Compositions and methods for regulating HBV and TTR expression

Abstract

This invention provides novel chemically modified antisense compounds that improve the strength and effectiveness of existing antisense compounds. [Solution] A compound comprising a modified oligonucleotide and a conjugated group is provided, wherein the modified oligonucleotide consists of 8 to 80 linked nucleosides and has a nucleic acid base sequence that is at least 85%, 90%, 95%, or 100% complementary to a specific sequence encoding transthyretin (TTR).

Description

[Technical Field]

[0001] Sequence List This application has been filed electronically along with its sequence listing. This sequence listing is provided as a 16KB file named BIOL0248WOSEQ_ST25.txt, created on May 1, 2014. The electronic information 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, thereby regulating the quantity, activity, and / or function of the target nucleic acid. For example, in certain cases, antisense compounds result in changes in the transcription or translation of the target. Such regulation of expression can be achieved, for example, by inhibition based on the degradation or occupation of the target mRNA. One example of regulation of RNA target function by degradation is the RNase H-based degradation of target RNA upon hybridization with a DNA-like antisense compound. Another example of regulation of gene expression by targeted degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing via a mechanism utilizing the RNA-induced silencing complex (RISC). Further examples of regulation of RNA target function are by occupation-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 that microRNA from binding to its messenger RNA target, and therefore interferes with the function of the microRNA. MicroRNA mimetics can enhance the innate function of microRNAs. Certain antisense compounds alter premRNA splicing. Regardless of the specific mechanism, sequence specificity makes antisense compounds attractive as a means of target validation and gene functionalization, as well as therapeutic agents that selectively modulate the expression of genes involved in disease development.

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

[0004] Novel chemical modifications improve the strength and efficacy of antisense compounds, opening up the possibility of oral delivery, enhancing subcutaneous administration, reducing the potential for side effects, and improving patient convenience. Chemical modifications that increase the strength of antisense compounds enable lower dose administration, reduce the potential for toxicity, and lower overall treatment costs. Modifications that increase resistance to degradation lead to slower elimination from the body, allowing for reduced dosing frequency. Different types of chemical modifications can be combined within a single compound to further optimize the compound's efficacy. [Overview of the project]

[0005] In certain embodiments, the disclosure provides a conjugated antisense compound. In certain embodiments, the disclosure provides an antisense oligonucleotide complementary to a nucleic acid transcript. The disclosure provides a conjugated antisense compound containing an antisense oligonucleotide. In a particular embodiment, the disclosure provides a method comprising contacting cells with a conjugated antisense compound containing an antisense oligonucleotide complementary to a nucleic acid transcript. In a particular embodiment, the disclosure provides a method comprising contacting cells with a conjugated antisense compound containing an antisense oligonucleotide and reducing the amount or activity of nucleic acid transcripts within the cells.

[0006] The asialoglycoprotein receptor (ASGP-R) has been previously described. See, for example, Park et al., PNAS vol.102, No.47, pp17125-17129 (2005). Such receptors are expressed on hepatocytes, specifically on hepatocytes. Furthermore, compounds containing a cluster of three N-acetylgalactosamine (GalNAc) ligands have been shown to bind to ASGP-R, resulting in the uptake of the compound into the cell. See, for example, Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp5216-5231 (May 2008). Thus, conjugates containing such GalNAc clusters are used to promote the uptake of certain compounds into hepatocytes, specifically hepatocytes. For example, certain GalNAc-containing conjugates have been shown to increase the activity of double-stranded siRNA compounds in hepatocytes in vivo. In such cases, the GalNAc-containing conjugate typically binds to the sense strand of the siRNA double-stranded compound. Because the sense strand is disposed of before the antisense strand ultimately hybridizes with the target nucleic acid, there is little concern that the conjugate will interfere with activity. Typically, the conjugate is attached to the 3' end of the sense strand of the siRNA. See, for example, U.S. Patent No. 8,106,022. Certain conjugated groups described herein are more active and / or easier to synthesize than those previously described.

[0007] In certain embodiments of the present invention, the conjugate is bound to single-stranded antisense compounds, including but not limited to RNase H-based antisense compounds and antisense compounds that alter the splicing of premRNA target nucleic acids. In such embodiments, the conjugate should remain bound to the antisense compound for a sufficient period to provide the benefit (improved uptake into cells), but should either be cleaved or not interfere with subsequent steps necessary for activity, such as hybridization to the target nucleic acid related to splicing or splicing regulation and interaction with RNase H or enzymes. This balance of properties is more important in the context of single-stranded antisense compounds than in siRNA compounds, and the conjugate may simply be bound to the sense strand. Conjugated single-stranded antisense compounds that exhibit improved strength in hepatocytes in vivo compared to the same antisense compound without a conjugate are disclosed herein. Given the required balance of properties of these compounds, such improvement in strength is remarkable.

