[0079] As used herein, "antisense compound" means a compound comprising or consisting of an oligonucleotide, at least a portion of which is complementary to a target nucleic acid to which it can hybridize, and which provides at least one antisense activity.

[0080] As used herein, "antisense activity" refers to any detectable and / or measurable change resulting from the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity includes regulating the amount or activity of target nucleic acid transcripts (e.g., mRNA). In certain embodiments, antisense activity includes regulating the splicing of pre-mRNA.

[0081] As used herein, "RNase H-based antisense compound" means an antisense compound in which at least part of the antisense activity of the antisense compound results from hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.

[0082] As used herein, "RISC-based antisense compound" means an antisense compound where at least part of the antisense activity of the antisense compound is attributable to the RNA-induced silencing complex (RISC).

[0083] As used herein, "detection" or "measurement" means that a test or assay for detection or measurement is performed. Such detection and / or measurement may result in a value of zero. Thus, if a test for detection or measurement results in a finding of no activity (zero activity), a step of detecting or measuring activity has nevertheless been performed.

[0084] As used herein, "detectable and / or measurable activity" means a statistically significant activity that is not zero.

[0085] As used herein, "essentially unchanged" means that there is little or no change in a particular parameter, especially compared to another parameter that changes significantly. In certain embodiments, when a parameter changes by less than 5%, the parameter is essentially unchanged. In certain embodiments, when a parameter changes by less than 2 times, the parameter is essentially unchanged, while another parameter changes by at least 10 times. For example, in certain embodiments, antisense activity is the change in the amount of target nucleic acid. In certain such embodiments, if the change in the amount of non-target nucleic acid is much smaller than the change in the amount of target nucleic acid, it is essentially unchanged, but the change does not need to be zero.

[0086] As used herein, "expression" refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modifications (e.g., splicing, polyadenylation, addition of a 5'-cap), and translation.

[0087] As used herein, "target nucleic acid" means a nucleic acid molecule to which an antisense compound is intended to hybridize to provide the desired antisense activity. Antisense oligonucleotides have sufficient complementarity to their target nucleic acid to allow hybridization under physiological conditions.

[0088] As used herein, "nucleobase complementarity" or "complementarity" in reference to nucleobase refers to a nucleobase that can base pair with another nucleobase.For example, in DNA, adenine (A) is complementary to thymine (T).For example, in RNA, adenine (A) is complementary to uracil (U).In certain embodiments, complementary nucleobase refers to the nucleobase of an antisense compound that can base pair with the nucleobase of its target nucleic acid.For example, if the nucleobase at a specific position of an antisense compound can hydrogen bond with the nucleobase at a specific position of a target nucleic acid, the hydrogen bond position between the oligonucleotide and the target nucleic acid is considered to be complementary in the nucleobase pair.Nucleobases that contain certain modifications can maintain the ability to pair with corresponding nucleobases, and therefore can still have nucleobase complementarity.

[0089] As used herein, "non-complementary" with respect to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

[0090] As used herein, "complementary" with respect to an oligomeric compound (e.g., linked nucleosides, oligonucleotides, or nucleic acids) refers to the ability of such an oligomeric compound or a region thereof to hybridize to another oligomeric compound or a region thereof through nucleobase complementarity. Complementary oligomeric compounds need not have nucleobase complementarity at every nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.

[0091] As used herein, "mismatch" refers to a nucleobase of a first oligomeric compound that cannot pair with a nucleobase at a corresponding position in a second oligomeric compound when the first and second oligomeric compounds are aligned. Either or both of the first and second oligomeric compounds can be oligonucleotides.

[0092] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common pairing mechanisms involve hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementary nucleobases.

[0093] As used herein, "specifically hybridize" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.

[0094] As used herein, "fully complementary" with respect to an oligonucleotide or a portion thereof means that each nucleobase of the oligonucleotide or portion thereof can pair with a nucleobase of a complementary nucleic acid or a contiguous portion thereof. Thus, the fully complementary region does not contain mismatched or non-hybridized nucleobases on either strand.

[0095] As used herein, "percent complementarity" refers to the percentage of nucleobases of an oligomeric compound that are complementary to equal-length portions of a target nucleic acid. Percent complementarity refers to the number of nucleobases of an oligomeric compound that are complementary to nucleobases at corresponding positions of a target nucleic acid, expressed as a percentage of the total number of nucleobases in the oligomeric compound. It is calculated by dividing by the length.

[0096] As used herein, "percent identity" means the number of nucleobases of a first nucleic acid that are of the same type (independent of chemical modification) as the nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0097] As used herein, "modulation" refers to a change in the quantity or quality of a molecule, function, or activity compared to the quantity or quality of the molecule, function, or activity before modulation. For example, modulation includes either an increase (stimulation or induction) or decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection in pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice variant compared to the amount in the absence of modulation. As used herein, "chemical motif" refers to a pattern of chemical modifications in an oligonucleotide or region thereof. A motif may be defined by modifications at certain nucleosides and / or certain linkage groups of the oligonucleotide.

[0098] As used herein, "nucleoside motif" refers to a pattern of nucleoside modifications in an oligonucleotide or region thereof. The linkages of such oligonucleotides can be modified or unmodified. Unless otherwise indicated, a motif that describes only nucleosides herein is intended to be a nucleoside motif. Thus, in such cases, the linkage is not limited.

[0099] As used herein, "sugar motif" refers to the pattern of sugar modifications in an oligonucleotide or region thereof.

[0100] As used herein, "linkage motif" refers to a pattern of linkage modifications in an oligonucleotide or a region thereof. The nucleosides of such oligonucleotides can be modified or unmodified. Unless otherwise indicated, motifs that describe only linkages herein are intended to be linkage motifs. Therefore, in such cases, the nucleosides are not limited.

[0101] As used herein, "nucleobase modification motif" means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, the nucleobase modification motif is independent of the nucleobase sequence.

[0102] As used herein, "sequence motif" refers to a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modification and therefore can have any combination of chemical modifications, including no chemical modifications.

[0103] As used herein, "type of modification" in reference to a nucleoside or a "type" of nucleoside refers to the chemical modification of the nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise indicated, a "nucleoside having a first type of modification" can be an unmodified nucleoside.

[0104] As used herein, "differentially modified" refers to chemical modifications or chemical substituents that differ from one another, including the absence of modification. Thus, for example, MOE nucleosides and unmodified DNA nucleosides are "differently modified" even though the DNA nucleosides are unmodified. Similarly, DNA and RNA are "differently modified" even though they both contain naturally occurring unmodified nucleosides. Nucleosides that are identical but contain different nucleobases are also "differently modified." Nucleosides are not differentially modified, e.g., a nucleoside comprising a 2'-OMe-modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2'-OMe-modified sugar and an unmodified thymine nucleobase are not differentially modified.

[0105] As used herein, " the same type of modification " refers to the same modification as each other, including the absence of modification.Thus, for example, two unmodified DNA nucleosides have " the same type of modification " even if the DNA nucleosides are not modified.Such nucleosides with the same type of modification can contain different nucleobases.

[0106] As used herein, "distinct region" means a portion of an oligonucleotide, wherein the chemical modifications or chemical modification motifs of any adjacent portions contain at least one difference that allows the distinct regions to be distinguished from one another.

[0107] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administration to an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0108] As used herein, the term "metabolic disorder" means a disease or condition characterized primarily by dysregulation of metabolism (the complex series of chemical reactions involved in breaking down food to produce energy).

[0109] As used herein, the term "cardiovascular disorder" means a disease or condition characterized primarily by dysfunction of the heart or blood vessels.

[0110] As used herein, the term "monocyclic or polycyclic ring system" is intended to include all ring systems selected from monocyclic or polycyclic radical ring systems, including single and mixed ring systems, wherein the rings are fused or linked and are independently selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, and heteroarylalkyl. Such monocyclic and polycyclic structures may contain rings each having the same level of saturation or each independently having varying degrees of saturation, including fully saturated, partially saturated, or fully unsaturated. Each ring may contain ring atoms selected from C, N, O, and S, to give rise to heterocyclic rings and rings containing only C ring atoms, which may occur in mixed motifs such as benzimidazole, where one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The monocyclic or polycyclic ring systems may be further substituted with substituents such as, for example, phthalimide, which has two =0 groups attached to one of the rings. The monocyclic or polycyclic ring systems may be attached to the parent molecule using a variety of strategies, such as direct attachment through a ring atom, fusion through multiple ring atoms, attachment through a substituent, or attachment through a bifunctional linking moiety.

[0111] As used herein, "prodrug" means an inactive or less active form of a compound that, when administered to a subject, is metabolized to form the active compound or a more active compound (e.g., a drug).

[0112] As used herein, "substituent" and "substituent group" refer to an atom or group that replaces a designated atom or group of a parent compound. For example, a substituent of a modified nucleoside is any atom or group that is different from an atom or group found in a naturally occurring nucleoside (e.g., a modified 2'-substituent is any atom or group at the 2' position of a nucleoside other than H or OH). Substituents may or may not be protected. In certain embodiments, Thus, the compounds of the present disclosure have a substituent at one position or at more than one position of the parent compound, which may be further substituted with other substituents and may be directly bonded to the parent compound or may be bonded via a linking group such as an alkyl or hydrocarbyl group.

[0113] Similarly, as used herein, a "substituent" with respect to a chemical functional group means an atom or group of atoms different from those normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen that replaces one of the hydrogen atoms of the unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)R- aa ), carboxyl (-C(O)OR -aa ), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-OR -aa ), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(R bb )(R cc )), Imino (=NR bb ), amide (-C(O)N(R bb )(R cc ) or -N(R bb )C(O)R aa ), azide (-N3), nitro (-NO2), cyano (-CN), carbamide (-OC(O)N(R bb )(R cc ) or -N(R bb )C(O)OR aa ), ureido(-N(R bb )C(O)N(R bb )(R cc )), thioureido (-N(R bb )C(S)N(R bb )(R cc )), guanidinyl (-N(R bb )C(=NR bb )N(R bb )(R cc )), amidinyl (-C(=NRbb )N(R bb )(R cc ) or -N(R bb )C(=NR bb )(R aa )), thiol (-SR bb ), sulfinyl (-S(O)R bb ), sulfonyl (-S(O)R bb ), and sulfonamidyl (-S(O)N(R bb )(R cc ) or -N(R bb )S-(O)2R bb ), but are not limited to, wherein each R aa , R bb , and R cc are independently H, an optionally linked chemical functionality, or a further substituent, with a preferred list including, but not limited to, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic, and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

[0114] As used herein, "alkyl" means a saturated straight or branched chain hydrocarbon radical containing up to 24 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. Alkyl groups typically contain from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1 to C6). 12 alkyl), more preferably having 1 to about 6 carbon atoms.

[0115] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain radical containing up to 24 carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally contain one or more additional substituents.

[0116] As used herein, "alkynyl" refers to a straight or branched chain hydrocarbon radical containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. As used herein, alkynyl groups may optionally include one or more further substituents. .

[0117] As used herein, "acyl" means a radical formed by removal of a hydroxyl group from an organic acid and has the general formula -C(O)-X, where X is typically aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, and the like. As used herein, acyl groups can optionally include further substituents.

[0118] As used herein, "alicyclic" refers to a cyclic ring system wherein the ring is aliphatic. The ring system can include one or more rings, with at least one ring being aliphatic. Preferred alicyclic groups include rings having from about 5 to about 9 carbon atoms in the ring. As used herein, alicyclic groups can optionally include further substituents.

[0119] As used herein, "aliphatic" refers to a straight- or branched-chain hydrocarbon radical containing up to 24 carbon atoms, with the saturation between any two carbon atoms being a single, double, or triple bond. Aliphatic groups preferably contain from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms, with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of the aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Such heteroatom-interrupted aliphatic groups include, but are not limited to, polyalkoxy groups, e.g., polyalkylene glycols, polyamines, and polyimines. The aliphatic groups used herein may optionally contain additional substituents.

[0120] As used herein, "alkoxy" refers to a radical formed between an alkyl group and an oxygen atom, which is used to attach the alkoxy group to the parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. Alkoxy groups used herein can optionally include further substituents.

[0121] As used herein, "aminoalkyl" refers to an amino-substituted C1-C 12 means an alkyl radical. The alkyl portion of said radical forms a covalent bond to the parent molecule. The amino group can be located at any position, and the aminoalkyl group can be substituted with further substituents on the alkyl and / or amino moieties.

[0122] As used herein, "aralkyl" and "arylalkyl" refer to C1-C 12It refers to an aromatic group covalently bonded to an alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, etc. As used herein, aralkyl groups can optionally include additional substituents attached to the alkyl, aryl, or both groups that form the radical group.

[0123] As used herein, "aryl" and "aromatic" refer to a monocyclic or polycyclic carbocyclic ring system radical having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl, and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituents.

[0124] As used herein, "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0125] As used herein, "heteroaryl" and "heteroaromatic" refer to a radical containing a monocyclic or polycyclic aromatic ring, ring system, or fused ring system, wherein at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl is also intended to include fused ring systems, including systems in which one or more of the fused rings does not contain a heteroatom. Heteroaryl groups typically contain one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. Heteroaryl radicals can be directly attached to a parent molecule or can be attached through a linking moiety such as an aliphatic group or a heteroatom. As used herein, heteroaryl groups may optionally include further substituents.

[0126] As used herein, "conjugate compound" refers to any atom, group of atoms, or bonded group of atoms suitable for use as a conjugate group. In certain embodiments, a conjugate compound may possess or impart one or more properties, including, but not limited to, pharmacodynamic properties, pharmacokinetic properties, binding properties, absorption properties, cellular distribution properties, cellular uptake properties, charge properties, and / or clearance properties.

[0127] As used herein, unless otherwise indicated or modified, the term "duplex" refers to two distinct oligomeric compounds that are hybridized to one another. Such double-stranded compounds may have one or more nucleosides or unhybridized nucleosides at one or both ends of one or both strands (overhangs) and / or one or more unhybridized internal nucleosides (mismatches), provided that sufficient complementarity exists to maintain hybridization under physiologically relevant conditions. B. A specific compound

[0128] In certain embodiments, the present invention provides conjugate antisense compounds comprising an antisense oligonucleotide and a conjugate.

[0129] a. Certain antisense oligonucleotides In certain embodiments, the present invention provides antisense oligonucleotide.Such antisense oligonucleotide comprises linked nucleosides, and each nucleoside comprises sugar moiety and nucleobase.The structure of such antisense oligonucleotide can be considered in terms of chemical characteristics (for example, modification and modification pattern) and nucleobase sequence (for example, the sequence, identity and sequence of antisense oligonucleotide and target nucleic acid).

[0130] i. Certain chemical characteristics In certain embodiments, antisense oligonucleotides comprise one or more modifications.In certain such embodiments, antisense oligonucleotides comprise one or more modified nucleosides and / or modified internucleoside linkages.In certain embodiments, modified nucleosides comprise modified sugar moieties and / or modified nucleobases.

[0131] 1. Certain sugar moieties In certain embodiments, compounds of the present disclosure comprise one or more modified nucleic acids containing a modified sugar moiety. Such compounds containing one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity to target nucleic acids, compared to oligonucleotides containing only nucleosides containing naturally occurring sugar moieties. In certain embodiments, the modified sugar moiety is a substituted sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions corresponding to the substitutions in the substituted sugar moiety.

[0132] In certain embodiments, the modified sugar moiety is a substituted sugar moiety containing one or more non-bridging sugar substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH ("OMe" or "O-methyl"), and 2'-O(CH)OCH ("MOE"). In certain embodiments, the sugar substituent at the 2' position is allyl, amino, azido, thio, O-allyl, O-C1-C2. 10 Alkyl, O-C1-C 10 substituted alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), wherein each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 and alkyl. Examples of sugar substituents at the 5'-position include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the substituted sugar comprises two or more non-bridging sugar substituents, such as a 2'-F-5'-methyl sugar moiety (see, e.g., PCT International Application No. WO2008 / 101157 for additional 5',2'-bis-substituted sugar moieties and nucleosides).

[0133] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In certain embodiments, 2'-substituted nucleosides include halo, allyl, amino, azido, SH, CN, OCN, CF, OCF, O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S, or N(R m )-alkynyl; O-alkylenyl-O-alkyl, alkynyl, aralkyl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ); or O-CH2-C(=O)-N(R m )(R n ), wherein each R m and Rn are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0134] In certain embodiments, 2'-substituted nucleosides are F, NH, N, OCF, O-CH, O(CH)NH, CH-CH=CH, O-CH-CH=CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (O-CH2-C(=O)-N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 It is alkyl.

[0135] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, OCF, O-CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(CH), -O(CH)O(CH)N(CH), and O-CH-C(=O)-N(H)CH.

[0136] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, O-CH3, and OCH2CH2OCH3.

[0137] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring to provide a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents include -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(R a R b )-N(R)-O-, or -C(R a R b )-ON(R)-; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2'(cEt); and 4'-CH(CHOCH3)-O-2'; and analogs thereof (see, e.g., U.S. Patent No. 7,399,845 issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., U.S. Patent No. 7,399,845 issued July 15, 2009). See International Publication No. WO 2009 / 006478, published January 8; 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., International Publication No. WO 2008 / 150729, published December 11, 2008); 4'-CH2-ON(CH3)-2' (see, e.g., US 2004 / 0171570, published September 2, 2004); 4'-CH2-ON(R)-2'; and 4'-CH2-N(R)-O-2'- (wherein each R is independently H, a protecting group, or a C1-C 12 alkyl); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12alkyl, or a protecting group) (see U.S. Pat. No. 7,427,672 issued Sep. 23, 2008); 4'-CH2-C(H)(CH3)-2' (e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and analogs thereof (see PCT International Application No. WO2008 / 154401 published Dec. 8, 2008).

[0138] In certain embodiments, such 4' to 2' bridges are independently -[C(R a )(R b )] n -, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x - and -N(R a )-, and During the ceremony, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 cycloaliphatic radical, substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each J1 and J2 is independently H, C1 to C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0139] Nucleosides containing a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include the following: (A) α-L-methyleneoxy(4'-CH2-O-2') BNA, (B) β-D-methyleneoxy(4'-CH2-O-2') BNA (also known as locked nucleic acid or LNA), (C) ethyleneoxy(4'-(CH2) 2-O-2') BNA, (D) aminooxy (4'-CH2-ON(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-O-2') BNA, (F) methyl (methyleneoxy) (4'-CH(CH3)-O-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, and (J) propylene carbocyclic (4'-(CH2)3-2') BNA, [ka] [ka] wherein Bx is a nucleobase moiety and R is independently H, a protecting group, or C1-C 12 It is alkyl.

[0140] Additional bicyclic sugar moieties are known in the art and are described, for example, in Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al. al., Tetrahedron,1998,54,3607-3630, Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638, Kumar et al.,Bioorg.Med.Chem.Lett.,1998,8,2219-2222, Singh et al. al., J.Org.Chem.,1998,63,10035-10039, Srivastava et al.,J.Am.Chem.Soc.,129(26)8362-8379(Jul.4,2007), Elayadi et al.,Curr.Opinion Invens.Drugs,2001,2,558-561, Braasch et al. al.,Chem.Biol.,2001,8,1-7,Orum et al.,Curr.Opinion Mol.Ther.,2001,3,239-243, U.S. Patent No. 7,053,207, U.S. Patent No. 6,268,490, U.S. Patent No. 6,770,748, U.S. Patent No. 6,794,499, U.S. Patent No. 7,034,133, 6,525,191, 6,670,461, and 7,399,845, International Publication No. WO2004 / 106356, WO1994 / 14226, WO2005 / 021570 No. WO2007 / 134181, U.S. Patent Publication Nos. US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618, U.S. Patent Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,99 Nos. 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844, and PCT International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922.

[0141] In certain embodiments, the bicyclic sugar moiety and the nucleoside that incorporates such a bicyclic sugar moiety are further defined by their isomeric configuration.For example, the nucleoside that comprises a 4'-2' methylene-oxy bridge can be in α-L configuration or β-D configuration.Previously, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleoside has been incorporated into antisense oligonucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0142] In certain embodiments, the substituted sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted and 4'-2'-bridging sugars) (see PCT International Application No. WO2007 / 134181, published November 22, 2007, in which LNAs are substituted, e.g., with 5'-methyl or 5'-vinyl groups).

[0143] In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain such embodiments, the oxygen atom of a naturally occurring sugar is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such modified sugar moieties also include the bridging and / or non-bridging substituents described above. For example, certain sugar surrogates include a 4'-sulfur atom and substitutions at the 2'-position (see, e.g., U.S. Patent Application No. US2005 / 0130923, published June 16, 2005) and / or the 5'-position. As a further example, carbocyclic bicyclic nucleosides with a 4'-2' bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0144] In certain embodiments, the sugar surrogate comprises a ring having more than five atoms. For example, in certain embodiments, the sugar surrogate comprises a morpholino. Morpholino compounds and their use in oligomeric compounds have been reported in numerous patents and published articles (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510, and U.S. Patent Nos. 5,698,685, 5,166,315, 5,185,444, and 5,034,506). As used herein, the term "morpholino" refers to a sugar surrogate having the following structure: [ka]

[0145] In certain embodiments, morpholinos can be modified, for example, by adding or altering various substituents from the morpholino structures described above. Such sugar surrogates are referred to herein as "modified morpholinos."

[0146] As another example, in certain embodiments, the sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro-HNA (F-HNA), and compounds having the following formula VI: [ka] wherein, independently, for each of said at least one tetrahydropyran nucleoside analog of Formula VI: Bx is a nucleobase moiety; T3 and T4 are each independently an internucleoside linking group that attaches a tetrahydropyran nucleoside analog to an antisense compound, or one of T3 and T4 is an internucleoside linking group that attaches a tetrahydropyran nucleoside analog to an antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linkage conjugate group, or a 5'- or 3'-terminal group; q1, q2, q3, q4, q5, q6, and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; Each of R1 and R2 is independently selected from hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where X is O, S, or NJ1, and each J1, J2, and J3 is independently H or C1-C6 alkyl.