[0008] In certain embodiments, the conjugated group as herein includes a cleavable moiety. As described above, the conjugate should remain in the compound for a sufficiently long period to provide enhanced uptake without being bound by a mechanism, but it is logical that thereafter, part of the conjugate, or ideally all of it, be cleaved to release the parent compound (e.g., the antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments make good use of an endogenous nuclease in the cell by attaching the remainder of the conjugate (cluster) to an antisense oligonucleotide via one or more cleavable bonds, such as a phosphodiester bond, to the nucleoside. In certain embodiments, the cluster is attached to the cleavable nucleoside by a phosphodiester bond. In certain embodiments, the cleavable nucleoside is attached to an antisense oligonucleotide (antisense compound) by a phosphodiester bond. In the application, the conjugated group may contain two or three cleavable nucleosides. In such embodiments, such cleavable nucleosides are linked to one another, to an antisense compound, and / or to a cluster by cleavable bonds (such as phosphodiester bonds). Certain conjugates described herein do not contain cleavable nucleosides, but instead contain cleavable bonds. 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, a conjugated antisense compound is a prodrug. Such a prodrug is administered to an animal and ultimately metabolized to a more active form. For example, a conjugated antisense compound may be 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' end of the oligonucleotide. Certain such 5' conjugates are cleaved more efficiently than their counterparts having a similar conjugated group attached to the 3' end. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, the effectiveness of oligonucleotides with a conjugate at the 5' end is higher than that of oligonucleotides with a conjugate at the 3' end (see, e.g., Examples 56, 81, 83, and 84). Furthermore, the 5' bond allows for simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3'-to-5' direction. To produce 3'-conjugated oligonucleotides, a pre-conjugated 3' nucleoside is typically attached to a solid support, and then the oligonucleotide is constructed as usual. However, attaching the conjugated nucleoside to the solid support complicates the synthesis. Furthermore, by using this means, the conjugate may then be present throughout the synthesis of the oligonucleotide and may be degraded in subsequent steps, or it may limit the types of reactants and reagents that can be used. By using the 5'-conjugated oligonucleotide structures and techniques described herein, oligonucleotides can be synthesized using standard automated techniques to introduce a conjugate with the final (most 5') nucleoside, or oligonucleotides can be synthesized after they have been cleaved from a solid support.

[0011] In consideration of the art and this disclosure, a person skilled in the art can readily 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, and is therefore less expensive, offering advantages in production. For example, the synthesis of certain conjugated groups consists of fewer synthesis steps compared to previously described conjugated groups, resulting in increased yield. Conjugated groups 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 more chemical intermediates. Thus, these and other conjugates described herein are more advantageous than previously described compounds when used in conjunction with any oligonucleotides containing either single-stranded or double-stranded oligonucleotides (e.g., siRNA).

[0012] Similarly, conjugated groups having only one or two GalNAc ligands are disclosed herein. As shown, such conjugated groups improve the activity of antisense compounds. Such compounds are easier to prepare than conjugates containing three GalNAc ligands. Conjugated groups containing one or two GalNAc ligands are used with single-stranded oligonucleotides. It can be bound to any antisense compound containing either strand of a double-stranded oligonucleotide (e.g., siRNA).

[0013] In certain embodiments, the conjugates described herein do not substantially alter tolerability to a certain extent. For example, it is shown herein that the immunogenicity of a conjugated antisense compound is lower than that of an unconjugated parent compound. Embodiments in which tolerability remains the same (or remains the same even if tolerability decreases only slightly compared to the increase in strength) have improved therapeutic properties.

[0014] In certain embodiments, conjugation allows for the modification of antisense compounds in a manner that would have less attractive results in the absence of conjugation. For example, in certain embodiments, substituting one or more phosphorothioate bonds in a complete phosphorothioate antisense compound with phosphodiester bonds results in some degree of improved tolerability. For instance, in certain cases, the immunogenicity of such an antisense compound having one or more phosphodiester bonds is lower than that of the same compound where each bond is a phosphorothioate bond. However, in certain cases, as shown in Example 26, a similar substitution of one or more phosphorothioate bonds with phosphodiester bonds also results in decreased cellular uptake and / or loss of strength. In certain embodiments, the conjugated antisense compounds described herein tolerate such bond changes with little or no loss of uptake and strength compared to their complete phosphorothioate-conjugated counterparts. In fact, in certain embodiments, for example in Examples 44, 57, 59, and 86, oligonucleotides containing a conjugate and at least one phosphodiester nucleoside linkage actually exhibit increased in vivo strength even when compared to complete phosphorothioate counterparts containing the same conjugate. Furthermore, since conjugation results in a substantial increase in uptake / strength, a slight loss of that substantial increase may be acceptable to achieve improved tolerability. Therefore, in certain embodiments, the conjugated antisense compound contains at least one phosphodiester linkage.