[0147] In certain embodiments, modified THP nucleosides of formula VI are provided, wherein q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, THP nucleosides of formula VI are provided, wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro, R2 is H, R1 is methoxy, R2 is H, R1 is methoxyethoxy, and R2 is H.

[0148] Many other bicyclic and tricyclic sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0149] 2'-F-5'-methyl substituted nucleosides (see PCT International Application No. WO2008 / 101157, published August 21, 2008, for other disclosed 5',2'-bis substituted nucleosides), as well as substitution of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see U.S. Patent Application No. US2005-002257, published June 16, 2005). Combinations of modifications are also provided, such as, but not limited to, 5'-substituted bicyclic nucleic acids (see PCT International Application No. WO2007 / 134181, published November 22, 2007), or alternatively 5'-substituted bicyclic nucleic acids (4'-CH2-O-2' bicyclic nucleosides further substituted at the 5' position with a 5'-methyl or 5'-vinyl group, see PCT International Application No. WO2007 / 134181, published November 22, 2007). Oligomerization and biochemical studies of carbocyclic bicyclic nucleosides, as well as their synthesis and preparation, have also been described (e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0150] In certain embodiments, the present disclosure provides oligonucleotides containing modified nucleosides. These modified nucleotides may contain modified sugars, modified nucleobases, and / or modified linkages. Specific modifications are selected so that the resulting oligonucleotides have desirable characteristics. In certain embodiments, the oligonucleotides contain one or more RNA-like nucleosides. In certain embodiments, the oligonucleotides contain one or more DNA-like nucleotides.

[0151] 2. Certain nucleobase modifications In certain embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

[0152] In certain embodiments, the modified nucleobase is selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines include 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3)uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azo Included are uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Additionally, modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).Modified nucleobases can also include bases in which purine or pyrimidine bases are replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Furthermore, nucleobases include the bases disclosed in U.S. Patent No. 3,687,808, and in The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859. These include bases disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288.

[0153] Representative United States patents that teach certain preparations of the above-described modified nucleobases, as well as other modified nucleobases, include U.S. Pat. Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552, ,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096, certain of which are commonly owned with the present application and each of which is incorporated herein by reference in its entirety.

[0154] 3. Certain internucleoside bonds In certain embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides can be linked together using any internucleoside linkage. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (PO), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (PS). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkages can be used to change, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester linkages.In certain embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or as separate enantiomers.Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates.Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0155] The oligonucleotides described herein contain one or more asymmetric centers and may therefore be (R) or (S), in terms of absolute stereochemistry, a or β, in the case of sugar anomers, etc. or amino acids, etc., which may be defined as (D) or (L). The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0156] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 ((3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'). Additionally, neutral internucleoside linkages include siloxanes (dialkylsiloxanes), carboxylic acid esters, carboxamides, Included are nonionic linkages containing sulfides, sulfonate esters, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additionally, neutral internucleoside linkages include nonionic linkages containing mixed N, O, S, and CH2 moieties.

[0157] 4. A specific motif In certain embodiments, antisense oligonucleotide comprises one or more modified nucleosides (such as the nucleosides that comprise modified sugar and / or modified nucleobase) and / or one or more modified internucleoside linkages.The pattern of such modification in oligonucleotide is herein referred to as motif.In certain embodiments, sugar, nucleobase and linkage motif are independent of each other.

[0158] a. Certain glycomotifs In certain embodiments, oligonucleotides contain one or more types of modified and / or naturally occurring sugar moieties arranged along the oligonucleotide or regions thereof in defined patterns or sugar modification motifs, which may include any of the sugar modifications discussed herein and / or other known sugar modifications.

[0159] In certain embodiments, an oligonucleotide comprises or consists of a region having a gapmer sugar motif, which comprises two external regions or "wings" and a central or internal region or "gap." The three regions of the gapmer sugar motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides, and at least a portion of the sugar moieties of the nucleosides in each of these wings differ from at least a portion of the sugar moieties of the nucleosides in the gap. Specifically, at least the sugar moieties of the nucleosides in each wing closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from the sugar moieties of the adjacent gap nucleosides, thus defining the boundary between the wing and the gap. In certain embodiments, the sugar moieties within the gap are identical to each other. In certain embodiments, the gap contains one or more nucleosides having sugar moieties that differ from the sugar moieties of one or more other nucleosides in the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric sugar gapmers). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric sugar gapmers).

[0160] i. Certain 5'-wing In certain embodiments, the 5'-wing of the gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 1 to 7 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5'-wing of the gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of one or two linked nucleosides. The 5'-wing of a gapmer consists of two to four linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of two or three linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of three or four linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of one nucleoside. In certain embodiments, the 5'-wing of a gapmer consists of two linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of three linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of four linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of five linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of six linked nucleosides.

[0161] In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside in the 5'-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside in the 5'-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside in the 5'-wing of a gapmer is an LNA nucleoside.

[0162] In certain embodiments, the 5'-wing of the gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside in the 5'-wing of the gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside in the 5'-wing of the gapmer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside in the 5'-wing of the gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside in the 5'-wing of the gapmer is a 2'-OMe nucleoside.

[0163] In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-deoxynucleoside. In certain embodiments, each nucleoside in the 5'-wing of the gapmer is a 2'-deoxynucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside in the 5'-wing of the gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides in the 5'-wing is an RNA-like nucleoside.

[0164] In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0165] In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0166] ii. Certain 3'-wings In certain embodiments, the 3'-wing of the gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 1 to 7 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of one or two linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of two to four linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of two or three linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of three or four linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of one nucleoside. In certain embodiments, the 3'-wing of the gapmer consists of two linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of three linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of four linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of five linked nucleosides. In certain embodiments, the 3'-wing of the gapmer consists of 6 linked nucleosides.

[0167] In certain embodiments, the 3'-wing of the gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside in the 3'-wing of the gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside in the 3'-wing of the gapmer is a constrained ethyl nucleoside. In certain embodiments, the gapmer Each nucleoside in the 3'-wing of is an LNA nucleoside.

[0168] In certain embodiments, the 3'-wing of the gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of the gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of the gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside in the 3'-wing of the gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside in the 3'-wing of a gapmer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside in the 3'-wing of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside in the 3'-wing of a gapmer is a 2'-OMe nucleoside.

[0169] In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-deoxynucleoside. In certain embodiments, each nucleoside in the 3'-wing of the gapmer is a 2'-deoxynucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside in the 3'-wing of the gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides in the 5'-wing is an RNA-like nucleoside.

[0170] In certain embodiments, the 3'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0171] In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0172] In certain embodiments, the 3'-wing of the gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In embodiments, the 3'-wing of the gapmer comprises at least one LNA nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one LNA nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one LNA nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one LNA nucleoside and at least one 2'-deoxynucleoside.

[0173] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside.

[0174] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside.

[0175] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside.

[0176] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside.

[0177] iii. A specific central region (gap) In certain embodiments, the gapmer gap consists of 6 to 20 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 to 15 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 to 12 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 to 15 linked nucleosides. The gapmer gap consists of 6 to 10 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 to 9 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 to 8 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 or 7 linked nucleosides. In certain embodiments, the gapmer gap consists of 7 to 10 linked nucleosides. In certain embodiments, the gapmer gap consists of 7 or 8 linked nucleosides. In certain embodiments, the gapmer gap consists of 8 to 10 linked nucleosides. In certain embodiments, the gapmer gap consists of 8 or 9 linked nucleosides. In certain embodiments, the gapmer gap consists of 6 linked nucleosides. In certain embodiments, the gapmer gap consists of 7 linked nucleosides. In certain embodiments, the gapmer gap consists of 8 linked nucleosides. In certain embodiments, the gapmer gap consists of 9 linked nucleosides. In certain embodiments, the gapmer gap consists of 10 linked nucleosides. In certain embodiments, the gapmer gap consists of 11 linked nucleosides. In certain embodiments, the gapmer gap consists of 12 linked nucleosides.

[0178] In certain embodiments, each nucleoside of the gapmer gap is a 2'-deoxynucleoside. In certain embodiments, the gap contains one or more modified nucleosides. In certain embodiments, each nucleoside of the gapmer gap is a 2'-deoxynucleoside or a "DNA-like" modified nucleoside. In such embodiments, "DNA-like" means that the nucleoside has characteristics similar to DNA, such that a duplex comprising a gapmer and an RNA molecule can activate RNase H. For example, under certain conditions, 2'-(ara)-F has been shown to support RNase H activation and is therefore DNA-like. In certain embodiments, one or more nucleosides of the gapmer gap are not 2'-deoxynucleosides or DNA-like. In certain such embodiments, the gapmer still supports RNase H activation (e.g., due to the number or placement of non-DNA nucleosides).

[0179] In certain embodiments, the gap comprises a stretch of unmodified 2'-deoxynucleosides interrupted by one or more modified nucleosides, thus resulting in three subregions (two stretches of one or more 2'-deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, any stretch of unmodified 2'-deoxynucleosides is shorter than 5, 6, or 7 nucleosides. In certain embodiments, such a short stretch is achieved by using a short gap region. In certain embodiments, a short stretch is achieved by interrupting a longer gap region.

[0180] In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, the gap comprises one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. In certain embodiments, the gap comprises one modified nucleoside. In certain embodiments, the gap comprises a 5'-substituted sugar moiety selected from among 5'-Me and 5'-(R)-Me. In certain embodiments, the gap comprises two modified nucleosides. In certain embodiments, the gap comprises three modified nucleosides. In certain embodiments, the gap comprises four modified nucleosides. In certain embodiments, In certain embodiments, the gap comprises two or more modified nucleosides, each modified nucleoside being the same. In certain embodiments, the gap comprises two or more modified nucleosides, each modified nucleoside being different.

[0181] In certain embodiments, the gap comprises one or more modified bonds. In certain embodiments, the gap comprises one or more methylphosphonate bonds. In certain embodiments, the gap comprises two or more modified bonds. In certain embodiments, the gap comprises one or more modified bonds and one or more modified nucleosides. In certain embodiments, the gap comprises one modified bond and one modified nucleoside. In certain embodiments, the gap comprises two modified bonds and two or more modified nucleosides.

[0182] b. A specific internucleoside linkage motif In certain embodiments, an oligonucleotide comprises modified internucleoside linkages arranged along the oligonucleotide or a region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, an oligonucleotide comprises a region with alternating internucleoside linkage motifs. In certain embodiments, an oligonucleotide of the present disclosure comprises a region of uniformly modified internucleoside linkages. In certain such embodiments, an oligonucleotide comprises a region uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of an oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of an oligonucleotide is selected from phosphodiester and phosphorothioate, and at least one internucleoside linkage is phosphorothioate.

[0183] In certain embodiments, an oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 7 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 9 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 11 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 12 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 13 phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least 14 phosphorothioate internucleoside linkages.

[0184] In certain embodiments, an oligonucleotide comprises at least one block of at least 6 contiguous phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least 7 contiguous phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least 8 contiguous phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least 9 contiguous phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least 10 contiguous phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide comprises at least one block of at least 12 contiguous phosphorothioate internucleoside linkages. The oligonucleotide comprises at least one block of consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3'-end of the oligonucleotide. In certain such embodiments, at least one such block is located within three nucleosides of the 3'-end of the oligonucleotide. In certain embodiments, the oligonucleotide comprises fewer than 15 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises fewer than 14 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises fewer than 13 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises fewer than 12 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises fewer than 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises fewer than 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises fewer than 9 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 7 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 5 phosphorothioate internucleoside linkages.

[0185] c. A specific nucleobase modification motif In certain embodiments, the oligonucleotide comprises chemical modifications to the nucleobases arranged along the oligonucleotide or its region in a defined pattern or nucleobase modification motif.In certain such embodiments, the nucleobase modifications are arranged in a gap motif.In certain embodiments, the nucleobase modifications are arranged in an alternating motif.In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

[0186] In certain embodiments, the oligonucleotide comprises a block of modified nucleobases. In certain such embodiments, the block is located at the 3' end of the oligonucleotide. In certain such embodiments, the block is located within 3 nucleotides of the 3' end of the oligonucleotide. In certain such embodiments, the block is located at the 5' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleotides of the 5' end of the oligonucleotide.

[0187] In certain embodiments, nucleic acid base modification is the function of the natural base at specific position of oligonucleotide.For example, in certain embodiments, each purine or each pyrimidine of oligonucleotide is modified.In certain embodiments, each adenine is modified.In certain embodiments, each guanine is modified.In certain embodiments, each thymine is modified.In certain embodiments, each cytosine is modified.In certain embodiments, each uracil is modified.

[0188] In certain embodiments, some or all of the cytosine moieties of oligonucleotide are 5-methylcytosine moieties, or none of them are 5-methylcytosine moieties.In this specification, 5-methylcytosine is not " modified nucleobase ".Therefore, unless otherwise indicated, unmodified nucleobase comprises both the cytosine residue with 5-methyl and the cytosine residue that does not have 5-methyl.In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.

[0189] In certain embodiments, the chemical modification to the nucleobase is the addition of certain conjugate groups to the nucleic acid. In certain embodiments, each purine or each pyrimidine of an oligonucleotide may be optionally modified to include a conjugate group.

[0190] d. A specific overall length In certain embodiments, the present disclosure provides oligonucleotides of any of a variety of lengths. In certain embodiments, the oligonucleotide consists of X to Y linked nucleosides, where X represents the minimum number of nucleosides in the range and Y represents the maximum number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, provided that X is less than or equal to Y. For example, in certain embodiments, the oligonucleotide may be any of the following: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9- 15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25 5, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12 ~16, 12~17, 12~18, 12~19, 12~20, 12~21, 12~22, 12~23, 12~24, 12~25, 12~26, 12~27, 12~28, 12~29, 12~30, 13~14, 13~15, 13~16, 13~17, 13~18, 13~19, 13~20, 13~21, 13~22, 13~23, 13~24, 13~25, 13~26,13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 1 8-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22 ~24, 22~25, 22~26, 22~27, 22~28, 22~29, 22~30, 23~24, 23~25, 23~26, 23~27, 23~28, 23~29, 23~30, 24~25, 24~26, 24~27, 24~28, 24~29, 24~30, 25~26, 25~27, 25~28, 25~29, 25~30, 26~27, 26~28, 26~29, 26~30, 27~28, 27~29, 27~30, 28~29, 28~30, or 29~30, It may consist of linked nucleosides. Regardless of a range or a specific number, in embodiments in which the number of nucleosides in the oligonucleotide of a compound is limited, this compound may still further comprise other substituents. For example, an oligonucleotide containing 8 to 30 nucleosides excludes an oligonucleotide having 31 nucleosides, but unless otherwise indicated, such an oligonucleotide may further comprise, for example, one or more conjugated groups, terminal groups, or other substituents.

[0191] Furthermore, when an oligonucleotide is described by a total length range and a region having a specific length, and the sum of the specific lengths of those regions is less than the upper limit of the total length range, the oligonucleotide may have additional nucleosides beyond the length of the specified region, provided that the total number of nucleosides does not exceed the upper limit of the total length range.

[0192] 5. Chemical Motifs of Certain Antisense Oligonucleotides In certain embodiments, the chemical structural characteristics of antisense oligonucleotides are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif, and total length.In certain embodiments, these parameters are each independent of each other.Therefore, each internucleoside linkage of an oligonucleotide with a gapmer sugar motif can be modified or unmodified, and can follow or not follow the gapmer modification pattern of sugar modification.Therefore, the internucleoside linkages in the wing region of the sugar gapmer can be identical or different from each other, and can be identical or different from the internucleoside linkages in the gap region.Similarly, such sugar gapmer oligonucleotides can contain one or more modified nucleobases, independent of the gapmer pattern of sugar modification.Those skilled in the art will recognize that such motifs can be combined to create various oligonucleotides.

[0193] In certain embodiments, the selection of internucleoside linkages and nucleoside modifications are not independent of each other.

[0194] i. A specific sequence and target In certain embodiments, the present invention provides antisense oligonucleotides having a sequence complementary to a target nucleic acid. Such antisense compounds can hybridize to the target nucleic acid and exhibit at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acids. In certain embodiments, specifically hybridizing antisense compounds have a nucleobase sequence that includes a region that has sufficient complementarity to the target nucleic acid to allow hybridization and to exhibit antisense activity, and insufficient complementarity to any non-target nucleic acid sequences to avoid or reduce non-specific hybridization to non-target nucleic acid sequences under conditions where specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic use, and under conditions where the assay is performed in the case of an in vitro assay). In certain embodiments, both the target and non-target sequences contain the target sequence, but the oligonucleotide is selective between the target and non-target. In such embodiments, selectivity can be due to the relative proximity of the target region of one nucleic acid molecule compared to another nucleic acid molecule.

[0195] In certain embodiments, the present disclosure provides antisense compounds comprising oligonucleotides that are completely complementary to target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, the oligonucleotide is 99% complementary to target nucleic acid.In certain embodiments, the oligonucleotide is 95% complementary to target nucleic acid.In certain embodiments, such oligonucleotide is 90% complementary to target nucleic acid.

[0196] In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, the antisense compounds are fully complementary to the target nucleic acid and comprise a region that is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is 6 to 14 nucleobases in length.

[0197] In certain embodiments, the oligonucleotide comprises a hybridizing region and a terminal region. In certain such embodiments, the hybridizing region consists of 12 to 30 linked nucleosides and is fully complementary to the target nucleic acid. In certain embodiments, the hybridizing region contains one mismatch compared to the target nucleic acid. In certain embodiments, the hybridizing region contains two mismatches compared to the target nucleic acid. In certain embodiments, the hybridizing region contains three mismatches compared to the target nucleic acid. In certain embodiments, the terminal region consists of one to four terminal nucleosides. In certain embodiments, the terminal nucleoside is located at the 3' end. In certain embodiments, one or more of the terminal nucleosides are not complementary to the target nucleic acid.

[0198] Antisense mechanisms include any mechanism involving hybridization of an oligonucleotide with a target nucleic acid, which hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy, for example, with concomitant inhibition or stimulation of cellular machinery involved in translation, transcription, or splicing of the target nucleic acid.

[0199] One type of antisense mechanism that involves the degradation of target RNA is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that "DNA-like" single-stranded antisense compounds induce RNase H activity in mammalian cells. Thus, the activation of RNase H leads to the cleavage of RNA targets, thereby significantly improving the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

[0200] In certain embodiments, the conjugate group comprises a cleavable moiety. In certain embodiments, the conjugate group comprises one or more cleavable bonds. In certain embodiments, the conjugate group comprises a linker. In certain embodiments, the linker comprises a protein-binding moiety. In certain embodiments, the conjugate group comprises a cell-targeting moiety (also referred to as a cell-targeting group). In certain embodiments, the cell-targeting moiety comprises a branching group. In certain embodiments, the cell-targeting moiety comprises one or more tethers. In certain embodiments, the cell-targeting moiety comprises a carbohydrate or carbohydrate cluster.

[0201] ii. Certain cleavable parts In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety is attached to the antisense oligonucleotide. In certain such embodiments, the cleavable moiety is attached directly to the cell targeting moiety. In certain such embodiments, the cleavable moiety is attached to a conjugated linker. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog is a purine. The cleavable moiety comprises an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside that is linked to the 3' position of the antisense oligonucleotide by a phosphodiester bond and to the linker by a phosphodiester or phosphorothioate bond. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine that is linked to the 3' position of the antisense oligonucleotide by a phosphodiester bond and to the linker by a phosphodiester or phosphorothioate bond. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine attached to the 3' position of the antisense oligonucleotide by a phosphodiester bond and to the linker by a phosphodiester bond.

[0202] In certain embodiments, the cleavable moiety is attached to the 3' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 2' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester bond. In certain embodiments, the cleavable moiety is attached to the linker by either a phosphodiester bond or a phosphorothioate bond. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester bond. In certain embodiments, the conjugate group does not comprise a cleavable moiety.

[0203] In certain embodiments, the cleavable moiety is cleaved only after it has been internalized by the target cell and the complex is administered to an animal. The cleavable moiety is cleaved in the cell, thereby releasing the active antisense oligonucleotide. Without wishing to be bound by theory, it is believed that the cleavable moiety is cleaved in the cell by one or more nucleases. In certain embodiments, one or more nucleases cleave the phosphodiester bond between the cleavable moiety and the linker. In certain embodiments, the cleavable moiety has a structure selected from the following: [ka] wherein each of Bx, Bx1, Bx2, and Bx3 is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following: [ka]

[0204] iii. Certain linkers In certain embodiments, the conjugate group comprises a linker. In certain such embodiments, the linker is covalently attached to a cleavable moiety. In certain such embodiments, the linker is covalently attached to the antisense oligonucleotide. In certain embodiments, the linker is covalently attached to a cell-targeting moiety. In certain embodiments, the linker further comprises a covalent bond to a solid support. In certain embodiments, the linker further comprises a covalent bond to a protein-binding moiety. In certain embodiments, the linker further comprises a covalent bond to a solid support and further comprises a covalent bond to a protein-binding moiety. In certain embodiments, the linker comprises multiple positions for attachment of a tethered ligand. In certain embodiments, the linker comprises multiple positions for attachment of a tethered ligand and is not attached to a branching group. In certain embodiments, the linker further comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not comprise a linker.