[0015] In certain embodiments, the conjugation of antisense compounds as described herein results in increased delivery, uptake, and activity in hepatocytes. Thus, more compounds are delivered to liver tissue. However, in certain embodiments, such increased delivery alone does not manifest an overall increase in activity. In certain embodiments, more compounds enter the hepatocytes. In certain embodiments, even such increased hepatocyte uptake does not manifest an overall increase in activity. In such embodiments, the productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of the GalNAc-containing conjugate increase the enrichment of antisense oligonucleotides in hepatocytes compared to non-parenchymal cells. This enrichment is beneficial for oligonucleotides that target genes expressed in hepatocytes.

[0016] In certain embodiments, the conjugated antisense compounds described herein result in reduced renal exposure. For example, as shown in Example 20, the concentration of an antisense oligonucleotide containing a specific embodiment of the GalNAc-containing conjugate is lower in the kidney than the concentration of an antisense oligonucleotide lacking the GalNAc-containing conjugate. This has several beneficial therapeutic implications. In therapeutic targets where renal activity is not required, renal exposure carries the risk of nephrotoxicity without commensurate benefit. Furthermore, high concentrations in the kidney typically result in the loss of the compound into the urine, leading to more rapid clearance. Therefore, in the case of non-renal targets, accumulation in the kidney is undesirable.

[0017] In a particular embodiment, this disclosure relates to a conjugate antisense represented by the following formula. Provides compounds, [ka] During the ceremony, A is an antisense oligonucleotide, B is a part that can be cut, C is a conjugate linker, D is a branching base, Each E is a tether, Each F is an ligand, q is an integer between 1 and 5.

[0018] In the figures described above and similar figures in this specification, the branching group "D" branches as many times as necessary to accommodate the number of (E~F) groups indicated by "q". Therefore, when q=1, the equation is as follows: [ka] When q=2, the equation is as follows: [ka] When q=3, the equation is as follows: [ka] When q=4, the equation is as follows: [ka] When q=5, the formula is as follows: [ka]

[0019] In a particular embodiment, a conjugated antisense compound having the following structure is provided. [ka]

[0020] In a particular embodiment, a conjugated antisense compound having the following structure is provided. [ka]

[0021] In a particular embodiment, a conjugated antisense compound having the following structure is provided. [ka]

[0022] In a particular embodiment, a conjugated antisense compound having the following structure is provided. [ka]

[0023] In embodiments having two or more of a particular set of variables (for example, two or more "m" or "n"), each such particular variable is selected independently unless otherwise indicated. Therefore, in the case of a structure having two or more n, each n is selected independently and can be either identical or non-identical to one another. [Modes for carrying out the invention]

[0024] It should be understood that both the general description above and the detailed description below are illustrative and descriptive only and do not limit the disclosure. In this specification, the use of the singular includes the plural unless otherwise explicitly stated. As used herein, the use of "or" means "and / or" unless otherwise explicitly stated. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" include both multiple elements and multiple components containing one unit and multiple elements and multiple components containing two or more subunits, unless otherwise explicitly stated.

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

[0026] Unless otherwise specified, the scientific names, procedures, and techniques used herein in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry are well known and commonly used in the art. Standard techniques are chemical synthesis... It can be used in chemical analysis. Certain such techniques and procedures include, for example, "Carbohydrate Modifications in Antisense." 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 ndThese can be found in Edition, Cold Spring Harbor Laboratory Press, 1989, and are incorporated herein by reference for all purposes. Where permitted, all patents, applications, published applications, and other publications, as well as other data, referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0027] Unless otherwise specified, the following terms have the following meanings:

[0028] As used herein, “nucleoside” means a compound comprising a nucleic acid base 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” means a chemical difference of a compound compared to its naturally occurring counterpart. Oligonucleotide chemical modifications include nucleoside modifications (including partial sugar modifications and nucleic acid base modifications) and nucleoside bond modifications. With respect to oligonucleotides, chemical modifications do not involve differences only in the nucleic acid base sequence.

[0030] As used herein, "furanosyl" refers to a structure containing a five-membered ring comprising 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 substitute.