[0205] In certain embodiments, the linker comprises at least one linear group comprising a group selected from an alkyl, amide, disulfide, polyethylene glycol, ether, thioether (-S-), and hydroxylamino (-ON(H)-) group. In certain embodiments, the linear group comprises a group selected from an alkyl, amide, and ether group. In certain embodiments, the linear group comprises a group selected from an alkyl and ether group. In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group comprises at least one neutral linking group. In certain embodiments, the linear group is covalently linked to the cell targeting moiety and the cleavable moiety. In certain embodiments, the linear group is covalently linked to the cell targeting moiety and the antisense oligonucleotide. In certain embodiments, the linear group is covalently linked to the cell targeting moiety, the cleavable moiety, and the solid support. In certain embodiments, the linear group is covalently linked to the cell targeting moiety, the cleavable moiety, the solid support, and the protein-binding moiety. In certain embodiments, a linear group includes one or more cleavable bonds.

[0206] In certain embodiments, the linker comprises a linear group covalently bonded to the scaffold group. In certain embodiments, the scaffold comprises a branched aliphatic group comprising a group selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the scaffold comprises a branched aliphatic group comprising a group selected from alkyl, amide, and ether groups. In certain embodiments, the scaffold comprises at least one monocyclic or polycyclic ring system. In certain embodiments, the scaffold comprises at least two monocyclic or polycyclic ring systems. In certain embodiments, the linear group is covalently bonded to the scaffold group, and the scaffold group comprises a cleavable moiety and a linker. In certain embodiments, the linear group is covalently bonded to a scaffold group, which is covalently bonded to a cleavable moiety, a linker, and a solid support. In certain embodiments, the linear group is covalently bonded to a scaffold group, which is covalently bonded to a cleavable moiety, a linker, and a protein-binding moiety. In certain embodiments, the linear group is covalently bonded to a scaffold group, which is covalently bonded to a cleavable moiety, a linker, a protein-binding moiety, and a solid support. In certain embodiments, the scaffold group comprises one or more cleavable bonds.

[0207] In certain embodiments, the linker comprises a protein-binding moiety, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomolytic components, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid), or lipids, including, but not limited to, cationic lipids. In certain embodiments, the protein-binding moiety is a C16-C22 long-chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane, or 1-pentafluoropropyl.

[0208] In certain embodiments, the linker has a structure selected from the following: [ka] wherein each n is independently 1 to 20, and p is 1 to 6.

[0209] In certain embodiments, the linker has a structure selected from the following: [ka] wherein each n is independently 1 to 20.

[0210] In certain embodiments, the linker has a structure selected from the following: [ka] In the formula, n is 1 to 20.

[0211] In certain embodiments, the linker has a structure selected from the following: [ka] wherein each L is independently a phosphorus linking group or a neutral linking group; Each n is independently 1 to 20.

[0212] In certain embodiments, the linker has a structure selected from the following: [ka] [ka]

[0213] In certain embodiments, the linker has a structure selected from the following: [ka]

[0214] In certain embodiments, the linker has a structure selected from the following: [ka]

[0215] In certain embodiments, the linker has a structure selected from the following: [ka] In the formula, n is 1 to 20.

[0216] In certain embodiments, the linker has a structure selected from the following: [ka]

[0217] In certain embodiments, the linker has a structure selected from the following: [ka]

[0218] In certain embodiments, the linker has a structure selected from the following: [ka]

[0219] In certain embodiments, the conjugated linker has the following structure: [ka]

[0220] In certain embodiments, the conjugated linker has the following structure: [ka]

[0221] In certain embodiments, the linker has a structure selected from the following: [ka]

[0222] In certain embodiments, the linker has a structure selected from the following: [ka] wherein each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

[0223] iv. A specific cell-targeting moiety In certain embodiments, the conjugate group comprises a cell targeting moiety. Certain such cell targeting moieties increase the cellular uptake of antisense compounds. In certain embodiments, the cell targeting moiety comprises a branching group, one or more tethers, and one or more ligands. In certain embodiments, the cell targeting moiety comprises a branching group, one or more tethers, one or more ligands, and one or more cleavable bonds.

[0224] 1. Certain branched groups In certain embodiments, the conjugate group comprises a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group connects the conjugated linker. In certain embodiments, the branching group connects a cleavable moiety. In certain embodiments, the branching group connects the antisense oligonucleotide. In certain embodiments, the branching group is covalently bonded to each of the linker and the tethered ligand. In certain embodiments, the branching group comprises a branched aliphatic group comprising a group selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the branching group comprises a group selected from alkyl, amide, and ether groups. In certain embodiments, the branching group comprises a group selected from alkyl and ether groups. In certain embodiments, the branching group comprises a monocyclic or polycyclic ring system. In certain embodiments, the branching group comprises one or more cleavable bonds. In certain embodiments, the conjugated group does not comprise a branching group.

[0225] In certain embodiments, the branching group has a structure selected from the following: [ka] [ka] wherein each n is independently 1 to 20; j is 1 to 3, m is 2 to 6.

[0226] In certain embodiments, the branching group has a structure selected from the following: [ka] each n is independently 1 to 20; m is 2 to 6.

[0227] In certain embodiments, the branching group has a structure selected from the following: [ka] [ka]

[0228] In certain embodiments, the branching group has a structure selected from the following: [ka] wherein each A1 is independently O, S, C=O, or NH; Each n is independently 1 to 20.

[0229] In certain embodiments, the branching group has a structure selected from the following: [ka] wherein each A1 is independently O, S, C=O, or NH; Each n is independently 1 to 20.

[0230] In certain embodiments, the branching group has a structure selected from the following: [ka] wherein A1 is O, S, C=O, or NH; Each n is independently 1 to 20.

[0231] In certain embodiments, the branching group has a structure selected from the following: [ka]

[0232] In certain embodiments, the branching group has a structure selected from the following: [ka]

[0233] In certain embodiments, the branching group has a structure selected from the following: [ka]

[0234] 2. A specific tether In certain embodiments, the conjugated group comprises one or more tethers covalently bonded to the branching group. In certain embodiments, the conjugated group comprises one or more tethers covalently bonded to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide, and polyethylene glycol groups, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester, and polyethylene glycol groups, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, and amide groups, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether, and amide groups, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one linker. It contains a cyclic or neutral linking group.

[0235] In certain embodiments, the tether comprises one or more cleavable bonds. In certain embodiments, the tether is attached to the branching group through either an amide group or an ether group. In certain embodiments, the tether is attached to the branching group through a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or a neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide group or an ether group. In certain embodiments, the tether is attached to the ligand through either an amide group or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.

[0236] In certain embodiments, each tether comprises a chain length of about 8 to about 20 atoms between the ligand and the branching group. In certain embodiments, each tether comprises a chain length of about 10 to about 18 atoms between the ligand and the branching group. In certain embodiments, each tether comprises a chain length of about 13 atoms.

[0237] In certain embodiments, the tether has a structure selected from the following: [ka] wherein each n is independently 1 to 20; Each p is from 1 to about 6.

[0238] In certain embodiments, the tether has a structure selected from the following: [ka]

[0239] In certain embodiments, the tether has a structure selected from the following: [ka] wherein each n is independently 1 to 20.

[0240] In certain embodiments, the tether has a structure selected from the following: [ka] wherein L is either a phosphorus linking group or a neutral linking group; Z1 is C(=O)O-R2, Z2 is H, C1-C6 alkyl, or substituted C1-C6 alkyl; R2 is H, C1-C6 alkyl, or substituted C1-C6 alkyl; Each m1 is independently 0 to 20, and at least one m1 is greater than 0 for each tether.

[0241] In certain embodiments, the tether has a structure selected from the following: [ka]

[0242] In certain embodiments, the tether has a structure selected from the following: [ka] wherein Z2 is H or CH3; Each m1 is independently 0 to 20, and at least one m1 is greater than 0 for each tether.

[0243] In certain embodiments, the tether has a structure selected from the following: [ka] or [ka] , wherein each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

[0244] In certain embodiments, the tether comprises a phosphorus linking group. In certain embodiments, the tether does not comprise any amide bonds. In certain embodiments, the tether comprises a phosphorus linking group and does not comprise any amide bonds.

[0245] 3. A specific ligand In certain embodiments, the present disclosure provides ligands, each of which is covalently attached to a tether. In certain embodiments, each ligand is selected to have affinity for at least one receptor on a target cell. In certain embodiments, a ligand is selected to have affinity for at least one receptor on the surface of a mammalian liver cell. In certain embodiments, a ligand is selected to have affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is independently selected from galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose. In certain embodiments, each ligand is N-acetylgalactosamine (GalNAc). In certain embodiments, the targeting moiety includes 2 to 6 ligands. In certain embodiments, the targeting moiety includes 3 ligands. In certain embodiments, the targeting moiety includes 3 N-acetylgalactosamine ligands.

[0246] In certain embodiments, the ligand is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a polyvalent carbohydrate cluster, a polysaccharide, a modified polysaccharide, or a polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For example, the amino sugar can be selected from any number of compounds known in the art, such as glucosamine, sialic acid, α-D-galactosamine, N-acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, and N-sulfo-D-glucosamine, and N-glycoloyl-α-neuraminic acid. For example, the thiosugar may be selected from the group consisting of 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

[0247] In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. In certain embodiments, "N-acetylgalactosamine" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, including both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, the terms β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably. Thus, in structures where one form is shown, these structures are intended to include the other form as well. For example, the α-form: 2-(acetylamino)-2-deoxy- When the structure of D-galactopyranose is shown, it is intended to include other forms as well. In certain preferred embodiments, the β form: 2-(acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment. [ka] [ka] [ka]

[0248] In certain embodiments, one or more ligands have a structure selected from the following: [ka] wherein each R1 is selected from OH and NHCOOH.

[0249] In certain embodiments, one or more ligands have a structure selected from the following: Has. [ka]

[0250] In certain embodiments, one or more of the ligands has a structure selected from the following: [ka]

[0251] In certain embodiments, one or more of the ligands has a structure selected from the following: [ka]

[0252] i. Certain conjugates In certain embodiments, the conjugate group comprises the structural features described above. In certain such embodiments, the conjugate group has the structure: [ka] wherein each n is independently 1 to 20.

[0253] In certain such embodiments, the conjugate group has the structure: [ka]

[0254] In certain such embodiments, the conjugate group has the structure: [ka] wherein each n is independently 1 to 20; Z is H or a bound solid support; Q is an antisense compound; X is O or S; Bx is a heterocyclic base moiety.

[0255] In certain such embodiments, the conjugate group has the structure: [ka]

[0256] In certain such embodiments, the conjugate group has the structure: [ka]

[0257] In certain such embodiments, the conjugate group has the structure: [ka]

[0258] In certain such embodiments, the conjugate group has the structure: [ka]

[0259] In certain such embodiments, the conjugate group has the structure: [ka]

[0260] In certain such embodiments, the conjugate group has the structure: [ka]

[0261] In certain such embodiments, the conjugate group has the structure: [ka]

[0262] In certain such embodiments, the conjugate group has the structure: [ka]

[0263] In certain embodiments, the conjugate does not comprise pyrrolidine.

[0264] In certain such embodiments, the conjugate group has the structure: [ka]

[0265] In certain such embodiments, the conjugate group has the structure: [ka]

[0266] In certain such embodiments, the conjugate group has the structure: [ka]

[0267] In certain such embodiments, the conjugate group has the structure: [ka]

[0268] In certain such embodiments, the conjugate group has the structure: [ka]

[0269] In certain such embodiments, the conjugate group has the structure: [ka]

[0270] In certain such embodiments, the conjugate group has the structure: [ka]

[0271] In certain such embodiments, the conjugate group has the structure: [ka]

[0272] In certain such embodiments, the conjugate group has the structure: [ka]

[0273] In certain such embodiments, the conjugate group has the structure: [ka]

[0274] In certain such embodiments, the conjugate group has the structure: [ka]

[0275] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 6 to 11 consecutive bonded atoms.

[0276] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 10 consecutive bond atoms.

[0277] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 4 to 11 contiguous bond atoms, said tether including exactly one amide bond.

[0278] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] In the formula, Y and Z are C1 to C 12 They are independently selected from substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, or groups containing ethers, ketones, amides, esters, carbamates, amines, piperidines, phosphates, phosphodiesters, phosphorothioates, triazoles, pyrrolidines, disulfides, or thioethers.

[0279] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] In the formula, Y and Z are C1 to C 12are independently selected from substituted or unsubstituted alkyl groups, or groups containing exactly one ether or exactly two ethers, amides, amines, piperidines, phosphates, phosphodiesters, or phosphorothioates.

[0280] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: , [ka] In the formula, Y and Z are C1 to C 12 are independently selected from substituted or unsubstituted alkyl groups.

[0281] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0282] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.

[0283] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 4 to 13 contiguous bonded atoms, and X does not include an ether group.

[0284] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 8 consecutive bond atoms, and X does not include an ether group.

[0285] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 4 to 13 consecutive bond atoms, said tether containing exactly one amide bond, and X does not contain an ether group.

[0286] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein X is a substituted or unsubstituted tether of 4 to 13 consecutive bond atoms, said tether comprising an amide bond and a substituted or unsubstituted C2 to C 11 It consists of an alkyl group.

[0287] In certain embodiments, the cell targeting portion of the conjugate group has the structure: [ka] In the formula, Y is C1 to C 12 It is selected from substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, or groups containing ethers, ketones, amides, esters, carbamates, amines, piperidines, phosphates, phosphodiesters, phosphorothioates, triazoles, pyrrolidines, disulfides, or thioethers.

[0288] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] In the formula, Y is C1 to C 12It is selected from substituted or unsubstituted alkyl groups, or groups containing ethers, amines, piperidines, phosphates, phosphodiesters, or phosphorothioates.

[0289] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] In the formula, Y is C1 to C 12 It is selected from substituted or unsubstituted alkyl groups.

[0290] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0291] In certain such embodiments, the cell targeting portion of the conjugate group has the structure: [ka] wherein n is 4, 5, 6, 7, or 8.

[0292] b. Certain conjugated antisense compounds In certain embodiments, the conjugate is attached to a nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside. In certain embodiments, the conjugate antisense compound has the structure: [ka] During the ceremony, A is an antisense oligonucleotide, B is a cleavable moiety, C is a conjugated linker; D is a branched group; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0293] In certain embodiments, the conjugated antisense compound has the structure: [ka] During the ceremony, A is an antisense oligonucleotide, C is a conjugated linker; D is a branched group; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0294] In certain such embodiments, the conjugated linker comprises at least one cleavable bond.

[0295] In certain such embodiments, the branching group comprises at least one cleavable bond.

[0296] In certain embodiments, each tether comprises at least one cleavable bond.

[0297] In certain embodiments, the conjugate is attached to a nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside.

[0298] In certain embodiments, the conjugated antisense compound has the structure: [ka] During the ceremony, A is an antisense oligonucleotide, B is a cleavable moiety, C is a conjugated linker; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0299] In certain embodiments, the conjugate is attached to a nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside. In certain embodiments, the conjugate antisense compound has the structure: [ka] During the ceremony, A is an antisense oligonucleotide, C is a conjugated linker; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0300] In certain embodiments, the conjugated antisense compound has the structure: [ka] During the ceremony, A is an antisense oligonucleotide, B is a cleavable moiety, D is a branched group; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0301] In certain embodiments, the conjugated antisense compound has the structure: [ka] During the ceremony, A is an antisense oligonucleotide, D is a branched group; Each E is a tether, each F is a ligand; q is an integer of 1 to 5.

[0302] In certain such embodiments, the conjugated linker comprises at least one cleavable bond.

[0303] In certain embodiments, each tether comprises at least one cleavable bond.

[0304] In certain embodiments, the conjugated antisense compound has a structure selected from among the following: [ka]

[0305] In certain embodiments, the conjugated antisense compound has a structure selected from among the following: [ka]

[0306] In certain embodiments, the conjugated antisense compound has a structure selected from among the following: [ka]

[0307] Representative United States patents, United States patent application publications, and international patent application publications that teach certain preparations of the above-described conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties, and other modifications include, but are not limited to, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, 2006 / 0148740, 2011 / 0123520, International Publication Nos. WO2013 / 033230, and WO2012 / 037254, each of which is incorporated herein by reference in its entirety.

[0308] Representative publications teaching certain preparations of the above-described conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties, and other modifications include BIESSEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent," J. Med. Chem. (1995) 38:1846-1852; BIESSEN et al., "Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor," J. Med. Chem. (1995) 38:1538-1546; LEE et al., "New and d more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes”Bioorganic & Medicinal Chemistry(2011)19:2494-2500, RENSEN et al.,“Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo”J.Biol.Chem.(2001)276(40):37577-37584, RENSEN et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteinss to the Hepatic Asialoglycoproteins Receptor” J. Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1999) 42:609-618, and Valentijn et al. al., “Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor,” Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated herein by reference in its entirety.

[0309] In certain embodiments, the conjugate antisense compound comprises an RNase H-based oligonucleotide (such as a gapmer) or a splice control oligonucleotide (such as a fully modified oligonucleotide), and an optional conjugate group containing at least one, two, or three GalNAc groups. In certain embodiments, the conjugate antisense compound comprises the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J,1987,4,317-328、Toyokuni et al.,Tetrahedron Lett,1990,31,2673-2676、Biessen et al.,J Med Chem,1995,38,1538-1546、Valentijn et al.,Tetrahedron,1997,53,759-770、Kim et al.,Tetrahedron Lett,1997,38,3487-3490、Lee et al.,Bioconjug Chem,1997,8,762-765、Kato et al.,Glycobiol,2001,11,821-829、Rensen et al.,J Biol Chem,2001,276,37577-37584、Lee et al.,Methods Enzymol,2003,362,38-43、et al.con,Westerlind J,2004,21,227-241、Lee et al.,Bioorg Med Chem Lett,2006,16(19),5132-5135、Maierhofer et al.,Bioorg Med Chem,2007,15,7661-7676ov Med、K Chem,2008,16,5216-5231、Lee et al.,Bioorg Med Chem,2011,19,2494-2500、Kornilova et al.,Analyt Biochem,2012,425,43-46、Pujol et al.,Angew Chemie Int Ed Engl,2012,51,7445-7448、Biessen et al.,J Med Chem,1995,38,1846-1852、Sliedre et al.,JMegtd. Chem,1999,42,609-618、Rensen et al.,J Med Chem,2004,47,5798-5808、Rensen et al.,Arterioscler Thromb Vasc Biol,2006,26,169-175、van Rossenberg et al., Ther,2004,11,457-464、Sato et al.,J Am Chem Soc,2004,126,14013-14022、Lee et al.,J Org Chem,2012,77,7564-7571、Biessen et al.,FASEB J,2000,14,1784-1792、Rajur et al.,Bioconjug Chem,1997,8,935-940、Duff et al.,Methods Enzymol,2000,313,297-321、Maier et al.,Bioconjug Chem,2003,14,18-29、Jayaprakash et al.,Org Lett,2010,12,5410-5413、Manoharan,Antisense Nucleic Acid Drug Dev,2002,12,103-128、Merwin et al.,Biocon. Chem,1994,5,612-620、Tomiya et al.,Bioorg Med Chem,2013,21,5275-5281, International Publication Nos. WO1998 / 013381, WO2011 / 038356, WO1997 / 046098, WO2008 / 098788, WO2004 / 101619, WO2012 / 037254, WO2011 / 120053, WO2011 / 100131, WO2011 / 163121, WO2012 / 177947, WO2013 / 033230, WO2013 / 075035, WO 2012 / 083185, WO2012 / 083046, WO2009 / 082607, WO2009 / 134487, WO2010 / 144740, WO2010 / 148013, WO1997 / 020 563, WO2010 / 088537, WO2002 / 043771, WO2010 / 129709, WO2012 / 068187, WO2009 / 126933, WO2004 / 024757, WO Nos. 2010 / 054406, WO2012 / 089352, WO2012 / 089602, WO2013 / 166121, WO2013 / 165816, U.S. Patent Nos. 4,751,219, 8,552,163, 6,908,903, 7,262,177, 5,994,517, 6,300,319, 8,106,022, 7,491,805, 7,491,805, 7,582,744, and 8,137 , 695, 6,383,812, 6,525,031, 6,660,720, 7,723,509, 8,541,548, 8,344,125, 8,313,772, 8,349,308, No. 8,450,467, No. 8,501,930, No. 8,158,601, No. 7,262,177, No. 6,906,182, No. 6,620,916, No. 8,435,491, No. 8,404,862, No. 7,851,615; Published U.S. Patent Application Publication Nos. US2011 / 0097264, US2011 / 0097265, US2013 / 0004427, US2005 / 0164235, US2006 / 0148740, US2008 / 0281044, US2010 / 0240730, US2003 / 0119724, US2006 / 0183886 No., US2008 / 0206869, US2011 / 0269814, US2009 / 0286973, US2011 / 0207799, US2012 / 0136042, US2012 / 0165393, US2008 / 0281041, US2009 / 0203135, US2012 / 0035115, US2012 / 009, 5075, US2012 / 0101148, US2012 / 0128760, US2012 / 0157509, US2012 / 0230938, US2013 / 0109817, US2013 / 0121954, US2013 / 0178512, US2013 / 0236968, US2011 / 0123520, US2003 / 0077829, US2008 / 0108801, and US2009 / 0203132, each of which is incorporated by reference in its entirety. C. Certain Uses and Features

[0310] In certain embodiments, conjugated antisense compounds exhibit potent reduction of target RNA in vivo. In certain embodiments, unconjugated antisense compounds accumulate in the kidney. In certain embodiments, conjugated antisense compounds accumulate in the liver. In certain embodiments, conjugated antisense compounds are well tolerated. Such properties make conjugated antisense compounds particularly useful for inhibiting many target RNAs, including, but not limited to, target RNAs involved in metabolic, cardiovascular, and other diseases, disorders, or conditions. Thus, provided herein are methods for treating such diseases, disorders, or conditions by contacting liver tissue with a conjugated antisense compound that targets an RNA associated with such a disease, disorder, or condition. Thus, also provided are methods for ameliorating any of various metabolic, cardiovascular, and other diseases, disorders, or conditions using the conjugated antisense compounds of the present invention.

[0311] In certain embodiments, conjugated antisense compounds are more potent than their unconjugated counterparts at certain tissue concentrations. Without wishing to be bound by any theory or mechanism, in certain embodiments, conjugates may allow conjugated antisense compounds to enter cells more efficiently or more efficiently. For example, in certain embodiments, conjugated antisense compounds may exhibit higher target reduction compared to their unconjugated counterparts, and both the conjugated antisense compounds and their unconjugated counterparts are present in tissues at the same concentration. For example, in certain embodiments, conjugated antisense compounds may exhibit higher target reduction compared to their unconjugated counterparts, and both the conjugated antisense compounds and their unconjugated counterparts are present in the liver at the same concentration.

[0312] Productive and non-productive uptake of oligonucleotides has been discussed previously (e.g., Geary, R.S., E. Wancewicz, et al. (2009). “Effect of Dose and Plasma Concentration on Liver Uptake and Pharmacologic Activity of a 2'-Methoxyethyl Modified Chimeric Antisense Oligonucleotide Targeting PTEN.” Biochem. Pharmacol. 78(3):284-91, and Koller, E., T.M. Vincent, et al. (2011). “Mechanisms of single-stranded phosphorothioate (See, "Modified antisense oligonucleotide accumulation in hepatocytes." Nucleic Acids Res. 39(11):4795-807). The conjugate groups described herein can improve productive uptake.

[0313] In certain embodiments, the conjugate groups described herein may further improve potency by increasing the affinity of the conjugated antisense compound for particular cell types or tissues. In certain embodiments, the conjugate groups described herein may further improve potency by increasing the recognition of the conjugated antisense compound by one or more cell surface receptors. In certain embodiments, the conjugate groups described herein may further improve potency by increasing the affinity of the conjugated antisense compound for particular cell types or tissues. Potency can be further improved by promoting endocytosis of the antisense compound.

[0314] In certain embodiments, the cleavable moiety may further improve potency by allowing the conjugate to be cleaved from the antisense oligonucleotide after the conjugated antisense compound has entered the cell. Thus, in certain embodiments, the conjugated antisense compound may be administered at a lower dose than that required for the unconjugated antisense oligonucleotide.

[0315] Phosphorothioate bonds have previously been incorporated into antisense oligonucleotides. Such phosphorothioate bonds improve the stability of the oligonucleotides by providing resistance to nucleases. Furthermore, phosphorothioate bonds also bind to certain proteins, leading to accumulation of antisense oligonucleotides in the liver. Oligonucleotides with fewer phosphorothioate bonds accumulate less in the liver and more in the kidney (see, e.g., Geary, R., "Pharmacokinetics of Antisense Oligonucleotides," vol. 1, no. 1, pp. 111-114, 2003). (See, "Properties of 2'-O-(2-Methoxyethyl)-Modified Oligonucleotide Analogs in Rats," Journal of Pharmacology and Experimental Therapeutics, Vol. 296, No. 3, pp. 890-897, and "Pharmacological Properties of 2'-O-Methoxyethyl Modified Oligonucleotides in Antisense a Drug Technology," Chapter 10, Crooke, ST, ed., 2008). In certain embodiments, oligonucleotides with fewer phosphorothioate internucleoside linkages and more phosphodiester internucleoside linkages accumulate less in the liver and more in the kidney. When treating liver diseases, this is undesirable for several reasons: (1) less drug reaches the desired site of action (liver), (2) drug is lost in the urine, and (3) the kidneys are exposed to relatively high concentrations of drug that may be toxic to the kidney. Thus, in the case of liver diseases, phosphorothioate linkages provide an important advantage.

[0316] However, in certain embodiments, administration of oligonucleotides uniformly linked by phosphorothioate internucleoside linkages induces one or more proinflammatory responses (see, e.g., J Lab Clin Med. 1996 Sep;128(3):329-38. "Amplification of antibody production by phosphorothioate oligodeoxynucleotides". Branda et al.; see also, e.g., Toxicologic Properties in Antisense a Drug Technology, Chapter 12, pages 342-351, Crooke, ST, ed., 2008). In certain embodiments, administration of oligonucleotides in which many of the internucleoside linkages comprise phosphorothioate internucleoside linkages induces one or more proinflammatory responses.

[0317] In certain embodiments, the extent of the pro-inflammatory effect may depend on several variables (e.g., backbone modifications, off-target effects, nucleobase modifications, and / or nucleoside modifications) (e.g., Toxicological Properties in Antisense Oligonucleotides). (See Drug Technology, Chapter 12, pages 342-351, Crooke, ST, ed., 2008). In certain embodiments, the degree of pro-inflammatory effect can be reduced by adjusting one or more variables. For example, the degree of pro-inflammatory effect of a given oligonucleotide can be reduced by replacing any number of phosphorothioate internucleoside linkages with phosphodiester internucleoside linkages. This can be alleviated by reducing the total number of phosphorothioate internucleoside linkages.

[0318] In certain embodiments, it is desirable to reduce the number of phosphorothioate bond, and thus can be reduced without losing stability or changing the distribution from liver to kidney.For example, in certain embodiments, the number of phosphorothioate bond can be reduced by replacing phosphorothioate bond with phosphodiester bond.In such embodiments, the antisense compound with fewer phosphorothioate bond and more phosphodiester bond induces lower or no pro-inflammatory reaction.Although the antisense compound with fewer phosphorothioate bond and more phosphodiester bond induces lower pro-inflammatory reaction, the antisense compound with fewer phosphorothioate bond and more phosphodiester bond does not accumulate in liver, and compared with the antisense compound with more phosphorothioate bond, the antisense compound with fewer phosphorothioate bond and more phosphodiester bond may be less effective at the same or similar dose.Therefore, in certain embodiments, it is desirable to design the antisense compound with multiple phosphodiester bond and multiple phosphorothioate bond, but also has stability and good distribution to liver.

[0319] In certain embodiments, even when some of the phosphorothioate linkages are replaced with less proinflammatory phosphodiester internucleoside linkages, conjugated antisense compounds accumulate more in the liver and less in the kidneys than their unconjugated counterparts. In certain embodiments, even when some of the phosphorothioate linkages are replaced with less proinflammatory phosphodiester internucleoside linkages, conjugated antisense compounds accumulate more in the liver and are not excreted in the urine as much as their unconjugated counterparts. In certain embodiments, the use of conjugates makes it possible to design more potent and better tolerated antisense drugs. In fact, in certain embodiments, conjugated antisense compounds have a larger therapeutic index than their unconjugated counterparts. This allows conjugated antisense compounds to be administered at higher absolute doses due to a lower risk of proinflammatory responses and a lower risk of nephrotoxicity. This higher dose allows for less frequent dosing because elimination (metabolism) is expected to be similar. Furthermore, as noted above, the more potent the compounds are, the lower the concentration can be before the next dose without loss of therapeutic activity, allowing for longer periods between doses.

[0320] In certain embodiments, the inclusion of some phosphorothioate linkages is still desirable. For example, because terminal linkages are vulnerable to exonucleases, in certain embodiments, these linkages are phosphorothioate or other modified linkages. Because internucleoside linkages connecting two deoxynucleosides are vulnerable to endonucleases, in certain embodiments, these linkages are phosphorothioate or other modified linkages. Because internucleoside linkages between modified nucleosides on the 5' side of the linking deoxynucleoside are vulnerable to endonucleases, in certain embodiments, these linkages are phosphorothioate or other modified linkages. Because internucleoside linkages between certain types of two modified nucleosides and between certain types of deoxynucleosides and modified nucleosides where the modified nucleoside is on the 5' side of the linkage are sufficiently resistant to nuclease digestion, this linkage can be phosphodiester.

[0321] In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound contains fewer than 16 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound contains fewer than 15 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 14 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 13 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 12 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 11 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 10 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 9 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 8 phosphorothioate linkages.

[0322] In certain embodiments, antisense compounds comprising one or more conjugate groups described herein have increased activity and / or potency and / or tolerability compared to parent antisense compounds lacking such one or more conjugate groups. Therefore, in certain embodiments, the attachment of such conjugate groups to oligonucleotides is desirable. Such conjugate groups can be attached to the 5'-end and / or 3'-end of an oligonucleotide. In certain cases, attachment at the 5'-end is synthetically desirable. Typically, oligonucleotides are synthesized by attaching the 3'-end nucleoside to a solid support and sequentially coupling nucleosides from 3' to 5' using techniques well known in the art. Thus, if a conjugate group is desired at the 3'-end, (1) the conjugate group can be attached to the 3'-end nucleoside, and then the conjugated nucleoside can be attached to a solid support for subsequent preparation of the oligonucleotide, or (2) the conjugate group can be attached to the 3'-end nucleoside of the complete oligonucleotide after synthesis. Neither of these methods is very efficient, and therefore both are expensive. Specifically, the attachment of conjugated nucleosides to solid supports, as demonstrated in the examples herein, is an inefficient process. In certain embodiments, attaching a conjugated group to the 5'-terminal nucleoside is synthetically easier than attachment at the 3'-terminus. Using well-characterized standard reactions, unconjugated 3'-terminal nucleosides can be attached to solid supports to prepare the oligonucleotides. Then, in the final coupling step, only the 5'-nucleoside bearing the conjugated group needs to be attached. In certain embodiments, this is more efficient than the direct attachment of conjugated nucleosides to solid supports, which is typically performed to prepare 3'-conjugated oligonucleotides. The examples herein demonstrate attachment at the 5'-terminus. In addition, certain conjugated groups have synthetic advantages. For example, certain conjugated groups containing phosphorus linking groups are synthetically simpler and more efficiently prepared than other conjugated groups, including previously reported conjugated groups (e.g., International Publication No. WO / 2012 / 037254).

[0323] In certain embodiments, a conjugated antisense compound is administered to a subject. In such embodiments, an antisense compound containing one or more conjugation groups described herein has increased activity and / or potency and / or tolerability compared to a parent antisense compound lacking such one or more conjugation groups. Without being bound by mechanism, it is believed that the conjugation group aids in distribution, delivery, and / or uptake into target cells or tissues. In certain embodiments, upon entry into target cells or tissues, it is desirable to cleave all or a portion of the conjugation group to release the active oligonucleotide. In certain embodiments, it is not necessary for all conjugation groups to be cleaved from the oligonucleotide. For example, in Example 20, conjugated oligonucleotides were administered to mice, and several different species were detected, each containing a different portion of the conjugation group remaining on the oligonucleotide (Table 10a). This conjugated antisense compound exhibited good potency (Table 10). Thus, in certain embodiments, such metabolite profiles of multiple partial cleavage of the conjugation group can be analyzed. The 5'-terminal conjugate group is likely to result in complete metabolism of the conjugate group. Without being constrained by mechanism, endogenous enzymes involved in metabolism of the 5'-terminus (e.g., 5'-nucleases) may be more active / efficient than their 3'-terminus counterparts. In certain embodiments, these conjugate groups are more amenable to metabolism to a single active species. In certain cases, when multiple forms of an active compound are found, it may be necessary to determine the relative amount and activity of each form. In certain embodiments, when regulatory review is required (e.g., USFDA or equivalent), it is desirable to have a single (or primarily single) active species. In certain such embodiments, it is desirable for such a single active species to be an antisense oligonucleotide lacking any portion of the conjugate group. In certain embodiments, a conjugate group at the 5'-terminus is more likely to result in complete metabolism of the conjugate group. Without being constrained by mechanism, endogenous enzymes involved in metabolism of the 5'-terminus (e.g., 5'-nucleases) may be more active / efficient than their 3'-terminus counterparts. In certain embodiments, these conjugate groups are more amenable to metabolism to a single active species. In certain embodiments, certain conjugate groups are more amenable to metabolism to an oligonucleotide.

[0324] D. Antisense In certain embodiments, the oligomeric compound of the present invention is an antisense compound. In such embodiments, the oligomeric compound is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is RNA. In certain embodiments, the target nucleic acid is non-coding RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain embodiments, the target nucleic acid is selected from mRNA, pre-mRNA, microRNA, non-coding RNA including small non-coding RNA, and promoter-directed RNA. In certain embodiments, the oligomeric compound is at least partially complementary to two or more target nucleic acids. For example, the oligomeric compound of the present invention is a microRNA mimic, which typically binds to multiple targets.

[0325] In certain embodiments, an antisense compound comprises a portion having a nucleobase sequence at least 70% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, an antisense compound comprises a portion having a nucleobase sequence at least 80% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, an antisense compound comprises a portion having a nucleobase sequence at least 90% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, an antisense compound comprises a portion having a nucleobase sequence at least 95% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, an antisense compound comprises a portion having a nucleobase sequence at least 98% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, an antisense compound is at least 70%, 80%, 90%, 95%, 98%, or 100% complementary to the nucleobase sequence of a target nucleic acid over the entire length of the antisense compound.

[0326] Antisense mechanisms include any mechanism involving hybridization of an oligomeric compound with a target nucleic acid, which hybridization results in a biological effect. In certain embodiments, such hybridization results in either degradation or occupancy of the target nucleic acid, for example, with the concomitant inhibition or stimulation of cellular machinery involved in translation, transcription, or polyadenylation of the target nucleic acid or of a nucleic acid with which the target nucleic acid may otherwise interact.

[0327] One type of antisense mechanism that involves degradation of target RNA is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that "DNA-like" single-stranded antisense compounds induce RNase H activity in mammalian cells. Thus, RNase H Activation of results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

[0328] Antisense mechanism also includes, but is not limited to, the RNAi mechanism that utilizes RISC pathway.Such RNAi mechanism includes, but is not limited to, siRNA, ssRNA and microRNA mechanism.Such mechanism includes the creation of microRNA mimics and / or anti-microRNA.

[0329] Antisense mechanism also includes, but is not limited to, the mechanism of hybridizing or mimicking the non-coding RNA other than microRNA or mRNA.Such non-coding RNA includes, but is not limited to, promoter-directed RNA and short RNA and long RNA that cause the transcription or translation of one or more nucleic acids.

[0330] In certain embodiments, the oligonucleotide comprising the conjugate described herein is an RNAi compound. In certain embodiments, the oligomeric oligonucleotide comprising the conjugate described herein is an ssRNA compound. In certain embodiments, the oligonucleotide comprising the conjugate described herein is paired with a second oligomeric compound to form an siRNA. In certain such embodiments, the second oligomeric compound also comprises a conjugate. In certain embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In certain embodiments, the oligonucleotide comprising the conjugate described herein is the antisense strand in an siRNA compound. In certain embodiments, the oligonucleotide comprising the conjugate described herein is the sense strand in an siRNA compound. In embodiments where the conjugate oligomeric compound is a double-stranded siRNA, the conjugate can be present in the sense strand, the antisense strand, or both the sense strand and the antisense strand. D. Target Nucleic Acids, Regions, and Segments

[0331] In certain embodiments, the conjugated antisense compound targets any nucleic acid. In certain embodiments, the target nucleic acid encodes a clinically relevant target protein. In such embodiments, modulation of the target nucleic acid results in clinical benefit. Certain target nucleic acids include, but are not limited to, the target nucleic acids illustrated in Table 1. [Table 1]

[0332] The targeted process usually involves determination of at least one target region, segment, or site within the target nucleic acid at which an antisense interaction will occur, resulting in a desired effect.

[0333] In certain embodiments, the target region is a structurally defined region of a nucleic acid. For example, in certain such embodiments, the target region may include a 3'UTR, a 5'UTR, an exon, an intron, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid region or target segment.

[0334] In certain embodiments, a target segment is at least about an 8-nucleobase portion of a target region to which a conjugated antisense compound is targeted. A target segment may comprise a DNA or RNA sequence comprising at least 8 contiguous nucleobases from the 5' end of one of the target segments (the remaining nucleobases begin immediately upstream of the 5' end of the target segment and extend beyond the 5' end of the target segment). A target segment is a contiguous stretch of identical DNA or RNA that continues until the DNA or RNA contains about 8 to about 30 nucleobases. A target segment can also be represented by a DNA or RNA sequence that contains at least 8 contiguous nucleobases from the 3' end of one of the target segments (the remaining nucleobases are contiguous stretches of identical DNA or RNA that begin immediately downstream of the 3' end of the target segment and continue until the DNA or RNA contains about 8 to about 30 nucleobases). A target segment can also be represented by a DNA or RNA sequence that contains at least 8 contiguous nucleobases from an internal portion of the target segment's sequence, and can extend in either or both directions until the conjugated antisense compound contains about 8 to about 30 nucleobases.

[0335] In certain embodiments, antisense compounds targeting the nucleic acids listed in Table 1 may be modified as described herein. In certain embodiments, antisense compounds may have modified sugar moieties, unmodified sugar moieties, or a mixture of modified and unmodified sugar moieties as described herein. In certain embodiments, antisense compounds may have modified internucleoside linkages, unmodified internucleoside linkages, or a mixture of modified and unmodified internucleoside linkages as described herein. In certain embodiments, antisense compounds may have modified nucleobases, unmodified nucleobases, or a mixture of modified and unmodified nucleobases as described herein. In certain embodiments, antisense compounds may have motifs as described herein.

[0336] In certain embodiments, antisense compounds targeted to the nucleic acids listed in Table 1 can be conjugated as described herein.

[0337] 1. Hepatitis B (HBV) Hepatitis B is a viral disease transmitted parenterally through contaminated materials such as blood and blood products, contaminated needles, and sexually and vertically from infected or carrier mothers to their offspring. The World Health Organization estimates that over 2 billion people worldwide are infected, with approximately 4 million acute cases and 1 million deaths per year, and 350 to 400 million chronic carriers (World Health Organization: Geographic Prevalence of Hepatitis B Prevalence, 2004. http: / / www.who.int / vaccines-surveillance / graphics / htmls / hepbprev.htm).

[0338] The virus HBV is a double-stranded, hepatotropic virus that infects only humans and nonhuman primates. Viral replication occurs primarily in the liver and, to a lesser extent, in the kidneys, pancreas, bone marrow, and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64:2000;51-68). Viral and immune markers are detectable in the blood, and characteristic antigen-antibody patterns evolve over time. The first detectable viral marker is HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA. Titers can be high during the incubation period, but HBV DNA and HBeAg levels begin to decline at the onset of disease and can become undetectable at the peak of clinical disease (Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med. 350:2004;1118-1129). HBeAg is a viral marker detectable in the blood that correlates with active viral replication and thus high viral load and infectivity (Hepatitis B antigen—the dangerous end game of hepatitis BN Engl J Med.347:2002;208-210). The presence of anti-HBsAb and anti-HBcAb (IgG) is a predictor of recovery and immunity in previously infected individuals. Shows infectious power.

[0339] Therapies currently recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) for the Treatment of Chronic HBV Infection include interferon α (IFNα), pegylated interferon α-2a (Peg-IFN2a), entecavir, and tenofovir. The nucleoside and nucleobase therapies, entecavir and tenofovir, successfully reduce viral load, but the HBeAg seroconversion and HBsAg loss rates are even lower than those achieved with IFNα therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir, are also used, but resistance development generally limits the therapeutic efficacy of nucleoside / nucleobase therapies.

[0340] Therefore, there is a need in the art for the discovery and development of new antiviral therapies. Furthermore, there is a need for new anti-HBV therapies that can increase the seroconversion rate of HBeAg and HBsAg. Recent clinical studies have demonstrated that HBeAg seroconversion and reduction (Fried et al. (2008) Hepatology 47:428) and a decrease in HBsAg (Moucari et al (2009) Hepatology 49:1151). Because high levels of antigen are thought to induce immunological tolerance, a decrease in antigen levels may have enabled immunological control of HBV infection. Current nucleoside therapy for HBV can dramatically reduce serum HBV levels but has little effect on HBeAg and HBsAg levels.

[0341] The antisense compound that targets HBV has been previously disclosed in International Publication No. WO2011 / 047312, WO2012 / 145674 and WO2012 / 145697, each of which is incorporated herein by reference in its entirety.Clinical research is planned to evaluate the effect of the antisense compound that targets HBV in patients.However, there is still a need to provide patients with further and more effective treatment options.

[0342] Certain conjugated antisense compounds targeting HBV nucleic acid In certain embodiments, the conjugate antisense compound targets an HBV nucleic acid having the sequence of GENBANK® Accession Number U95551.1, incorporated herein as SEQ ID NO: 1. In certain such embodiments, the conjugate antisense compound targeting SEQ ID NO: 1 is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 1.

[0343] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 3. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 3.

[0344] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 4. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 4.

[0345] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 5. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 5.

[0346] In certain embodiments, conjugate antisense compounds targeted to SEQ ID NO: 1 comprise at least an 8 contiguous nucleobase sequence of SEQ ID NO: 6. A conjugate antisense compound targeting SEQ ID NO:1 comprises the nucleobase sequence of SEQ ID NO:6.

[0347] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 7. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 7.

[0348] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 8. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 8.

[0349] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 9. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 9.

[0350] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 10. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 10.