[0034] As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to, furanosyls with 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 having 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 in a 2'-substituted sugar moiety is another origin of the furanosyl ring). (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 with fluorine at the 2' position. Unless otherwise indicated, the fluorine in 2'-F nucleosides is located at the ribo position (substituting the OH group of the natural ribose).

[0038] As used herein, the term “sugar substitute” means a structure that does not contain furanosyl and can substitute the naturally occurring sugar moiety of a nucleoside so that the resulting nucleoside subunits can bond together and / or to other nucleosides to form an oligomeric compound that can hybridize into a complementary oligomeric compound. Such structures include rings with a different number of atoms than furanosyl (e.g., a 4, 6, or 7-membered ring), substitution of the oxygen of furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen), or both a change in the number of atoms and substitution of oxygen. Such structures may also include substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar substitutes optionally containing further substituents). Sugar substitutes also include more complex sugar substitutions (e.g., acyclic systems of peptide nucleic acids). Sugar substitutes include, but are not limited to, morpholinos, cyclohexenyls, and cyclohexitols.

[0039] As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4- to 7-membered ring (including, but not limited to, a furanosyl) that includes a bridge connecting two atoms of a 4- to 7-membered ring to form a second ring, thereby 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 4'-carbon of the furanosyl.

[0040] As used herein, “nucleotide” means a nucleoside that further contains a phosphate-binding group. As used herein, “linked nucleoside” may or may not be linked by a phosphate bond and therefore includes, but is not limited to, “linked nucleotides.” As used herein, “linked nucleoside” is a nucleoside linked in a contiguous sequence (i.e., no further nucleosides are present between the linked sequences).

[0041] As used herein, “nucleic acid base” means a group of atoms capable of forming a nucleoside that can be linked to a sugar moiety and incorporated into an oligonucleotide, which can then be bound to another oligonucleotide or a naturally occurring nucleic acid base that is complementary to the nucleic acid. Nucleic acid bases may be naturally occurring or may be modified.

[0042] As used herein, the terms “unmodified nucleic acid base” or “naturally occurring nucleic acid base” mean naturally occurring heterocyclic nucleic acid bases of RNA or DNA, where purines are based on adenine (A) and guanine (G), and pyrimidines are based on thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

[0043] As used herein, “modified nucleic acid base” means any nucleic acid base that is not naturally occurring.

[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 nucleic acid base.

[0045] As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside containing a bicyclic sugar moiety.

[0046] As used herein, “restricted ethyl nucleoside” or “cEt” means a nucleoside containing a bicyclic sugar moiety with a 4'-CH(CH3)-O-2' bridge.

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

[0048] As used herein, “2'-substituted nucleoside” means a nucleoside having a substituent at the 2' position other than H or OH. Unless otherwise indicated, 2'-substituted nucleosides are not bicyclic nucleosides.

[0049] As used herein, “deoxynucleoside” means a nucleoside containing a 2'-H furanosyl sugar moiety found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleic acid base or an RNA nucleic acid base (e.g., uracil).

[0050] As used herein, “olivian oligonucleotide” means a compound comprising multiple linked nucleosides. In certain embodiments, the 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 nucleoside bonds contain a phosphorus atom. As used herein, oligonucleotide includes oligonucleosides.

[0052] As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified nucleoside bond.

[0053] As used herein, “bond” or “bonding group” means a group of atoms that bond together two or more other groups of atoms.

[0054] As used herein, “nucleoside bond” means a covalent bond between adjacent nucleosides in an oligonucleotide.

[0055] As used herein, “naturally occurring nucleoside bond” means a 3'-to-5' phosphodiester bond.

[0056] As used herein, “modified nucleoside bond” means any nucleoside bond other than naturally occurring nucleoside bonds.

[0057] As used herein, “terminal nucleoside bond” means a bond between the last two nucleosides of an oligonucleotide or its defined region.

[0058] As used herein, "phosphorus bond group" means a bond group containing a phosphorus atom. A phosphorus bond group includes, but is not limited to, groups having the following formulas: [ka] During the ceremony, R a and R d Each of these is independently O, S, CH2, NH, or NJ1, where J1 is a C1-C6 alkyl or substituted C1-C6 alkyl. R b is either O or S, R c These are OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino, or substituted amino. J1 is R b It is either O or S.

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

[0060] As used herein, "internucleoside phosphate bond" means a phosphate bond that directly bonds two nucleosides.

[0061] As used herein, “non-nucleoside phosphorus group” means a phosphorus group that does not directly bond two nucleosides. In certain embodiments, a non-nucleoside phosphorus group bonds a nucleoside to a non-nucleoside group. In certain embodiments, a non-nucleoside phosphorus group bonds two groups, neither of which are nucleosides.