[0351] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 11. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 11. [Table 2]

[0352] In certain embodiments, the compound comprises or consists of ISIS 505358 and a conjugate group. ISIS 505358 has the formula: Ges mCes Aes A modified oligonucleotide having the formula: Ges Aes Gds Gds Tds Gds Ads Ads Gds mCds Gds Ads Aes Ges Tes Ges mCe, A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0353] In certain embodiments, the compound comprises or consists of ISIS 509934 and a conjugate group. ISIS 509934 has the formula: mCes mCes Ae s Aes Tes Tds Tds Ads Tds Gds mCds mCds Tds Ads mCds Aes Ges mCes mCes Te, wherein A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0354] In certain embodiments, the compound comprises or consists of ISIS 510100 and a conjugate group. ISIS 510100 has the formula: Ges Ges mCes A modified oligonucleotide having Ads Tds Ads Gds mCds Ads Gds mCds Ads Gds Ges Aes Tes Ge, wherein A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0355] In certain embodiments, the compound comprises or consists of ISIS 552023 and a conjugate group. ISIS 552023 is a modified oligonucleotide having the formula: Aes Ges Ges Aes Ges Tes Tds mCds mCds Gds mCds Ads Gds Tds Ads Tds Ges Ges Aes Te, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0356] In certain embodiments, the compound comprises or consists of ISIS 552024 and a conjugate group. ISIS 552024 has the formula: Ges Tes Ges Aes Aes Ges mCds Gds Ads Ads Gds Tds Gds mCdsAdsmCdsAesmCesGesGe, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0357] In certain embodiments, the compound comprises ISIS 552032 and a conjugate group. ISIS 552032 is of the formula: Ges Tes Ges mCes Aes Ges Ads Gds Gds Tds Gds Ads Ads A modified oligonucleotide having the formula: Gds mCds Gds Aes Aes Ges Te, A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0358] In certain embodiments, the compound comprises or consists of ISIS 552859 and a conjugate group. ISIS 552859 has the formula: Aes Gks Gks Tds Gds Ads Ads Gds mCds Gds Ads Ads Gds A modified oligonucleotide having Tks Gks mCe, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; k is a cEt-modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0359] In certain embodiments, the compound comprises or consists of ISIS 552925 and a conjugate group. ISIS 552925 is a modified oligonucleotide having the formula: Tes mCks mCds Gds mCds Ads Gds Tds Ads Tds Gds Gds Aks Tes mCks Ge, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; k is a cEt-modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage. s is a phosphorothioate internucleoside linkage.

[0360] In certain embodiments, the compound comprises or consists of ISIS 577119 and a conjugate group. ISIS 577119 is a modified oligonucleotide having the formula: Aks Ads Tks Tds Tks Ads Tds Gds mCds mCds Tds Ads mCds Aes Ges mCes mCes Te, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; k is a cEt-modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0361] In certain embodiments, the compound having the following chemical structure comprises or consists of ISIS 505358 having a 5'-X, where X is a conjugate group as described herein. [ka]

[0362] In certain embodiments, the compound comprises or consists of ISIS 712408, which has the following chemical structure: [ka]

[0363] In certain embodiments, the compound comprises or consists of ISIS 695324, which has the following chemical structure: [ka]

[0364] In certain embodiments, the compound comprises or consists of SEQ ID NO:3, 5'-GalNAc, and a chemical modification represented by the following chemical structure: [ka] In the formula, R 1 is -OCH2CH2OCH3(MOE), and R 2 is H or R 1 and R 2 together to form a bridge, where R 1 is -O- and R 2 is —CH—, —CH(CH)—, or —CHCH—, and the resulting bridge is selected from —O—CH—, —O—CH(CH)—, and —O—CHCH—; 1 and R 2 are directly connected, On the same ring, R of each ring 3 and R 4 For each pair, independently for each ring, R 3 is selected from H and —OCHCHOCH, and R 4 is H or R 3 and R 4 together to form a bridge, where R 3 is -O- and R 4 is —CH—, —CH(CH)—, or —CHCH—, and the resulting bridge is selected from —O—CH—, —O—CH(CH)—, and —O—CHCH—; 3 and R 4 are directly connected, R 5 is selected from H and -CH3; Z is S - and O - is selected from.

[0365] In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2012 / 145697, the entire contents of which are incorporated herein by reference, and a conjugate group as described herein. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2012 / 145697, the entire contents of which are incorporated herein by reference, and a conjugate group as described herein. The compound comprises an antisense oligonucleotide having any of the nucleobase sequences of SEQ ID NOS: 14-22, 1288-1350, 1364-1372, 1375, 1376, and 1379, and a conjugate group described herein. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO 2011 / 047312, the entirety of which is incorporated herein by reference, and a conjugate group described herein. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO 2012 / 145674, the entirety of which is incorporated herein by reference, and a conjugate group described herein. In certain embodiments, the compound comprises an antisense oligonucleotide having a nucleobase sequence of any one of SEQ ID NOS: 18-35 disclosed in International Publication No. WO 2012 / 145674. In certain embodiments, the compound comprises a double-stranded oligonucleotide disclosed in International Publication No. WO 2013 / 159109, the entire contents of which are incorporated herein by reference, and a conjugate group as described herein. In certain embodiments, the compound comprises a double-stranded oligonucleotide, one strand of which has a nucleobase sequence of any one of SEQ ID NOS: 30-125 disclosed in International Publication No. WO 2013 / 159109. All nucleobase sequences of the referenced SEQ ID NOS are incorporated herein by reference.

[0366] HBV treatment index In certain embodiments, the present invention provides methods for modulating expression of HBV in a subject using conjugated antisense compounds that target HBV nucleic acid. In certain embodiments, expression of HBV is reduced.

[0367] In certain embodiments, the present invention provides a method for treating a subject using a conjugated antisense compound targeting HBV nucleic acid in a pharmaceutical composition. In certain embodiments, the subject has an HBV-related condition. In certain embodiments, the HBV-related condition includes, but is not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, hepatitis, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. In certain embodiments, the HBV-related condition may be accompanied by a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or a positive test for the presence of antibodies specific to hepatitis B virus antigen, and symptoms may include any or all of the following: influenza-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain in the liver region of the body, clay-colored or gray stools, itching throughout the body, and dark urine. In certain embodiments, the subject is at risk for an HBV-related condition. The subject includes a subject with one or more risk factors for developing an HBV-associated condition, including sexual exposure to an individual infected with hepatitis B virus, living with an individual with lifelong hepatitis B virus infection, exposure to human blood infected with hepatitis B virus, injecting illicit drugs, contact with a person with hemophilia, and visiting an area where hepatitis B is common. In certain embodiments, the subject has been identified as needing treatment for an HBV-associated condition.

[0368] Certain embodiments provide a method for reducing HBV DNA and / or HBV antigen levels in an animal infected with HBV, comprising administering to the animal a conjugated antisense compound that targets HBV nucleic acid. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, the amount of HBV antigen can be sufficiently reduced to result in seroconversion.

[0369] In certain embodiments, the present invention provides a conjugate antisense targeting HBV nucleic acid. A method for preparing a medicament using the compound is provided.

[0370] In certain embodiments, the present invention provides conjugated antisense compounds or pharmaceutically acceptable salts thereof that target HBV nucleic acids for use in therapy.

[0371] Certain embodiments provide conjugated antisense compounds targeting HBV nucleic acids for use in treating HBV-associated conditions, including, but not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, hepatitis, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia.

[0372] Certain embodiments provide conjugated antisense compounds targeting HBV nucleic acids for use in reducing HBV DNA and / or HBV antigen levels in animals infected with HBV, comprising administering to the animal a conjugated antisense compound targeting HBV nucleic acids. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, the amount of HBV antigen can be sufficiently reduced to result in seroconversion.

[0373] It will be understood that any of the compounds described herein can be used in the above-mentioned methods and uses.For example, in certain embodiments, the conjugate antisense compound targeting HBV nucleic acid in the above-mentioned methods and uses includes: a conjugate antisense compound targeting SEQ ID NO: 1, which comprises at least 8 consecutive nucleobase sequences of any of SEQ ID NOs: 3-11; ... ISIS 505358, ISIS 509934, ISIS 510100, ISIS 552023, ISIS 552024, ISIS 552032, ISIS 552859, ISIS 552925, or ISIS and a compound comprising an antisense oligonucleotide disclosed in International Publication No. WO2012 / 145697, the entire contents of which are incorporated herein by reference, and a conjugate group; a compound comprising an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOS: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379, as disclosed in International Publication No. WO2012 / 145697, and a conjugate group as described herein; These may include, but are not limited to, compounds comprising an antisense oligonucleotide having a nucleobase sequence of any one of SEQ ID NOS: 14 to 22 disclosed in WO2011 / 047312 and a conjugate group described herein; compounds comprising an antisense oligonucleotide having a nucleobase sequence of any one of SEQ ID NOS: 18 to 35 disclosed in International Publication No. WO2012 / 145674; or compounds comprising a double-stranded oligonucleotide in which one strand has a nucleobase sequence of any one of SEQ ID NOS: 30 to 125 disclosed in International Publication No. WO2013 / 159109.

[0374] 2. Transthyretin (TTR) TTR (also known as prealbumin, hyperthyroxinemia, prealbuminopathy, thyroxine; senile systemic amyloidosis, amyloid polyneuropathy, amyloidosis I, PALB; transthyretinopathy, HST2651; TBPA; prealbuminopathy euthyroid hyperthyroxinemia) is a serum / plasma and cerebrospinal fluid protein involved in the transport of thyroxine and retinol (Sakaki et al., Mol Biol Med. 1989, 6:161-8). Structurally, TTR is a homotetramer, and point mutations and misfolding of the protein lead to the deposition of amyloid fibrils, resulting in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy. It is associated with disorders such as familial amyloidosis (FAP), and familial amyloidosis cardiovascular disease (FAC).

[0375] TTR is synthesized primarily by the liver and cerebral choroid plexus, and to a lesser extent by the human retina (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300). While transthyretin synthesized in the liver is secreted into the blood, transthyretin derived from the choroid plexus is destined for the CSF. In the choroid plexus, transthyretin synthesis accounts for approximately 20% of total local protein synthesis and 25% of total CSF protein (Dickson et al., J Biol Chem, 1986, 261, 3475-3478).

[0376] Using genetic and immunohistochemical diagnostic tests, patients with TTR amyloidosis are being identified worldwide. Recent studies have shown that TTR amyloidosis is not a rare, endemic disease as previously thought, but may affect as many as 25% of the elderly population (Tanskanen et al., Ann Med. 2008;40(3):232-9).

[0377] At the biochemical level, TTR was identified as the major protein component in amyloid deposits in patients with FAP (Costa et al., Proc. Natl. Acad. Sci. USA 1978, 75:4499-4503), and later, a substitution of methionine with valine at position 30 of the protein was found to be the most common molecular defect causing the disease (Saraiva et al., J. Clin. Invest. 1984, 74:104-119). In FAP, widespread systemic extracellular deposition of TTR aggregates and amyloid fibrils occurs throughout connective tissues, particularly in the peripheral nervous system (Sousa and Saraiva, Prog. Neurobiol. 2003, 71:385-400). Following TTR deposition, axonal degeneration occurs, beginning in small-diameter unmyelinated and myelinated fibers and ultimately leading to neuronal loss at ganglionic sites.

[0378] Antisense compounds targeting TTR have been previously disclosed in U.S. Patent No. US2005 / 0244869, International Publication Nos. WO2010 / 017509, and WO2011 / 139917, each of which is incorporated herein by reference in its entirety. Antisense oligonucleobases targeting TTR, ISIS-TTR Rx is currently in Phase II / III clinical trials to test its effectiveness in treating subjects with familial amyloidotic polyneuropathy. However, there remains a need to provide patients with additional, more potent treatment options.

[0379] Certain conjugated antisense compounds targeting TTR nucleic acids In certain embodiments, the conjugate antisense compound targets a TTR nucleic acid having the sequence of GENBANK® Accession Number NM_000371.3, incorporated herein as SEQ ID NO: 2. In certain such embodiments, the conjugate antisense compound targeting SEQ ID NO: 2 is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

[0380] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 2 comprises at least an 8-contiguous nucleobase sequence of any one of SEQ ID NOs: 12-19. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 2 comprises at least an 8-contiguous nucleobase sequence of any one of SEQ ID NOs: 12-19.

[0381] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 12. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:12.

[0382] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 13. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:13.

[0383] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 14. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:14.

[0384] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 15. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:15.

[0385] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 16 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 78. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO: 16 comprises the nucleobase sequence of SEQ ID NO: 78.

[0386] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 17. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:17.

[0387] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 18. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:18.

[0388] In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises at least an 8-contiguous nucleobase sequence of SEQ ID NO: 19. In certain embodiments, a conjugate antisense compound targeted to SEQ ID NO:2 comprises the nucleobase sequence of SEQ ID NO:19. [Table 3]

[0389] In certain embodiments, the compound comprises or consists of ISIS 420915 and a conjugate group. ISIS 420915 has the formula: Tes mCes Tes a modified oligonucleotide having Tes Ges Gds Tds Tds Ads mCds Ads Tds Gds Ads Ads Aes Tes mCes mCes mCe, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0390] In certain embodiments, the compound comprises or consists of ISIS 304299 and a conjugate group. ISIS 304299 has the formula: mCes Tes Tes a modified oligonucleotide having Ges Ges Tds Tds Ads mCds Ads Tds Gds Ads Ads Ads Tes mCes mCes mCes Ae, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0391] In certain embodiments, the compound comprises or consists of ISIS 420921 and a conjugate group. ISIS 420921 is a modified oligonucleotide having the formula: Ges Ges Aes Aes Tes Ads mCds Tds mCds Tds Tds Gds Gds Tds Tds Aes mCes Aes Tes Ge, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0392] In certain embodiments, the compound comprises or consists of ISIS 420922 and a conjugate group. ISIS 420922 is a modified oligonucleotide having the formula: Tes Ges Ges Aes Aes Tds Ads mCds Tds mCds Tds Tds Gds Gds Tds Tes Aes mCes Aes Te, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0393] In certain embodiments, the compound comprises or consists of ISIS 420950 and a conjugate group. ISIS 420950 is a modified oligonucleotide having the formula: Tes Tes Tes Tes Aes Tds Tds Gds Tds mCds Tds mCds Tds Gds mCds mCes Tes Ges Ges Ae, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0394] In certain embodiments, the compound comprises or consists of ISIS 420955 and a conjugate group. ISIS 420955 is a modified oligonucleotide having the formula: Ges Aes Aes Tes Ges Tds Tds Tds Tds Ads Tds Tds Gds Tds mCds Tes mCes Tes Ges mCe, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0395] In certain embodiments, the compound comprises or consists of ISIS 420957 and a conjugate group. ISIS 420957 is a modified oligonucleotide having the formula: Aes Ges Ges Aes Aes Tds Gds Tds Tds Tds Tds Ads Tds Tds Gds Tes mCes Tes mCes Te, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0396] In certain embodiments, the compound comprises or consists of ISIS 420959 and a conjugate group. ISIS 420959 has the formula: Aes mCes Aes Ges Ges Ads Ads Tds Gds Tds Tds Tds Tds A modified oligonucleotide having Ads Tds Tes Ges Tes mCes Te, wherein: A is adenine, mC is 5'-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside; d is a 2'-deoxynucleoside; s is a phosphorothioate internucleoside linkage.

[0397] In certain embodiments, the compound having the following chemical structure comprises or consists of ISIS 420915 having a 5'-X, where X is a conjugate group as described herein. [ka]

[0398] In certain embodiments, the compound comprises or consists of ISIS 682877, which has the following chemical structure: [ka]

[0399] In certain embodiments, the compound comprises or consists of ISIS 682884, which has the following chemical structure: [ka]

[0400] In certain embodiments, the compound comprises or consists of SEQ ID NO: 12, 5'-GalNAc, and a chemical modification represented by the following chemical structure: [ka] In the formula, R 1is -OCH2CH2OCH3(MOE), and R 2 is H or R 1 and R 2 together to form a bridge, where R 1 is -O- and R 2 is —CH—, —CH(CH)—, or —CHCH—, and the resulting bridge is selected from —O—CH—, —O—CH(CH)—, and —O—CHCH—; 1 and R 2 are directly connected, On the same ring, R 3 and R 4 For each pair, independently for each ring, R 3 is selected from H and —OCHCHOCH, and R 4 is H or R 3 and R 4 together to form a bridge, where R 3 is -O- and R 4 is —CH—, —CH(CH)—, or —CHCH—, and the resulting bridge is selected from —O—CH—, —O—CH(CH)—, and —O—CHCH—; 3 and R 4 are directly connected, R 5 is selected from H and -CH3; Z is S - and O - is selected from.

[0401] In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2011 / 139917 or U.S. Patent No. US8,101,743, the entire contents of which are incorporated herein by reference, and a conjugate group. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2011 / 139917, the entire contents of which are incorporated herein by reference, and a conjugate group. The compounds include antisense oligonucleotides having the nucleobase sequence of any of SEQ ID NOS: 8-160, 170-177, and a conjugate group described herein. In certain embodiments, the compounds include antisense oligonucleotides having the nucleobase sequence of any of SEQ ID NOS: 12-89, as disclosed in U.S. Pat. No. 8,101,743, and a conjugate group described herein. In certain embodiments, the compounds include antisense oligonucleotides having a nucleobase sequence complementary to a preferred target segment of any of SEQ ID NOS: 90-133, as disclosed in U.S. Pat. No. 8,101,743, and a conjugate group described herein. All nucleobase sequences of the referenced SEQ ID NOS are incorporated herein by reference.

[0402] TTR treatment index In certain embodiments, the present invention provides methods for modulating the expression of TTR in a subject using conjugated antisense compounds targeted to a TTR nucleic acid. In certain embodiments, the expression of TTR is reduced.

[0403] In certain embodiments, the present invention provides a method for treating a subject using a conjugated antisense compound targeting a TTR nucleic acid in a pharmaceutical composition. In certain embodiments, the subject has a transthyretin-related disease, disorder, or condition, or symptoms thereof. In certain embodiments, the transthyretin-related disease, disorder, or condition is transthyretin amyloidosis. "Transthyretin-related amyloidosis" or "transthyretin amyloidosis" or "transthyretin amyloid disease," as used herein, refers to any pathology or disease associated with the dysfunction or dysregulation of transthyretin, which results in the formation of transthyretin-containing amyloid fibrils. Transthyretin amyloidosis includes, but is not limited to, hereditary TTR amyloidosis, leptomeningeal amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy, familial ocular leptomeningeal amyloidosis, senile cardiac amyloidosis, or senile systemic amyloidosis.

[0404] In certain embodiments, the present invention provides methods for preparing medicaments with conjugated antisense compounds targeted to TTR nucleic acids.

[0405] In certain embodiments, the present invention provides conjugated antisense compounds or pharmaceutically acceptable salts thereof targeted to a TTR nucleic acid for use in therapy.

[0406] Certain embodiments provide conjugated antisense compounds targeted to a TTR nucleic acid for use in treating a transthyretin-related disease, disorder, or condition, or a symptom thereof. In certain embodiments, the transthyretin-related disease, disorder, or condition is transthyretin amyloidosis.

[0407] It will be understood that any of the compounds described herein can be used in the aforementioned methods and uses.For example, in certain embodiments, the conjugate antisense compounds targeting TTR nucleic acids in the aforementioned methods and uses include conjugate antisense compounds targeting SEQ ID NO: 2, which comprises at least 8 consecutive nucleobase sequences of any one of SEQ ID NOs: 12-19; ​​... ISIS 420915, ISIS 304299, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, or ISIS 420959 and a conjugate group; an antisense oligonucleotide disclosed in International Publication No. WO2011 / 139917 or U.S. Pat. No. US8,101,743, the entireties of which are incorporated herein by reference, and a conjugate group; The present invention may include, but is not limited to, compounds comprising: an antisense oligonucleotide having any of the nucleobase sequences of SEQ ID NOS: 8-160, 170-177 disclosed in International Publication No. WO2011 / 139917, and a conjugate group as described herein; an antisense oligonucleotide having any of the nucleobase sequences of SEQ ID NOS: 12-89 disclosed in US Patent No. US8,101,743, and a conjugate group as described herein; or an antisense oligonucleotide having any of the nucleobase sequences complementary to the preferred target segments of SEQ ID NOS: 90-133 disclosed in US Patent No. US8,101,743, and a conjugate group as described herein. All of the nucleobase sequences of the above-referenced SEQ ID NOS are incorporated herein by reference. E. Certain Pharmaceutical Compositions

[0408] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising one or more antisense compounds. In certain embodiments, such pharmaceutical compositions comprise a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises a sterile saline solution and one or more antisense compounds. In certain embodiments, such pharmaceutical compositions consist of a sterile saline solution and one or more antisense compounds. In certain embodiments, the sterile saline is pharmaceutical-grade saline. In certain embodiments, the pharmaceutical composition comprises one or more antisense compounds and sterile water. In certain embodiments, the pharmaceutical composition consists of one or more antisense compounds and sterile water. In certain embodiments, the sterile saline is pharmaceutical-grade water. In certain embodiments, the pharmaceutical composition comprises one or more antisense compounds and phosphate-buffered saline (PBS). In certain embodiments, the pharmaceutical composition consists of one or more antisense compounds and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical-grade PBS.

[0409] In certain embodiments, antisense compounds can be mixed with pharmaceutically acceptable active and / or inactive substances to prepare pharmaceutical compositions or formulations. The methods for formulating compositions and pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0410] Pharmaceutical compositions comprising antisense compounds include any pharmaceutically acceptable salts, esters, or salts of such esters.In certain embodiments, pharmaceutical compositions comprising antisense compounds include one or more oligonucleotides that can provide (directly or indirectly) biologically active metabolites or residues thereof when administered to animals, including humans.Therefore, for example, the present disclosure is directed to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0411] Prodrugs may involve the incorporation of additional nucleosides at one or both ends of the oligonucleotide that are cleaved by endogenous nucleases in the body to form the active antisense oligonucleotide.

[0412] Lipid moieties have been used in nucleic acid therapy in a variety of ways. In certain such methods, nucleic acids are introduced into preformed liposomes or lipoplexes made from a mixture of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, the lipid moiety is selected to increase drug distribution to specific cells or tissues. In certain embodiments, the lipid moiety increases drug distribution to adipose tissue. In certain embodiments, the lipid moiety is selected to increase distribution of the agent to muscle tissue.

[0413] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, the excipient is selected from water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0414] In certain embodiments, the pharmaceutical compositions provided herein include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, including pharmaceutical compositions containing hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0415] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more tissue-specific delivery molecules designed to deliver one or more agents of the present disclosure to a particular tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

[0416] In certain embodiments, the pharmaceutical compositions provided herein include a cosolvent system. Certain such cosolvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such cosolvent systems are used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which is a solution of 3 wt.% benzyl alcohol, 8 wt.% of the nonpolar surfactant Polysorbate 80™, and 65 wt.% polyethylene glycol 300 in absolute ethanol. The proportions of such cosolvent systems can be varied significantly without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components can be varied; for example, other surfactants can be substituted for Polysorbate 80™, the fraction size of polyethylene glycol can be changed, other biocompatible polymers can replace polyethylene glycol, e.g., polyvinylpyrrolidone, and other sugars or polysaccharides can replace dextrose.

[0417] In certain embodiments, the pharmaceutical compositions provided herein are prepared for oral administration. In certain embodiments, the pharmaceutical compositions are prepared for buccal administration.

[0418] In certain embodiments, pharmaceutical compositions are prepared for administration by injection (e.g., intravenously, subcutaneously, intramuscularly, etc.). In certain such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients (e.g., ingredients that aid in solubility or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. Certain pharmaceutical compositions for injection are provided in unit dosage form, for example, in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending agents, stabilizing agents, and / or dispersing agents. Certain solvents suitable for use in injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions can be prepared in the presence of carboxymethyl The suspension may contain substances which increase the viscosity of the suspension, such as sodium cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents which increase the solubility of the drugs to allow for the preparation of highly concentrated solutions.

[0419] In certain embodiments, pharmaceutical compositions are prepared for transmucosal administration. In certain such embodiments, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0420] In certain embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of oligonucleotide.In certain embodiments, a therapeutically effective amount is sufficient to prevent, alleviate, or improve the symptoms of disease, or to prolong the life of the subject being treated.The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art.

[0421] In certain embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to a biologically, pharmaceutically, or therapeutically more active form of the oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain cases, the prodrug is more bioavailable (e.g., by oral administration) than the corresponding active form. In certain cases, the prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, the prodrug is less water-soluble than the corresponding active form. In certain cases, such prodrugs have excellent permeability across cell membranes where aqueous solubility impairs mobility. In certain embodiments, the prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In certain cases, the carboxylic acid-containing compound is the corresponding active form. In certain embodiments, the prodrug comprises a short peptide (polyamino acid) bound to an acid group. In certain such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0422] In certain embodiments, the present disclosure provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is present in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.

[0423] In certain embodiments, the present disclosure provides a method for administering pharmaceutical compositions comprising the oligonucleotides of the present disclosure to animals.Suitable administration routes include, but are not limited to, oral administration, rectal administration, transmucosal administration, enteral administration, intestinal administration, topical administration, suppository administration, inhalation administration, intrathecal administration, intraventricular administration, intraperitoneal administration, intranasal administration, intraocular administration, intratumoral administration, and parenteral (for example, intravenous, intramuscular, intramedullary and subcutaneous) administration.In certain embodiments, pharmaceutical intrathecal administration is administered to achieve local exposure rather than systemic exposure.For example, pharmaceutical compositions can be directly injected into desired affected area (for example, into the liver).

[0424] Non-limiting disclosure and incorporation by reference While certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, the following examples serve only to illustrate, and are not intended to limit, the compounds described herein. Each of the references, GenBank accession numbers, etc. listed in this application is incorporated herein by reference in its entirety. It can be enjoyed.

[0425] Certain compounds, compositions, and methods herein are referred to as "comprising" or "comprising" exactly a certain number of specified elements or features. Such a description is used to indicate that the compound, composition, or method may contain additional elements, but that the number of specified elements or features is the specified number. For example, a "conjugate comprising exactly one GalNAc" is a conjugate that contains only one GalNAc, but may also contain other elements in addition to that one GalNAc.

[0426] Although the sequence listing accompanying this application identifies each sequence as either "RNA" or "DNA" as necessary, in practice, these sequences may be modified with any combination of chemical modifications. Those skilled in the art will readily recognize that the designation "RNA" or "DNA" to describe modified oligonucleotides is arbitrary in certain instances. For example, an oligonucleotide containing a nucleoside containing a 2'-OH sugar moiety and a thymine base may be described as a DNA with a modified sugar (2'-OH in the case of the natural 2'-H in DNA) or an RNA with a modified base (thymine (methylated uracil) in the case of the natural uracil in RNA). Thus, the nucleic acid sequences provided herein, including but not limited to those in the Sequence Listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including, but not limited to, such nucleic acids with modified nucleobases. By way of further example and not limitation, an oligonucleotide having the nucleobase sequence "ATCGATCG" can be used with an RNA base, whether modified or unmodified, for example, an oligonucleotide having the sequence "AUCGAUCG," as well as oligonucleotides having some DNA bases and some RNA bases, such as "AUCGATCG," and oligonucleotides having the sequence "ATCGATCG." me The present invention encompasses any oligonucleotide having such a nucleobase sequence, including, but not limited to, such compounds including oligonucleotides with other modified bases such as "AACGAUCG", wherein: me C denotes a cytosine base containing a methyl group at the 5-position. [Example]

[0427] The following examples illustrate certain specific embodiments of the present disclosure, but do not limit them.Furthermore, when specific embodiments are provided, the inventors intend that these specific embodiments are generally applicable.For example, the disclosure of an oligonucleotide with a specific motif provides rational support for other oligonucleotides with this motif or similar motifs.Similarly, for example, when a specific high-affinity modification appears at a specific position, other high-affinity modifications at the same position are considered suitable unless otherwise indicated.

[0428] Example 1: General Method for the Preparation of Phosphoramidites (Compounds 1, 1a, and 2) [ka] Compounds 1, 1a, and 2 were prepared according to procedures known in the art and described herein (Seth et al., Bioorg. Med. Chem., 2011, 21). (4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554), as well as published PCT international applications (International Publication Nos. WO2011 / 115818, WO2010 / 077578, WO2010 / 036698, WO2009 / 143369, WO2009 / 006478, and WO2007 / 090071), and see also U.S. Pat. No. 7,569,686).

[0429] Example 2: Preparation of Compound 7 [ka] Compound 3 (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to a published procedure (Weber et al., J. Med. Chem., 1991, 34, 2692).

[0430] Example 3: Preparation of Compound 11 [ka] Compounds 8 and 9 are commercially available.

[0431] Example 4: Preparation of Compound 18 [ka] Compound 11 was prepared according to the procedure illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using a similar procedure reported by Rensen et al. (J. Med. Chem., 2004, 47, 5798-5808).

[0432] Example 5: Preparation of Compound 23 [ka] Compounds 19 and 21 are commercially available.

[0433] Example 6: Preparation of Compound 24 [ka] Compounds 18 and 23 were prepared according to the procedures illustrated in Examples 4 and 5.

[0434] Example 7: Preparation of Compound 25 [ka] Compound 24 was prepared according to the procedure illustrated in Example 6.

[0435] Example 8: Preparation of Compound 26 [ka] Compound 24 is prepared according to the procedure illustrated in Example 6.

[0436] Example 9: General preparation of conjugated ASO containing GalNAc3-1 at the 3' end (compound 29) [ka] [ka] The protected GalNAc3-1 has the following structure: [ka]

[0437] The GalNAc3 cluster portion of the conjugated group GalNAc3-1 (GalNAc3-1 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. a has the following formula: [ka]

[0438] Solid support-bound protected GalNAc3-1 (compound 25) was prepared according to the procedure illustrated in Example 7. Oligomeric compound 29 containing GalNAc3-1 at the 3' end was prepared using standard procedures for automated DNA / RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks (compounds 1 and 1a) were prepared according to the procedure illustrated in Example 1. Since other phosphoramidite building blocks can be used to prepare oligomeric compounds with defined sequences and compositions, the illustrated phosphoramidites are representative and not intended to be limiting. The order and amount of phosphoramidites added to the solid support can be adjusted to prepare the gapped oligomeric compounds described herein. Such gapped oligomeric compounds can have defined compositions and base sequences dictated by any given target.

[0439] Example 10: General preparation of conjugated ASO containing GalNAc3-1 at the 5' end (compound 34) [ka] [ka] Unylinker™ 30 is commercially available. Oligomeric compound 34 containing the 1 cluster was prepared using standard procedures for automated DNA / RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks (compounds 1 and 1a) were prepared according to the procedure illustrated in Example 1. Because other phosphoramidite building blocks can be used to prepare oligomeric compounds with defined sequences and compositions, the illustrated phosphoramidites are representative and not intended to be limiting. The order and amount of phosphoramidites added to the solid support can be adjusted to prepare the gapped oligomeric compounds described herein. Such gapped oligomeric compounds can have defined compositions and base sequences dictated by any given target.

[0440] Example 11: Preparation of Compound 39 [ka] [ka] Compounds 4, 13, and 23 were prepared according to the procedures illustrated in Examples 2, 4, and 5. Compound 35 was prepared using a similar procedure published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

[0441] Example 12: Preparation of Compound 40 [ka] Compound 38 is prepared according to the procedure illustrated in Example 11.

[0442] Example 13: Preparation of Compound 44 [ka] [ka] Compounds 23 and 36 are prepared according to the procedures illustrated in Examples 5 and 11. Compound 41 is prepared using a similar procedure published in International Publication No. WO2009082607.

[0443] Example 14: Preparation of Compound 45 [ka] Compound 43 is prepared according to the procedure illustrated in Example 13.

[0444] Example 15: Preparation of Compound 47 [ka] Compound 46 is commercially available.

[0445] Example 16: Preparation of Compound 53 [ka] Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared according to the procedures illustrated in Examples 4 and 15.

[0446] Example 17: Preparation of Compound 54 [ka] Compound 53 is prepared according to the procedure illustrated in Example 16.

[0447] Example 18: Preparation of Compound 55 [ka] Compound 53 is prepared according to the procedure illustrated in Example 16.

[0448] Example 19: General method for the preparation of conjugated ASOs containing GalNAc3-1 at the 3' position by solid phase techniques (preparation of ISIS 647535, 647536, and 651900) Unless otherwise specified, all reagents and solutions used in the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid supports are available from, for example, T, A, G, and m Used for the incorporation of nucleoside residues, including C residues. A 0.1 M solution of phosphoramidites in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleosides and 2′-MOE.

[0449] ASO synthesis was performed on an ABI 394 synthesizer (1-2 μmol scale) or a GE Healthcare Bioscience AeKTA Oligo Pilot Synthesizer (40-200 μmol scale) using the phosphoramidite coupling method on a VIMAD solid support (110 μmol / g, Guzaev et al., 2003) packed in a column with GalNAc3-1. For this coupling step, a four-fold excess of phosphoramidite was delivered relative to the loading of the solid support, and phosphoramidite condensation was carried out for 10 min. All other steps followed the standard protocol provided by the manufacturer. The dimethoxytrityl (DMT) group was removed from the 5'-hydroxyl group of the nucleotide using a solution of 6% dichloroacetic acid in toluene. 4,5-dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as the activating agent during the coupling step. Phosphorothioate linkages were introduced by sulfurization with a 0.1 M solution of xanthan hydride in 1:1 pyridine / CH3CN with a contact time of 3 min. A solution of 20% tert-butyl hydroperoxide in H3CN was used as the oxidizing agent, with a contact time of 12 minutes, to provide phosphodiester internucleoside linkages.

[0450] After the desired sequence was assembled, the cyanoethylphosphate protecting groups were deprotected using a 1:1 (v / v) mixture of triethylamine and acetonitrile with a contact time of 45 min. The solid support-bound ASO was suspended in aqueous ammonia (28–30 wt%) and heated at 55 °C for 6 h.

[0451] The unbound ASO was then filtered and the ammonia was boiled off. The residue was purified by high-pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0–40% B after 60 min, flow rate 14 mL / min, λ = 260 nm). The residue was desalted by HPLC on a reverse-phase column to give the desired ASO in 15–30% isolated yield based on initial loading on the solid support. The ASO was characterized by ion-pair HPLC coupled to MS analysis using an Agilent 1100 MSD system.

[0452] Conjugate-free antisense oligonucleotides were synthesized using standard oligonucleotide synthesis procedures well known in the art.

[0453] Using these methods, three distinct antisense compounds targeting ApoC III were prepared. As summarized in Table 4 below, each of the three antisense compounds targeting ApoC III has the same nucleobase sequence: ISIS 304801 is a 5-10-5 MOE gapmer with all phosphorothioate linkages; ISIS 647535 is identical to ISIS 304801 except that GalNAc3-1 is conjugated at its 3' end; and ISIS 647536 is identical to ISIS 647535 except that certain internucleoside linkages in the compound are phosphodiester linkages. Two distinct antisense compounds targeting SRB-1 were synthesized, as further summarized in Table 4: ISIS 440762 is a 2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages; and ISIS 651900 is identical to ISIS except that it contains GalNAc3-1 at its 3' end. It was identical to 440762. [Table 4]

[0454] The subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, and "k" indicates a 6'-(S)-CH3 bicyclic nucleoside. "S" indicates a phosphorothioate internucleoside linkage (PS), "o" indicates a phosphodiester internucleoside linkage (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. "GalNAc3-1" indicates a conjugate group having the structure shown above in Example 9. When GalNAc3-1 contains a cleavable adenosine linking the ASO to the remainder of the conjugate, "GalNAc3-1" a Note that this nomenclature is used in the tables above to refer to all nucleobase sequences that contain an adenosine that is part of a conjugate. do " can also be omitted to list a sequence ending in "GalNAc3-1." This convention of using the subscript "a" to indicate the portion of the conjugate group lacking a cleavable nucleoside or cleavable moiety will be used throughout these embodiments. This portion of the conjugate group lacking a cleavable moiety will be referred to herein as a "cluster" or "conjugate cluster" or "GalNAc3 cluster." In certain instances, it is convenient to describe the conjugate group by providing the cluster and its cleavable moiety separately.

[0455] Example 20: Dose-dependent antisense inhibition of human ApoC III in huApoC III transgenic mice ISIS 304801 and ISIS 647535, each of which targets human ApoC III and is described above, were tested separately and evaluated for their ability to inhibit human ApoC III in human ApoC III transgenic mice in a dose-dependent study.

[0456] process Human ApoCIII transgenic mice were maintained on a 12-hour light / dark cycle and fed Teklad laboratory diet ad libitum. Animals were allowed to acclimate for at least 7 days in the research facility before the start of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. For injection, ASOs were dissolved in 0.9% PBS.

[0457] Human ApoC III transgenic mice were intraperitoneally injected weekly for 2 weeks with ISIS 304801 or 647535 at 0.08, 0.25, 0.75, 2.25, or 6.75 μmol / kg, or with PBS as a control. Each treatment group consisted of four animals. 48 hours after the final dose, blood was collected from each mouse, and the mice were sacrificed and tissues were collected.

[0458] ApoC III mRNA analysis Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) were performed according to standard protocols. ApoC III mRNA levels were determined in the livers of mice using Ribogreen. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen) before normalizing to PBS-treated controls. The results below are presented as the mean percent of ApoC III mRNA levels for each treatment group normalized to PBS-treated controls and are denoted as "%PBS." The median effective concentration (ED) of each ASO was 1.25. 50 ) are also shown in Table 5 below.

[0459] As illustrated, both antisense compounds reduced ApoC III RNA compared to the PBS control. Furthermore, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801). [Table 5]

[0460] ApoC III protein analysis (turbidimetric assay) Plasma ApoC III protein analysis was determined using the procedure reported by Graham et al (Circulation Research), published online ahead of print on March 29, 2013.

[0461] Approximately 100 μL of plasma isolated from mice was analyzed undiluted using an Olympus clinical analyzer and a commercially available turbidimetric ApoC III assay (Kamiya, catalog number KAI-006, Kamiya Biomedical, Seattle, WA). The assay protocol was performed as described by the supplier.

[0462] As shown below in Table 6, both antisense compounds reduced ApoC III protein compared to the PBS control. Furthermore, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801). [Table 6]

[0463] Plasma triglycerides and cholesterol were measured by the method of Bligh and Dyer (Bligh, E G and Dyer, W J Can. J. Biochem. Physiol. 37:911-917, 1959) (Bligh, E and Dyer, W, Can. J. Biochem. Physiol. ,37,911-917,1959) and measured using a Beckmann Coulter clinical analyzer and commercially available reagents.

[0464] Triglyceride levels were measured relative to PBS-injected mice and are expressed as "% PBS." The results are presented in Table 7. As illustrated, both antisense compounds reduced triglyceride levels. Furthermore, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801). [Table 7]

[0465] Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and the amounts of different cholesterol fractions (HDL and LDL). The results are presented in Tables 8 and 9. As illustrated, both antisense compounds reduced total cholesterol levels, lowered LDL, and increased HDL. Furthermore, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801). The increase in HDL levels and the decrease in LDL levels are cardiovascular beneficial effects of antisense inhibition of ApoC III. [Table 8] [Table 9]

[0466] Pharmacokinetic analysis (PK) The PK of the ASO was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were analyzed by MSD1 utilizing IP-HPLC-MS. Tissue levels (μg / g) of full-length ISIS 304801 and 647535 were measured, and the results are provided in Table 10. As illustrated, the liver concentrations of the total full-length antisense compound were similar for these two antisense compounds. Thus, while the GalNAc3-1 conjugated antisense compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not present at significantly higher concentrations in the liver. In fact, the calculated EC 50 (provided in Table 10) confirm that the observed increase in potency of the conjugates is not entirely due to increased accumulation. This result suggests that the conjugates improved potency by a mechanism other than liver accumulation alone, perhaps by improving productive cellular uptake of the antisense compound.

[0467] The results also show that the concentration of GalNAc3-1-conjugated antisense compounds in the kidney is lower than that of antisense compounds lacking the GalNAc conjugate. This has several beneficial therapeutic implications. In therapeutic indications where renal activity is not required, exposure to the kidney carries the risk of nephrotoxicity without corresponding benefit. Furthermore, high concentrations in the kidney typically result in compound loss in the urine, resulting in more rapid clearance. Therefore, for non-renal targets, renal accumulation is undesirable. These data suggest that GalNAc3-1 conjugation reduces renal accumulation. [Table 10]

[0468] Metabolites of ISIS 647535 were also identified, and their masses were confirmed by high-resolution mass spectrometry. The cleavage sites and structures of the observed metabolites are shown below. The relative percentage of full-length ASO was calculated using standard procedures, and the results are presented in Table 10a. The major metabolite of ISIS 647535 was the full-length ASO (i.e., ISIS 304801) lacking all conjugates, resulting from cleavage at cleavage site A shown below. Additionally, additional metabolites derived from other cleavage sites were also observed. These results suggest that the introduction of other cleavable bonds, such as esters, peptides, disulfides, phosphoramidates, or acylhydrazones between the GalNAc3-1 sugar and the ASO, that can be cleaved by intracellular enzymes, in the reducing environment of the cytosol, or compatible with the acidic pH of endosomes and lysosomes, may also be useful. [Table 10a] [ka] [ka] [ka]

[0469] Example 21: Antisense inhibition of human ApoC III in human ApoC III transgenic mice in a single-dose study ISIS 304801, 647535, and 647536, each of which targets human ApoC III and are listed in Table 4, were further evaluated for their ability to inhibit human ApoC III in human ApoC III transgenic mice in a single-dose study.

[0470] process Human ApoCIII transgenic mice were maintained on a 12-h light-dark cycle and Tek The animals were fed lad experimental diet ad libitum. Animals were allowed to acclimate for at least 7 days in the research facility before the start of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. For injection, ASOs were dissolved in 0.9% PBS.

[0471] Human ApoC III transgenic mice were intraperitoneally injected once with ISIS 304801, 647535, or 647536 (as described above), or a PBS-treated control, at the doses indicated below. Treatment groups consisted of three animals, and the control group consisted of four animals. Blood was collected from each mouse before treatment and after the final dose, and plasma samples were analyzed. Mice were sacrificed 72 hours after the final dose.

[0472] Samples were collected and analyzed to determine ApoC III mRNA and protein levels in the liver, plasma triglycerides, and cholesterol, including HDL and LDL fractions, and were assessed as described above (Example 20). Data from these analyses are presented in Tables 11-15 below. Serum liver transaminase levels, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were measured relative to saline-injected mice using standard protocols. ALT and AST levels indicated that the antisense compound was well tolerated at all doses.

[0473] These results demonstrate improved potency for antisense compounds containing the 3'-terminal GalNAc3-1 conjugate (ISIS 647535 and 647536) compared with an antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801). Furthermore, ISIS 647536, which contains a GalNAc3-1 conjugate and several phosphodiester linkages, is as potent as ISIS 647535, which contains the same conjugate, but all internucleoside linkages within the ASO are phosphorothioate. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0474] These results confirm that GalNAc3-1 conjugates improve the potency of antisense compounds. These results also demonstrate comparable potency of GalNAc3-1 conjugated antisense compounds, which have mixed linkages (ISIS 647536, which has six phosphodiester linkages) and a fully phosphorothioate version of the same antisense compound (ISIS 647535).