[0062] As used herein, “neutral bond” means an uncharged bond. Neutral bond groups include, but are not limited to, phosphotriesters, methylphosphonates, MMI (-CH2-N(CH3)-O-), amide-3 (-CH2-C(=O)-N(H)-), amide-4 (-CH2-N(H)-C(=O)-), formacetal (-O-CH2-O-), and thioformacetal (-S-CH2-O-). Furthermore, neutral bond groups include nonionic bonds, including siloxanes (dialkylsiloxanes), carboxylic acid esters, carboxamides, sulfides, sulfonic acid esters, and amides (e.g., Carbohydrate Modifications in Antisense Research; YSSanghvi and PDCook). Eds.ACS Symposium Series 580;Chapters 3 See also and 4 (pp. 40-65). Furthermore, the neutral bonding group includes a nonionic bond containing a mixed N, O, S, and CH2 component moiety.

[0063] As used herein, "internucleoside neutral bond" means a neutral bond that directly bonds two nucleosides.

[0064] As used herein, “non-nucleoside-neutral bonding group” means a neutral bonding group that does not directly bond two nucleosides. In certain embodiments, a non-nucleoside-neutral bonding group bonds a nucleoside to a group other than a nucleoside. In certain embodiments, a non-nucleoside-neutral bonding group bonds two groups, neither of which are nucleosides.

[0065] As used herein, “oligomeric compound” means a polymeric structure comprising two or more substructures. In certain embodiments, the oligomeric compound comprises an oligonucleotide. In certain embodiments, the oligomeric compound comprises one or more conjugated groups and / or terminal groups. In certain embodiments, the oligomeric compound consists of an oligonucleotide. The oligomeric compound also comprises naturally occurring nucleic acids. In certain embodiments, the oligomeric compound comprises a skeleton of one or more linked monomeric subunits, each linked monomeric subunit being directly or indirectly bound to a heterocyclic base moiety. In certain embodiments, the oligomeric compound may also comprise monomeric subunits not bound to a heterocyclic base moiety, thereby providing a debasing site. In certain embodiments, the bonds linking the monomeric subunits, sugar moieties or substitutes, and heterocyclic base moieties may be modified independently. In certain embodiments, linked sugar units that may or may not contain a heterocyclic base may be replaced with mimics such as monomers in peptide nucleic acids.

[0066] As used herein, “end group” means one or more atoms bonded to either the 3' or 5' end of an oligonucleotide, or both. In certain embodiments, the end group is a conjugated group. In certain embodiments, the end group comprises one or more end group nucleosides.

[0067] As used herein, “conjugate” or “conjugated group” means an atom or group of atoms that is bound to an oligonucleotide or oligomeric compound. Generally, conjugated groups modify one or more properties of the compound to which they are bound, including but not limited to pharmacodynamic properties, pharmacokinetic properties, binding properties, absorption properties, cell distribution properties, cell uptake properties, charge properties, and / or clearance properties.

[0068] As used herein, “conjugated linker” or “linker” in relation to a conjugated group means a portion of a conjugated group that includes any atom or group of atoms, and which (1) covalently bonds an oligonucleotide to another portion of the conjugated group, or (2) covalently bonds two or more portions of the conjugated group.

[0069] The conjugated group, referred to herein as a radical, provides a bond for forming a covalent bond to an oligomeric compound, such as an antisense oligonucleotide. In certain embodiments, the bond site in the 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 bond site in the 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 a bond to the oligomeric compound is a cleavable bond. In certain such embodiments, such a cleavable bond constitutes all or part of a cleavable portion.

[0070] In certain embodiments, the conjugated group comprises a cleavable moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster moiety such as a GalNAc cluster moiety. Such a carbohydrate cluster moiety comprises a target moiety and, optionally, a conjugated linker. In certain embodiments, the carbohydrate cluster moiety is identified by the number and identity of ligands. For example, in certain embodiments, the carbohydrate cluster moiety comprises three GalNAc groups and is denoted as "GalNAc3". In certain embodiments, the carbohydrate cluster moiety comprises four GalNAc groups and is denoted as "GalNAc4". Specific carbohydrate cluster moieties (having specific tethers, branching, and conjugated linker groups) are described herein by Roman numerals followed by the subscript "a It is written as "GalNac3-1 a " refers to a specific carbohydrate cluster segment having three GalNac groups, as well as a conjugated group having specifically identified tether, branching, and binding groups. Such carbohydrate cluster segments are attached to the oligomeric compound via cleavable bonds or cleavable moieties such as cleavable nucleosides.