[0475] Phosphorothioate linkages provide several properties to antisense compounds. For example, they resist nuclease digestion, bind to proteins, and result in accumulation of the compound in the liver rather than the kidney / urine. These are particularly desirable properties when treating liver indications. However, phosphorothioate linkages have also been associated with inflammatory responses. Therefore, reducing the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, but it also reduces the concentration of the compound in the liver, increases the concentration in the kidney and urine, reduces stability in the presence of nucleases, and reduces overall potency. These results indicate that GalNAc3-1-conjugated antisense compounds in which certain phosphorothioate linkages are replaced with phosphodiester linkages are as potent against targets in the liver as their counterparts with full phosphorothioate linkages. Such compounds are expected to be less pro-inflammatory (see Example 24, which describes an experiment showing that reducing PS results in reduced inflammatory effects).

[0476] Example 22: Effects of GalNAc3-1 conjugate-modified ASO targeting SRB-1 in vivo ISIS 440762 and 651900, which each target SRB-1 and are listed in Table 4, were evaluated for their ability to inhibit SRB-1 in Balb / c mice in a dose-dependent study.

[0477] process Six-week-old male Balb / c mice (Jackson Laboratory, Bar Harbor, ME) were subcutaneously injected once with ISIS 440762, 651900, or a PBS-treated control at the doses indicated below. Each treatment group consisted of four animals. Forty-eight hours after the final dose, mice were sacrificed, and SRB-1 mRNA levels in the liver were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen) before normalizing to the PBS-treated control. The results below are presented as the average percent SRB-1 mRNA levels for each treatment group normalized to the PBS-treated control and are denoted "%PBS."

[0478] As illustrated in Table 16, both antisense compounds reduced SRB-1 mRNA levels. Furthermore, the antisense compound containing the GalNAc3-1 conjugate (ISIS 651900) was much more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 440762). These results demonstrate that the potency benefit of the GalNAc3-1 conjugate can be observed using antisense oligonucleotides complementary to different targets and bearing different chemically modified nucleosides; in this case, the modified nucleosides contain a constrained ethyl sugar moiety (bicyclic sugar moiety). [Table 16]

[0479] Example 23: Human peripheral blood mononuclear cell (hPBMC) assay protocol The hPBMC assay was performed using the BD Vacutainer CPT tube method. Whole blood samples were obtained from volunteer donors who provided informed consent at the US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) and collected into four to fifteen BD Vacutainer CPT 8 mL tubes (VWR catalog number BD362753). The PBMC assay data sheet was used to record the approximate starting total whole blood volume in each donor's CPT tube.

[0480] The blood sample was mixed again immediately before centrifugation by gently inverting the tube 8-10 times. The CPT tubes were centrifuged at 1500-1800 RCF (2700 RPM, Beckman Allegra 6R) in a horizontal (swing-out) rotor at room temperature (18-25°C) for 30 minutes with the brake off. Cells were collected from the buffy coat interface (between the Ficoll and polymer gel layers) and transferred to a sterile 50 mL conical tube, pooling up to five CPT tubes / 50 mL conical tubes / donor. The cells were then resuspended in PBS (Ca ++ , Mg ++The tubes were filled to a maximum of 50 mL and mixed by inverting several times. The samples were then centrifuged at 330 x g (1215 RPM, Beckman Allegra 6R) for 15 minutes at room temperature, and as much of the supernatant as possible was aspirated without disturbing the pellet. The tubes were gently spun to remove the cell pellet, and the cells were resuspended in RPMI + 10% FBS + pen / strep (approximately 1 mL per 10 mL starting whole blood volume). 60 μL of the sample was pipetted into a sample vial (Beckman Coulter) containing 600 μL of VersaLyse reagent (Beckman Coulter, catalog number A09777) and gently vortexed for 10–15 seconds. The sample was allowed to incubate at room temperature for 10 minutes and mixed again before centrifugation. The cell suspension was counted in a Vicell XR cell viability analyzer (Beckman Coulter) using the PBMC cell type (a dilution factor of 1:11 was preserved for other parameters). Viable cells / mL and viability were recorded. The cell suspension was diluted to 1 x 10 in RPMI + 10% FBS + pen / strep. 7 Diluted to live PBMC / mL.

[0481] Cells were plated at 5 × 10 in 50 μL / well in 96-well tissue culture plates (Falcon Microtest). 5 Plated in RPMI + 10% FBS + pen / strep. 50 μL / well of 2x concentrated oligo / control diluted in RPMI + 10% FBS + pen / strep was added according to the experimental template (total 100 μL / well). Plates were placed on a shaker and allowed to mix for approximately 1 minute. After 24 hours of incubation at 37°C, 5% CO2, plates were centrifuged at 400 x g for 10 minutes, after which the supernatant was removed for MSD cytokine assays (i.e., human IL-6, IL-10, IL-8, and MCP-1).

[0482] Example 24: Evaluation of the pro-inflammatory effects of GalNAc3-1-conjugated ASOs in an hPBMC assay The antisense oligonucleotides (ASOs) listed in Table 17 were evaluated for pro-inflammatory effects in the hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for IL-6 release in this assay. hPBMCs were isolated from fresh volunteer donors and treated with ASOs at concentrations of 0, 0.0128, 0.064, 0.32, 1.6, 8, 40, and 200 μM.

[0483] IL-6 levels were used as the primary readout. EC 50 and E max The results were compared between the two donors. max / EC 50 It is expressed as the average ratio of max / EC 50 A lower ratio indicates a relative decrease in the pro-inflammatory response, and a higher ratio indicates a relative increase in the pro-inflammatory response.

[0484] Regarding the test compounds, the least pro-inflammatory compounds were PS / PO-binding ASOs (ISI The GalNAc3-1 conjugated ASO (ISIS 647535) was slightly less proinflammatory than its unconjugated counterpart (ISIS 304801). These results indicate that the incorporation of several PO linkages reduces the proinflammatory response, and that the addition of GalNAc3-1 conjugates does not increase the proinflammatory potential of the compound, but may reduce the proinflammatory response. Therefore, it would be expected that antisense compounds containing both mixed PS / PO linkages and GalNAc3-1 conjugates would result in a lower proinflammatory response compared to fully PS-linked antisense compounds (with or without GalNAc3-1 conjugates). These results support the conclusion that GalNAc 3- 1 conjugated antisense compounds, especially those with reduced PS content, are less pro-inflammatory.

[0485] Taken together, these results suggest that GalNAc3-1 conjugate compounds, especially those with reduced PS content, can be administered at higher doses than their fully PS-containing counterparts, which lack the GalNAc3-1 conjugate. Because the half-lives of these compounds are not expected to differ substantially, such higher doses would result in less frequent dosing. In practice, such dosing would be even less frequent because the GalNAc3-1 conjugate compounds are more potent (see Examples 20-22), requiring re-dosing once compound concentrations fall below a desired level, which is based on potency. [Table 17]

[0486] The subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, "k" indicates a 6'-(S)-CH3 bicyclic nucleoside (e.g., cEt), "s" indicates a phosphorothioate internucleoside linkage (PS), "o" indicates a phosphodiester internucleoside linkage (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. "A do’ -GalNAc3-1 a " indicates a conjugate having the structure GalNAc3-1 shown in Example 9 attached to the 3' end of an antisense oligonucleotide as shown. [Table 18]

[0487] Example 25: Effects of GalNAc3-1 conjugated modified ASO targeting human ApoC III in vitro The aforementioned ISIS 304801 and 647535 were tested in vitro. Primary hepatocytes from transgenic mice at a density of 25,000 cells / well were treated with modified oligonucleotides at concentrations of 0.03, 0.08, 0.24, 0.74, 2.22, 6.67, and 20 μM. After a treatment period of approximately 16 hours, RNA was isolated from the cells, and mRNA levels were measured by quantitative real-time PCR. hApoC III mRNA levels were adjusted according to total RNA content measured by RIBOGREEN.

[0488] IC using standard methods 50 was calculated and the results are presented in Table 19. As illustrated, a similar intensity was observed in cells treated with ISIS 647535 compared to the control ISIS 304801. [Table 19]

[0489] In this experiment, the large potency benefit of the GalNAc3-1 conjugate observed in vivo was not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro showed increased potency for oligonucleotides containing various GalNAc conjugates compared to oligonucleotides lacking the GalNAc conjugate (see Examples 60, 82, and 92).

[0490] Example 26: Effect of PO / PS binding on ApoC III ASO activity Human ApoC III transgenic mice were intraperitoneally injected with ISIS 304801 or ISIS 616468 (both as described above) or a PBS-treated control at 25 mg / kg once a week for 2 weeks. Treatment groups consisted of three animals, and the control group consisted of four animals. Blood was collected from each mouse before treatment and after the final dose, and plasma samples were analyzed. Mice were sacrificed 72 hours after the final dose.

[0491] Samples were collected and analyzed to determine ApoC III protein levels in the liver as described above (Example 20). Data from these analyses are presented below in Table 20.

[0492] These results show a decrease in potency of the antisense compound with PO / PS in the wings (ISIS 616468) compared to the intact PS (ISIS 304801). [Table 20]

[0493] Example 27: Compound 56 [ka] Compound 56 is commercially available from Glen Research or can be prepared according to the published procedure reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

[0494] Example 28: Preparation of Compound 60 [ka] Compound 4 was prepared according to the procedure illustrated in Example 2. Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.

[0495] Compound 57 is representative and not intended to be limiting, as phosphoramidites of defined composition can be prepared using other monoprotected substituted or unsubstituted alkyl diols, including but not limited to those presented herein. .

[0496] Example 29: Preparation of Compound 63 [ka] Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. J. Org. Chem., 2013, 3, 566-577, and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

[0497] Alternatively, compound 63 is prepared using procedures similar to those reported in the scientific and patent literature by Kim et al. (Synlett, 2003, 12, 1838-1840, and published PCT International Application No. WO2004063208 to Kim et al.).

[0498] Example 30: Preparation of Compound 63b [ka] Compound 63a is prepared using a procedure similar to that reported by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

[0499] Example 31: Preparation of Compound 63d [ka] Compound 63c is prepared using a procedure similar to that reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

[0500] Example 32: Preparation of Compound 67 [ka] Compound 64 was prepared according to the procedure illustrated in Example 2. Compound 65 is prepared using a procedure similar to that reported by Or et al., published PCT International Application No. WO2009003009. The protecting groups used in compound 65 are representative and not intended to be limiting, as other protecting groups can be used, including but not limited to those presented herein.

[0501] Example 33: Preparation of Compound 70 [ka] Compound 64 was prepared according to the procedure illustrated in Example 2. Compound 68 is commercially available. The protecting groups used in compound 68 are representative and not intended to be limiting, as other protecting groups can be used, including but not limited to those presented herein.

[0502] Example 34: Preparation of Compound 75a [ka] Compound 75 is prepared according to the published procedure reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

[0503] Example 35: Preparation of Compound 79 [ka] Compound 76 was prepared according to the published procedure reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

[0504] Example 36: Preparation of Compound 79a [ka] Compound 77 is prepared according to the procedure illustrated in Example 35.

[0505] Example 37: General method for the preparation of conjugated oligomeric compound 82 containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end on a solid support (Method I) [ka] [ka] GalNAc3-2 has the following structure: [ka]

[0506] The GalNAc3 cluster portion of the conjugated group GalNAc3-2 (GalNAc3-2 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. a has the following formula: [ka]

[0507] VIMAD-linked oligomeric compound 79b was prepared using standard procedures for automated DNA / RNA synthesis (Dupouy et al., Angew. Chem. Int. E (See, e.g., J. Am. Chem. Soc ...

[0508] Example 38: Alternative method for the preparation of oligomeric compound 82 containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' terminus (Method II) [ka] VIMAD-linked oligomeric compound 79b was prepared using standard procedures for automated DNA / RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc3-2 cluster phosphoramidite (compound 79) was prepared as illustrated in Example 35. This alternative method allows for the addition of a phosphodiester-linked GalNAc3-2 conjugate in the final step of the synthesis. This allows for the single-step introduction of 5'-terminal phosphodiester conjugates into oligomeric compounds. The illustrated phosphoramidites are representative and not intended to be limiting, as other phosphoramidite building blocks, including but not limited to those presented herein, can be used to prepare oligomeric compounds having phosphodiester conjugates at the 5' termini. The order and amounts of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds described herein with any predetermined sequence and composition.

[0509] Example 39: General method for the preparation of oligomeric compound 83h containing a GalNAc3-3 conjugate at the 5' end (GalNAc3-1 modified for 5' end attachment) on solid support [ka] [ka] Compound 18 was prepared according to the procedure illustrated in Example 4. Compounds 83a and 83b are commercially available. Oligomeric compound 83e containing a phosphodiester-linked hexylamine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia afforded 5'-GalNAc3-3 conjugated oligomeric compound (83h).

[0510] GalNAc3-3 has the following structure: [ka]

[0511] The GalNAc3 cluster portion of the conjugated group GalNAc3-3 (GalNAc3-3 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. a has the following formula: [ka]

[0512] Example 40: General method for the preparation of oligomeric compound 89 containing a phosphodiester-linked GalNAc3-4 conjugate at the 3' terminus on solid support [ka] [ka] [ka] GalNAc3-4 has the following structure: [ka]

[0513] wherein CM is a cleavable moiety. In certain embodiments, the cleavable moiety is: [ka]

[0514] The GalNAc3 cluster portion of the conjugated group GalNAc3-4 (GalNAc3-4 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. a has the following formula: [ka]

[0515] The protected Unylinker-functionalized solid support compound 30 is commercially available. Compound 84 is prepared using a procedure similar to that reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).

[0516] Phosphoramidite building blocks (compounds 60 and 79a) are prepared according to the procedures illustrated in Examples 28 and 36. The illustrated phosphoramidites are representative and not intended to be limiting, as other phosphoramidite building blocks can be used to prepare oligomeric compounds having phosphodiester bond conjugates at the 3' termini with predetermined sequences and compositions. The order and amounts of phosphoramidites added to the solid support can be adjusted to prepare oligomeric compounds described herein with any predetermined sequence and composition.

[0517] Example 41: General method for the preparation of ASOs containing a phosphodiester-linked GalNAc3-2 (see Example 37 where Bx is adenine) conjugate at the 5' position by solid-phase techniques (preparation of ISIS 661134) Unless otherwise specified, all reagents and solutions used in the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid supports are available from, for example, T, A, G, and m Used for the incorporation of nucleoside residues, including C residues. 5'-terminal phosphodiester-linked GalNAc3-2 conjugates were synthesized using phosphoramidite compounds 56 and 60. 0.1 M solutions of phosphoramidites in anhydrous acetonitrile were used for β-D-2'-deoxyribonucleosides and 2'-MOE.

[0518] The column was packed with VIMAD solid support (110 μmol / g, Guzaev et al. ASO synthesis was performed using the phosphoramidite coupling method in a 3D synthesis system (1-2 μmol scale) on an ABI 394 synthesizer or a GE Healthcare synthesizer (2003). The synthesis was performed on a Bioscience AeKTA Oligo Pilot Synthesizer (40–200 μmol scale). For this coupling step, a 4-fold excess of phosphoramidite was delivered relative to the initial loading of the solid support, and phosphoramidite coupling was performed for 10 min. All other steps followed the standard protocol provided by the manufacturer. The dimethoxytrityl (DMT) group was removed from the 5'-hydroxyl group of the nucleotide using a solution of 6% dichloroacetic acid in toluene. 4,5-dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as the activating agent during the coupling step. Phosphorothioate linkages were introduced by sulfurization with a 0.1 M solution of xanthan hydride in 1:1 pyridine / CH3CN for a contact time of 3 min. A solution of 20% tert-butyl hydroperoxide in CH3CN containing 6% water was used as the oxidizing agent for a contact time of 12 min to provide phosphodiester internucleoside linkages.

[0519] After the desired sequence was assembled, the cyanoethylphosphate protecting groups were deprotected using 20% ​​diethylamine in toluene (v / v) with a contact time of 45 min. The solid support-bound ASO was suspended in aqueous ammonia (28–30 wt%) and heated at 55 °C for 6 h.

[0520] The unbound ASO was then filtered and the ammonia was boiled off. The residue was purified by high-pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0–40% B after 60 min, flow rate 14 mL / min, λ = 260 nm). The residue was desalted by HPLC on a reverse-phase column to give the desired ASO in 15–30% isolated yield based on initial loading on the solid support. The ASO was characterized by ion-pair HPLC coupled to MS analysis using an Agilent 1100 MSD system. [Table 21]

[0521] The subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, "k" indicates a 6'-(S)-CH3 bicyclic nucleoside (e.g., cEt), "s" indicates a phosphorothioate internucleoside linkage (PS), "o" indicates a phosphodiester internucleoside linkage (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. GalNAc3-2 a The structure of is shown in Example 37.

[0522] Example 42: General method for the preparation of ASOs containing GalNAc3-3 conjugates at the 5' position by solid phase techniques (preparation of ISIS 661166) The synthesis of ISIS 661166 was carried out using procedures similar to those illustrated in Examples 39 and 41.

[0523] ISIS 661166 is a 5-10-5 MOE gapmer containing a GalNAc3-3 conjugate at the 5' position. The ASO was characterized by ion-pair HPLC coupled to MS analysis using an Agilent 1100 MSD system. [Table 21a]

[0524] The subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, "s" indicates a phosphorothioate internucleoside linkage (PS), "o" indicates a phosphodiester internucleoside linkage (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. The structure of "5'-GalNAc3-3a" is shown in Example 39.

[0525] Example 43: Dose-dependent study of phosphodiester-linked GalNAc3-2 at the 5' end targeting SRB-1 in vivo (see Examples 37 and 41 where Bx is adenine) ISIS 661134, which contains a phosphodiester-linked GalNAc3-2 conjugate at the 5' end (see Example 41), was tested for antisense inhibition of SRB-1 in mice in a dose-dependent study. Unconjugated ISIS 440762 and 651900 (GalNAc3-1 conjugates at the 3' end, see Example 9) were included in the study for comparison and are listed in Table 4 above.

[0526] process Six-week-old male Balb / c mice (Jackson Laboratory, Bar Harbor, ME) were subcutaneously injected once with ISIS 440762, 651900, 661134, or a PBS-treated control at the doses indicated below. Each treatment group consisted of four animals. Seventy-two hours after the final dose, mice were sacrificed, and SRB-1 mRNA levels in the liver were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen) before normalizing to the PBS-treated control. The results below are presented as the average percent SRB-1 mRNA levels for each treatment group normalized to the PBS-treated control and are denoted as "%PBS." ED was performed using a method similar to that described above. 50 were measured and are presented below.

[0527] As illustrated in Table 22, treatment with antisense oligonucleotides reduced SRB-1 mRNA levels in a dose-dependent manner. Indeed, antisense oligonucleotides containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end (ISIS 661134) or a GalNAc3-1 conjugate linked to the 3' end (ISIS 651900) showed significantly improved potency compared with unconjugated antisense oligonucleotides (ISIS 440762). Furthermore, ISIS 661134, which contains a phosphodiester-linked GalNAc3-2 conjugate at the 5' end, was equipotent compared with ISIS 651900, which contains a GalNAc3-1 conjugate at the 3' end. [Table 22]

[0528] The structures of 3'GalNAc3-1 and 5'GalNAc3-2 are described in Examples 9 and 37 above.

[0529] Pharmacokinetic analysis (PK) The PK of the high dose group (7 mg / kg) of ASO was tested and evaluated in a manner identical to that exemplified in Example 20. Liver samples were minced and extracted using standard protocols. Full-length metabolites of 661134 (5'GalNAc3-2) and ISIS 651900 (3'GalNAc3-1) were identified, and their masses were confirmed by high-resolution mass spectrometry. Results showed that the ASO containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end (ISIS The results showed that the major metabolite detected for the ASO (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites were observed at detectable levels. Unlike its counterpart, additional metabolites similar to those reported above in Table 10a were observed for the ASO (ISIS 651900) bearing a GalNAc3-1 conjugate at the 3' end. These results suggest that having phosphodiester-linked GalNAc3-1 or GalNAc3-2 conjugates may improve the PK profile of ASOs without compromising their potency.

[0530] Example 44: Effect of PO / PS conjugation on antisense inhibition of ASOs containing GalNAc3-1 conjugates (see Example 9) at the 3' end targeting SRB-1 ISIS containing GalNAc3-1 conjugates at the 3' end, each targeting SRB-1 655861 and 655862 were tested for their ability to inhibit SRB-1 in mice in a single-dose study. The parent unconjugated compound, ISIS 353382, was included in the study for comparison.

[0531] The ASO was a 5-10-5 MOE gapmer, in which the gap region contained 10 2'-deoxyribonucleosides and each wing region contained 5 2'-MOE modified nucleosides. The ASOs were prepared using methods similar to those illustrated in Example 19 above and are shown in Table 23 below. [Table 23]

[0532] The subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, "s" indicates a phosphorothioate internucleoside linkage (PS), "o" indicates a phosphodiester internucleoside linkage (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. The structure of "GalNAc3-1" is shown in Example 9.

[0533] process Six-week-old male Balb / c mice (Jackson Laboratory, Bar Harbor, ME) were subcutaneously injected once with ISIS 353382, 655861, 655862, or a PBS-treated control at the doses indicated below. Each treatment group consisted of four animals. Blood was collected from each mouse before treatment and after the final dose, and plasma samples were analyzed. Seventy-two hours after the final dose, mice were sacrificed, and SRB-1 mRNA levels in the liver were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen) before normalizing to the PBS-treated control. The results below are presented as the average percent SRB-1 mRNA levels for each treatment group normalized to the PBS-treated control and are denoted "%PBS." ED was performed using a method similar to that described above. 50 are measured and reported below.