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

[0072] As used herein, “cleavable bond” means any chemical bond that can be divided. In certain embodiments, the cleavable bond is selected from amides, polyamides, esters, ethers, phosphodiesters (one or both), phosphate esters, carbamates, disulfides, or peptides.

[0073] As used herein, “carbohydrate cluster” means a compound having one or more carbohydrate residues conjugated to a scaffold or linker group (see, for example, 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 entirety, as examples of carbohydrate-conjugated clusters).

[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 naturally occurring carbohydrates, modified carbohydrates, or carbohydrate derivatives.

[0076] As used herein, “protecting group” means any compound or protecting group known to those skilled in the art. Non-exclusive examples of protecting groups include “Protective Groups in "Organic Chemistry," TW Greene, PGMWuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc., New York, can be found therein and is incorporated herein by reference in its entirety.

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

[0078] As used herein, “double-stranded” means a single self-complementary oligomer compound that forms a pair or hairpin structure of oligomer compounds that hybridize with each other. In certain embodiments, the double-stranded oligomer compound is a first and second oligo Contains Mer compounds.

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

[0080] As used herein, “antisense activity” means 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 the modulation of the quantity or activity of a target nucleic acid transcript (e.g., mRNA). In certain embodiments, antisense activity includes the modulation of premRNA splicing.

[0081] As used herein, “RNase H-based antisense compound” means an antisense compound in which at least a portion of the antisense activity of the antisense compound is due to 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 in which at least a portion of the antisense activity is attributable to the RNA-induced silencing complex (RISC).

[0083] As used herein, “detection” or “measurement” means that a test or assay is performed for detection or measurement. Such detection and / or measurement may yield a value of zero. Therefore, if a test for detection or measurement yields a finding of no activity (zero activity), the step of detecting or measuring activity has still been performed.

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

[0085] As used herein, “essentially invariant” means that a particular parameter changes little or no, especially in comparison to another parameter that changes much more. In a particular embodiment, if a parameter changes by less than 5%, that parameter is essentially invariant. In a particular embodiment, if a parameter changes by less than twofold, that parameter is essentially invariant, while another parameter changes by at least tenfold. For example, in a particular embodiment, antisense activity is a change in the amount of target nucleic acid. In a particular such embodiment, 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 invariant, but the change does not have to be zero.

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

[0087] As used herein, “target nucleic acid” means a nucleic acid molecule that an antisense compound is intended to hybridize with to produce the desired antisense activity. Antisense oligonucleotides have sufficient complementarity to their target nucleic acids to enable hybridization under physiological conditions.

[0088] As used herein, “nucleic acid base complementarity” or “complementarity” means a nucleic acid base that can base-pair with another nucleic acid base. 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, a complementary nucleic acid base means a nucleic acid base of an antisense compound that can base-pair with a nucleic acid base of its target nucleic acid. For example, if a nucleic acid base at a particular position in an antisense compound can hydrogen-bond with a nucleic acid base at a particular position in the target nucleic acid, the positions of the hydrogen bonds between the oligonucleotide and the target nucleic acid are considered complementary in that nucleic acid base pair. A nucleic acid base with certain modifications may retain its ability to pair with the corresponding nucleic acid base and therefore may still have nucleic acid base complementarity.

[0089] As used herein, “non-complementary” with respect to nucleic acid bases means a pair of nucleic acid bases that do not form hydrogen bonds with each other.

[0090] As used herein, “complementary” with respect to an oligomeric compound (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the ability of such an oligomeric compound or region thereof to hybridize to another oligomeric compound or region thereof via nucleic acid base complementarity. Complementary oligomeric compounds do not need to have nucleic acid base complementarity at each nucleoside; rather, some mismatches are acceptable. In a particular embodiment, a complementary oligomeric compound or region is complementary at 70% of the nucleic acid bases (70% complementary). In a particular embodiment, a complementary oligomeric compound or region is 80% complementary. In a particular embodiment, a complementary oligomeric compound or region is 90% complementary. In a particular embodiment, a complementary oligomeric compound or region is 95% complementary. In a particular embodiment, a complementary oligomeric compound or region is 100% complementary.

[0091] As used herein, “mismatch” means a nucleic acid base of the first oligomer compound that cannot pair with the nucleic acid base of the second oligomer compound at the corresponding position when the first and second oligomer compounds are aligned. Either or both of the first and second oligomer compounds may be oligonucleotides.

[0092] As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a specific mechanism, the most common pairing mechanisms include hydrogen bonding, which may be Watson-Crick hydrogen bonds, Hoogsteen hydrogen bonds, or reverse Hoogsteen hydrogen bonds between complementary nucleic acid bases.