[0534] As illustrated in Table 24, treatment with antisense oligonucleotides reduced SRB-1 mRNA levels in a dose-dependent manner compared to PBS-treated controls. Indeed, antisense oligonucleotides containing a GalNAc3-1 conjugate at the 3' end (ISIS 655861 and 655862) showed significantly improved potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). Furthermore, ISIS 655862, which has mixed PS / PO linkages, showed improved potency compared to the full PS (ISIS 655861). [Table 24]

[0535] Serum liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured relative to saline-injected mice using standard protocols. Organ weights were also assessed. Results showed that no increase in transaminase levels (Table 25) or organ weights (data not shown) was observed in ASO-treated mice compared with PBS controls. Furthermore, an ASO with mixed PS / PO linkages (ISIS 655862) showed similar transaminase levels compared with an ASO with intact PS (ISIS 655861). [Table 25]

[0536] Example 45: Preparation of PFP ester (compound 110a) [ka] [ka] Compound 4 (9.5 g, 28.8 mmole) was treated with compound 103a or 103b (38 mmole), TMSOTf (0.5 equiv.) in dichloromethane (200 mL) and molecular sieves, respectively, and stirred at room temperature for 16 hours. At this point, the organic layer was filtered through Celite, then washed with sodium bicarbonate, water, and brine. The organic layer was then separated, dried over sodium sulfate, filtered, and reduced under reduced pressure. The resulting oil was purified by silica gel chromatography (2% to 10% methanol / dichloromethane) to give compounds 104a and 104b in greater than 80% yield. LCMS and proton NMR were consistent with the structures.

[0537] Compounds 104a and 104b were treated under the same conditions as compounds 100a-d (Example 47) to give compounds 105a and 105b in greater than 90% yield. LCMS and proton NMR were consistent with the structures.

[0538] Compounds 105a and 105b were treated separately with compound 90 under the same conditions as compounds 901a-d to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR were consistent with the structures.

[0539] Compounds 106a and 106b were treated under the same conditions as compounds 96a-d (Example 47) to give 107a (60%) and 107b (20%). LCMS and proton NMR were consistent with the structures.

[0540] Compounds 107a and 107b were treated under the same conditions as compounds 97a-d (Example 47) to give compounds 108a and 108b in 40-60% yield. NMR was consistent with the structure.

[0541] Compounds 108a (60%) and 108b (40%) were treated under the same conditions as compounds 100a-d (Example 47) to give compounds 109a and 109b in greater than 80% yield. LCMS and proton NMR were consistent with the structures.

[0542] Compound 109a was treated under the same conditions as compounds 101a-d (Example 47) to give compound 110a in 30-60% yield. LCMS and proton NMR were consistent with the structure. Alternatively, compound 110b can be prepared in a similar manner starting from compound 109b.

[0543] Example 46: General procedure for conjugation with PFP ester (oligonucleotide 111); preparation of ISIS 666881 (GalNAc3-10) 5'-hexylamino-modified oligonucleotides were synthesized and purified using standard solid-phase oligonucleotide procedures. The 5'-hexylamino-modified oligonucleotides were dissolved in 0.1 M sodium tetraborate (pH 8.5, 200 μL) and 3 equivalents of the selected PFP-esterified GalNAc3 cluster dissolved in DMSO (50 μL) were added. If the PFP ester precipitated upon addition to the ASO solution, DMSO was added until all the PFP ester was in solution. After approximately 16 hours of mixing at room temperature, the reaction was complete. The resulting solution was diluted with water to 12 mL and then spun at 3000 rpm onto a spin filter with a mass cutoff of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was then lyophilized to dryness, redissolved in concentrated aqueous ammonia, mixed at room temperature for 2.5 hours, and then concentrated in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified, desalted by RP-HPLC, and lyophilized to give the GalNAc3-conjugated oligonucleotide. [ka]

[0544] Oligonucleotide 111 is conjugated to GalNAc3-10. The GalNAc3 cluster portion of the conjugate group GalNAc3-10 (GalNAc3-10 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-A, as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc3-10 below. d -P(=O)(OH)-. GalNAc3-10 (GalNAc3-10 a The structure of methyltrimethylsilyl-2-methyl ... [ka]

[0545] ISIS 666881 was prepared according to this general procedure. A 5'-hexylamino-modified oligonucleotide (ISIS 660254) was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 μmol) was dissolved in 0.1 M sodium tetraborate (pH 8.5, 200 μL) and 3 equivalents of PFP ester (compound 110a) dissolved in DMSO (50 μL) was added. If the PFP ester precipitated upon addition to the ASO solution, additional DMSO (600 μL) was required to completely dissolve the PFP ester. After approximately 16 hours of mixing at room temperature, the reaction was complete. The solution was diluted with water to a total volume of 12 mL and sedimented at 3000 rpm onto a spin filter with a mass cutoff of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness, redissolved in concentrated aqueous ammonia, mixed at room temperature for 2.5 hours, and then concentrated in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified, desalted by RP-HPLC, and lyophilized to give ISIS 666881 (42 mg, 4.7 μmol) in 90% wt yield. [Table 25-2]

[0546] The capital letters indicate the nucleobase of each nucleoside, m C indicates 5-methylcytosine. Subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, "s" indicates a phosphorothioate internucleoside linkage (PS), "o" indicates a phosphodiester internucleoside linkage (PO), and "o'" indicates -OP(=O)(OH)-. Conjugated groups are shown in bold.

[0547] Example 47: Preparation of Oligonucleotide 102 Containing GalNAc3-8 [ka] [ka] [ka] Triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-diisopropylethylamine (12.35 mL, 72 mmol). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 mmol) was added dropwise under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added along with N,N-diisopropylethylamine (12.35 mL, 72 mmol), and the reaction was allowed to stir at room temperature for 16 hours. At this point, the DMF was reduced to >75% under reduced pressure, and the mixture was then dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water, and brine. The organic layer was then separated, dried over sodium sulfate, filtered, and reduced to an oil under reduced pressure. The resulting oil was purified by silica gel chromatography (2% to 10% methanol / dichloromethane) to give compounds 92a and 92b in approximately 80% yield. LCMS and proton NMR were consistent with the structures.

[0548] Compound 92a or 92b (6.7 mmol) was treated with 20 mL of dichloromethane and 20 mL of trifluoroacetic acid at room temperature for 16 hours. The resulting solution was evaporated, then dissolved in methanol and treated with Dowex-OH resin for 30 minutes. The resulting solution was filtered and reduced to an oil under reduced pressure to give compounds 93a and 93b in 85-90% yield.

[0549] Compound 7 or 64 (9.6 mmole) was treated with HBTU (3.7 g, 9.6 mmole) and N,N-diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compound 93a or 93b (3 mmole) and allowed to stir at room temperature for 16 hours. At this point, the DMF was reduced to >75% under reduced pressure, and then the mixture was cooled to room temperature. The mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water, and brine. The organic layer was then separated, dried over sodium sulfate, filtered, and reduced to an oil under reduced pressure. The resulting oil was purified by silica gel chromatography (5% to 20% methanol / dichloromethane) to give compounds 96a-d in 20-40% yield. LCMS and proton NMR were consistent with the structures.

[0550] Compounds 96a-d (0.75 mmole) were individually hydrogenated in ethanol (75 mL) over Raney nickel for 3 h. At this point, the catalyst was filtered off through Celite and the ethanol was removed under reduced pressure to give compounds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structures.

[0551] Compound 23 (0.32 g, 0.53 mmole) was treated with HBTU (0.2 g, 0.53 mmole) and N,N-diisopropylethylamine (0.19 mL, 1.14 mmole) in DMF (30 mL) for 15 minutes. Compounds 97a-d (0.38 mmole) were added individually and allowed to stir at room temperature for 16 hours. At this point, the DMF was reduced to >75% under reduced pressure, and the mixture was then dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water, and brine. The organic layer was then separated, dried over sodium sulfate, filtered, and reduced to an oil under reduced pressure. The resulting oil was purified by silica gel chromatography (2% → 20% methanol / dichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR were consistent with the structures.

[0552] Compound 99 (0.17 g, 0.76 mmole) was treated with HBTU (0.29 g, 0.76 mmole) and N,N-diisopropylethylamine (0.35 mL, 2.0 mmole) in DMF (50 mL) for 15 minutes. Compounds 97a-d (0.51 mmole) were added individually and allowed to stir at room temperature for 16 hours. At this point, the DMF was reduced to >75% under reduced pressure, and the mixture was then dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water, and brine. The organic layer was then separated, dried over sodium sulfate, filtered, and reduced to an oil under reduced pressure. The resulting oil was purified by silica gel chromatography (5% → 20% methanol / dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR were consistent with the structures.

[0553] Compounds 100a-d (0.16 mmole) were individually hydrogenated in methanol / ethyl acetate (1:1, 50 mL) over 10% Pd(OH) / C for 3 h. At this point, the catalyst was filtered off through Celite and the organics were removed under reduced pressure to give compounds 101a-d in 80-90% yield. LCMS and proton NMR were consistent with the structures.

[0554] Compounds 101a-d (0.15 mmole) were dissolved separately in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmole). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmole) was added dropwise under argon, and the reaction was allowed to stir at room temperature for 30 minutes. At this point, the DMF was reduced to over 75% under reduced pressure, and the mixture was then dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water, and brine. The organic layer was then separated, dried over sodium sulfate, filtered, and reduced to an oil under reduced pressure. The resulting oil was purified by silica gel chromatography (2% to 5% methanol / dichloromethane) to give compounds 102a-d in approximately 80% yield. LCMS and proton NMR were consistent with the structures. [ka]

[0555] Oligomeric compound 102 containing a GalNAc3-8 conjugate group was prepared using the general procedure illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3-8 (GalNAc3-8 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. In one preferred embodiment, the cleavable moiety is -P(=O)(OH)-A d -P(=O)(OH)-.

[0556] GalNAc3-8(GalNAc3-8 a The structure of methyltrimethylsilyl-2-methyl ... [ka]

[0557] Example 48: Preparation of Oligonucleotide 119 Containing GalNAc3-7 [ka] [ka] Compound 112 was synthesized according to the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

[0558] Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol / ethyl acetate (22 mL / 22 mL). Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 hours. The reaction mixture was filtered through a pad of Celite, and the pad was washed with 1:1 methanol / ethyl acetate. The filtrate and washings were combined and concentrated to dryness to give compound 105a (quantitative). The structure was confirmed by LCMS.

[0559] Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol), and DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL), and the reaction mixture was stirred at room temperature for 5 minutes. To this was added a solution of compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL). The reaction was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure to give an oil. The residue was dissolved in CHCl (100 mL) and washed with saturated aqueous NaHCO (100 mL) and brine (100 mL). The organic phase was separated, dried (NaSO), filtered, and concentrated. The residue was purified by silica gel column chromatography, eluting with 10-20% MeOH in dichloromethane, to give compound 114 (1.45 g, 30%). The structure was confirmed by LCMS and NMR (CDCl) NMR (CDCl) δ 1.01-1.02 (mM), 1.02-1.03 (mM), 1.04-1.05 (mM), 1.06-1.07 (mM), 1.08-1.09 (mM), 1.09-1.10 (mM), 1.09-1.11 (mM), 1.08-1.12 (mM), 1.09-1.13 (mM), 1.08-1.14 (mM), 1.08-1.15 ... 1 Confirmed by 1 H NMR analysis.

[0560] Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol / ethyl acetate (4 mL / 4 mL). Palladium on carbon (wet, 0.14 g) was added. The reaction mixture was flushed with hydrogen and stirred under hydrogen at room temperature for 12 hours. The reaction mixture was filtered through a pad of Celite. The reaction mixture was filtered. The Celite pad was washed with methanol / ethyl acetate (1:1). The filtrate and washings were combined and evaporated under reduced pressure to give compound 115 (quantitative). The structure was confirmed by LCMS and 1 Confirmed by 1 H NMR analysis.

[0561] Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol), and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL), and the reaction mixture was stirred at room temperature for 5 minutes. To this, a solution of compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added, and the reaction was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure, and the residue was dissolved in CHCl. ​​The organic layer was washed with saturated aqueous NaHCO and brine, dried over anhydrous NaSO, and filtered. The organic layer was concentrated to dryness, and the resulting residue was purified by silica gel column chromatography, eluting with 3-15% MeOH in dichloromethane, to give compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and NMR. 1 Confirmed by 1 H NMR analysis. [ka]

[0562] Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol / ethyl acetate (5 mL / 5 mL). Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was flushed with hydrogen and stirred under hydrogen at room temperature for 12 hours. The reaction mixture was filtered through a pad of Celite. The Celite pad was washed with methanol / ethyl acetate (1:1). The filtrate and washings were combined and evaporated under reduced pressure to give compound 117 (0.73 g, 98%). The structure was confirmed by LCMS and NMR (CDCl). 1 Confirmed by 1 H NMR analysis.

[0563] Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution, N,N-diisopropylethylamine (70 μL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours and poured into saturated aqueous NaHCO3 solution. The mixture was extracted with dichloromethane, washed with brine, and dried over anhydrous Na2SO4. This di The dichloromethane solution was concentrated to dryness and purified by silica gel column chromatography, eluting with 5-10% MeOH in dichloromethane, to give compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and 1 H and 1 H and 19 Confirmed by F NMR. [ka]

[0564] Oligomeric compound 119 containing a GalNAc3-7 conjugate group was prepared using the general procedure illustrated in Example 46. The GalNAc3 cluster portion of GalNAc3-7 (GalNAc3-7 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-A d -P(=O)(OH)-.

[0565] GalNAc3-7(GalNAc3-7 a The structure of methyltrimethylsilyl-2-methyl ... [ka]

[0566] Example 49: Preparation of Oligonucleotide 132 Containing GalNAc3-5 [ka] Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 minutes. The reaction mixture was cooled in an ice bath, and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added, and the reaction mixture was stirred under an argon atmosphere for 18 hours. The reaction was monitored by TLC (1:1 ethyl acetate:hexane; Rf = 0.47). The solvent was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with 1 M NaHSO (3 × 150 mL), saturated aqueous NaHCO (3 × 150 mL), and brine (2 × 100 mL). The organic layer was dried over NaSO. The drying agent was removed by filtration, and the organic layer was concentrated by rotary evaporation. The crude mixture was purified by silica gel column chromatography, eluting with 35-50% EtOAc in hexane to give compound 122 (15.50 g, 78.13%). The structure was confirmed by LCMS and 1 Confirmed by H NMR analysis. Mass (m / z) 589.3 [M+H] + .

[0567] A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled solution of compound 122 (7.75 g, 13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 minutes and monitored by TLC (1:1 EtOAc:hexanes). The reaction mixture was concentrated under reduced pressure to half its volume. The remaining solution was cooled in an ice bath and neutralized by the addition of concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL), and washed with brine (100 mL). An emulsion formed and was removed after standing overnight. The organic layer was separated and dried (NaSO 4) , filtered, and evaporated to give compound 123 (8.42 g). Residual salts are the likely cause of the excess mass. LCMS was consistent with the structure. This product was used without further purification. Calculated MW: 574.36, Found MW: 575.3 [M+H]+ . [ka]

[0568] Compound 126 was synthesized according to the procedure described in the literature (J. Am. Chem. Soc. 2011, 133, 958-963). [ka] [ka]

[0569] Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol), and compound 126 (6.33 g, 16.14 mmol) were dissolved in DMF (40 mL), and the resulting reaction mixture was cooled in an ice bath. To this was added N,N-diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol), followed by Bop coupling reagent (1.17 g, 2.66 mmol) under an argon atmosphere. The ice bath was removed, and the solution was allowed to warm to room temperature. The reaction was complete after 1 h, as determined by TLC (89:10:1 DCM:MeOH:AA). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1M NaHSO (3 x 100 mL), saturated aqueous NaHCO (3 x 100 mL), and brine (2 x 100 mL). The organic phase was separated, dried (NaSO), filtered, and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexane / EtOAC:100% EtOAc to give compound 127 (9.4 g) as a white foam. LCMS and 1 H NMR was consistent with the structure. Mass (m / z) 778.4 [M+H] + .

[0570] Trifluoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room temperature for 1 hour. The reaction mixture was co-evaporated to dryness with toluene (30 mL) under reduced pressure. The resulting residue was dissolved in acetonitrile (30 mL). L) and toluene (40 mL) twice to give compound 128 (1.67 g) as the trifluoroacetic acid salt, which was used in the next step without further purification. LCMS and 1 H NMR was consistent with the structure. Mass (m / z) 478.2 [M+H] + .

[0571] Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined in a round-bottom flask and dried over PO for 4 hours under reduced pressure. The mixture was then dissolved in anhydrous DMF (1 mL) and stirred for 5 minutes. To this was added a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-diisopropylethylamine (0.2 mL). The reaction mixture was stirred at room temperature under an argon atmosphere. After 30 minutes, the reaction was complete as determined by LCMS and TLC (7% MeOH / DCM). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO (3 × 20 mL), saturated aqueous NaHCO (3 × 20 mL), and brine (3 × 20 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography using 5-15% MeOH in dichloromethane to give compound 129 (96.6 mg). LC MS and 1 H NMR is consistent with the structure. Mass (m / z) 883.4 [M+2H] + .

[0572] Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in a 20 mL scintillation vial. To this was added a small amount of 10% Pd / C (0.015 mg), and the reaction vessel was flushed with H2 gas. The reaction mixture was stirred at room temperature for 18 hours under an H2 atmosphere. The reaction mixture was filtered through a Celite pad, and the Celite pad was washed with methanol. The filtrate washes were pooled together and concentrated under reduced pressure to give compound 130 (0.08 g). LCMS and 1 H NMR was consistent with the structure. The product was used without further purification. Mass (m / z) 838.3 [M+2H] + .

[0573] To a 10 mL sharp-nosed round-bottom flask was added compound 130 (75.8 mg, 0.046 mmol), 0.37 M pyridine / DMF (200 μL), and a stir bar. To this solution was added 0.7 M pentafluorophenyl trifluoroacetate / DMF (100 μL) dropwise with stirring. The reaction was complete after 1 h, as determined by LC / MS. The solvent was removed under reduced pressure, and the residue was dissolved in CHCl (approximately 10 mL). The organic layer was partitioned three times each with NaHSO (1 M, 10 mL), saturated aqueous NaHCO (10 mL), and brine (10 mL). The organic phase was separated, dried over NaSO, filtered, and concentrated to give compound 131 (77.7 mg). LC / MS was consistent with the structure. It was used without further purification. Mass (m / z) 921.3 [M+2H] + . [ka]

[0574] Oligomeric compound 132 containing a GalNAc3-5 conjugate group was prepared using the general procedure illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3-5 (GalNAc3-5 a ) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-A d-P(=O)(OH)-.

[0575] GalNAc3-5(GalNAc3-5 a The structure of methyltrimethylsilyl-2-methyl ... [ka]

[0576] Example 50: Preparation of oligonucleotide 144 containing GalNAc4-118 [ka] [ka] Synthesis of Compound 134. Aminomethyl VIMAD resin (2.5 g, 450 μmol / g), which had been washed with acetonitrile, dimethylformamide, dichloromethane, and acetonitrile, was added to a Merrifield flask. The resin was swollen in acetonitrile (4 mL). Compound 133 was preactivated in a 100 mL round-bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL), and DIEA (3.0 mmol, 0.5 mL)...

Claims

1. The chemical structure below: 【Chemistry 1】 (SEQ ID NO: 12), or a pharmaceutically acceptable salt thereof, In the formula, R １ Ga-OCH ２ CH ２ OCH ３ (MOE), and R ２ is H; or R １ and R ２ together form a bridge, where R １ is —O—, and R ２ Ga-CH ２ -, -CH(CH ３ ) - or -CH ２ CH ２ - and the resulting bridge is -O-CH ２ -, -O-CH(CH ３ )-, and -O-CH ２ CH ２ R is selected from １ and R ２ are directly connected; R on the same ring ３ and R ４ For each pair, independently for each ring, R ３ is H and -OCH ２ CH ２ OCH ３ and R ４ is H; or R ３ and R ４ together form a bridge, where R ３ is —O—, and R ４ Ga-CH ２ -, -CH(CH ３ ) - or -CH ２ CH ２ - and the resulting bridge is -O-CH ２ -, -O-CH(CH ３ )-, and -O-CH ２ CH ２ R is selected from ３ and R ４ are directly connected; R ５ is H and -CH ３ Selected from: Z is S － and O － A compound or salt selected from:

2. R １ Ga-OCH ２ CH ２ OCH ３ and R ２ The compound of claim 1 , wherein is H.

3. R on the same ring ３ and R ４ For each pair, independently for each ring, R ３ is H and -OCH ２ CH ２ OCH ３ and R ４ The compound of claim 1 , wherein is H.

4. R ５ Ga-CH ３ 2. The compound of claim 1, wherein:

5. The compound according to any one of claims 1 to 4, wherein the pharmaceutically acceptable salt is a sodium salt or a potassium salt.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or diluent.

7. 7. The pharmaceutical composition according to claim 6, comprising a compound according to any one of claims 1 to 5 and sterile saline or sterile water.

8. The compound has the following chemical structure: 【Chemistry 2】 8. The pharmaceutical composition of claim 7, which has the formula:

9. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable salt is a sodium salt.

10. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable salt is a potassium salt.

11. Use of a compound according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or 7 in the manufacture of a medicament for treating transthyretin amyloidosis.

12. The use according to claim 11, wherein the transthyretin amyloidosis is selected from hereditary transthyretin amyloidosis, leptomeningeal amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy, familial ocular leptomeningeal amyloidosis, senile cardiac amyloidosis, and senile systemic amyloidosis.

13. 13. The use according to claim 11 or 12, wherein the pharmaceutically acceptable salt is a sodium salt.

14. 13. The use according to claim 11 or 12, wherein the pharmaceutically acceptable salt is a potassium salt.

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