[0093] As used herein, "specifically hybridizes" means the ability of an oligomeric compound to hybridize to one nucleic acid site with a higher affinity than it would to hybridize to one nucleic acid site.

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

[0095] As used herein, "percent complementarity" refers to the proportion of nucleic acid bases in the oligomer compound that are complementary to the isolength portions of the target nucleic acid. Percent complementarity is the number of nucleic acid bases in the oligomer compound that are complementary to the nucleic acid bases at the corresponding positions of the target nucleic acid. It is calculated by dividing by the length.

[0096] As used herein, “percent identity” means the number of nucleic acid bases of the first nucleic acid that are identical (independent of chemical modifications) to the nucleic acid bases at the corresponding positions of the second nucleic acid, divided by the total number of nucleic acid bases of the first nucleic acid.

[0097] As used herein, “regulation” means 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 regulation. For example, regulation includes any change in gene expression that is either increased (stimulated or induced) or decreased (inhibited or reduced). As a further example, regulation of expression may include a change in splice site selection of premRNA treatment that results in a change in the absolute or relative quantity of a particular splice variant compared to the quantity in the absence of regulation. As used herein, “chemical motif” means a pattern of chemical modifications on an oligonucleotide or its region. A motif may be defined by modifications on a particular nucleoside and / or a particular binding group of an oligonucleotide.

[0098] As used herein, “nucleoside motif” means an oligonucleotide or a pattern of nucleoside modification within its region. Such oligonucleotide binding may be modified or unmodified. Unless otherwise indicated, motifs describing only nucleosides herein are intended to be nucleoside motifs. Therefore, in such cases, binding is not limited.

[0099] As used herein, “sugar motif” means a pattern of sugar modification in an oligonucleotide or its region.

[0100] As used herein, “binding motif” means a pattern of binding modification in an oligonucleotide or its region. The nucleoside of such oligonucleotides may be modified or unmodified. Unless otherwise indicated herein, motifs described solely by their binding are intended to be binding motifs. Thus, in such cases, the nucleoside is not limited.

[0101] As used herein, “nucleic acid base modification motif” means a pattern of modification of nucleic acid bases along an oligonucleotide. Unless otherwise indicated, nucleic acid base modification motifs are independent of the nucleic acid sequence.

[0102] As used herein, “sequence motif” means a pattern of nucleic acid bases aligned along an oligonucleotide or a portion thereof. Unless otherwise indicated, sequence motifs are independent of chemical modifications and therefore may have any combination of chemical modifications, including no chemical modifications.

[0103] As used herein, "type of modification" with respect to a nucleoside or a certain "type" of nucleoside means chemical modification of a nucleoside, and includes modified and unmodified nucleosides. Therefore, unless otherwise indicated, "nucleoside having the first type of modification" may be an unmodified nucleoside.

[0104] As used herein, “separately modified” means different chemical modifications or chemical substituents, including the absence of modification. Therefore, for example, a MOE nucleoside and an unmodified DNA nucleoside are “separately modified,” even if the DNA nucleoside is not modified. Similarly, DNA and RNA are “separately modified,” even if both are naturally occurring unmodified nucleosides. Nucleosides containing identical but different nucleic acid bases… Creosides are not modified separately. For example, a nucleoside containing a 2'-OMe modified sugar and an unmodified adenine nucleic acid base is not modified separately from a nucleoside containing a 2'-OMe modified sugar and an unmodified thymine nucleic acid base.

[0105] As used herein, “identical type of modification” refers to modifications that are identical to each other, including the absence of modification. Therefore, for example, two unmodified DNA nucleosides have “identical type of modification” even if the DNA nucleosides are not modified. Such nucleosides having identical type of modification may contain different nucleic acid bases.

[0106] As used herein, “separate regions” means a portion of an oligonucleotide, and any chemical modification or motif of chemical modification of any adjacent portion includes at least one difference that makes the separate regions distinguishable from one another.

[0107] As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administration to animals. 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 abnormal regulation of metabolism (a series of complex chemical reactions related to the breakdown of food into 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, wherein the rings are condensed or linked and include single-ring and mixed-ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, and heteroarylalkyl. Such monocyclic and polycyclic structures may each have the same level of saturation, or each independently may contain rings with 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 produce heterocyclic rings and rings containing only C ring atoms, which may be present in mixed motifs such as benzimidazole, for example; one ring may have only carbon ring atoms, and a fused ring may have two nitrogen atoms. Monocyclic or polycyclic ring systems can be further substituted with substituents, such as phthalimides having two =O groups bonded to one of the rings. Monocyclic or polycyclic ring systems can be bonded to the parent molecule using various strategies, including direct bonding via ring atoms, condensation via multiple ring atoms, bonding via substituents, or bonding via bifunctional bonding moieties.

[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 an active compound or a more active compound (e.g., a drug).

[0112] As used herein, “substituent” and “substituent group” mean an atom or group that substitutes for an atom or group of a designated parent compound. For example, a substituent of a modified nucleoside is any atom or group different from the atom or group found in a naturally occurring nucleoside (for example, 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... Furthermore, the compounds of the present disclosure have substituents at one position or two or more positions of the parent compound. The substituents can be further substituted with other substituents and can be directly bonded to the parent compound or can be bonded via a linking group such as an alkyl group or a hydrocarbyl group.

[0113] Similarly, as used herein, "substituent" with respect to a chemical functional group means an atom or group of atoms different from the atoms or groups of atoms normally present in the designated functional group. In certain embodiments, the 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 compliant for use as substituents include halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)R- aa ), carboxyl (-C(O)O-R -aa ), aliphatic group, alicyclic group, alkoxy, substituted oxy (-O-R -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)2R bb ), and sulfonamidyl (-S(O)2N(R bb )(R cc ) or -N(R bb )S-(O)2R bb This includes, but is not limited to, ) in the formula, each R aa , R bb , and R cc These are independently H, optionally linked chemical functional groups, or further substituents having a preferred list including, but not limited to, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic, and heteroarylalkyl groups. Selected substituents in the compounds described herein are present to a recursive degree.

[0114] As used herein, “alkyl” means a saturated linear or branched 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, and dodecyl. Alkyl groups typically contain 1 to about 24 carbon atoms, and more typically 1 to about 12 carbon atoms (C1-C24). 12 It contains alkyl groups, and more preferably has 1 to about 6 carbon atoms.

[0115] As used herein, “alkenyl” means a linear 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, and dienes such as 1,3-butadiene. Alkenyl groups typically contain 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, and more preferably 2 to about 6 carbon atoms. Alkenyl groups as used herein may optionally contain one or more further substituents.

[0116] As used herein, “alkynyl” means a linear or branched 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, and 1-butynyl. An alkynyl group typically contains 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, and more preferably 2 to about 6 carbon atoms. As used herein, an alkynyl group may optionally contain one or more further substituents. .

[0117] As used herein, "acyl" means a radical formed by the removal of a hydroxyl group from an organic acid, having the general formula -C(O)-X, where X is typically aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, and the like. The acyl groups used herein may optionally contain further substituents.

[0118] As used herein, “alicyclic” means a cyclic ring system, wherein the ring is aliphatic. The ring system may contain one or more rings, at least one of which is aliphatic. Preferred alicyclic groups contain rings having about 5 to about 9 carbon atoms. Alicyclic groups as used herein may optionally contain further substituents.

[0119] As used herein, “aliphatic” means a linear or branched hydrocarbon radical containing up to 24 carbon atoms, wherein the saturation between any two carbon atoms is a single, double, or triple bond. Aliphatic groups preferably contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms, and more preferably 1 to about 6 carbon atoms. The linear or branched chain of an aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Such aliphatic groups interrupted by heteroatoms include, but are not limited to, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally contain further substituents.

[0120] As used herein, "alkoxy" means a radical formed between an alkyl group and an oxygen atom, and the oxygen atom is used to bond 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, and n-hexoxy. The alkoxy groups used herein may optionally contain further substituents.

[0121] As used herein, "aminoalkyl" refers to amino-substituted C1-C1 12 This refers to an alkyl radical. The alkyl portion of the radical forms a covalent bond with 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 portion.

[0122] As used herein, "aralkyl" and "arylalkyl" refer to C1-C12. 12This 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 and phenethyl. The aralkyl groups used herein may optionally include further substituents bonded to the alkyl, aryl, or both groups that form the radical group.

[0123] As used herein, "aryl" and "aromatic" mean monocyclic or polycyclic carbocyclic radicals having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and idenyl. Preferred aryl ring systems have about 5 to about 20 carbon atoms in one or more rings. The aryl groups used herein may optionally contain further substituents.

[0124] As used herein, “halo” and “halogen” mean an atom selected from fluorine, chlorine, bromine, and iodine.

[0125] As used herein, “heteroaryl” and “heteroaromatic” mean radicals comprising monocyclic or polycyclic aromatic rings, ring systems, or fused ring systems, wherein at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryls are also intended to include fused ring systems in which one or more of the fused rings do not contain heteroatoms. 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, etc. Heteroaryl radicals may be directly bonded to the parent molecule or bonded via a bonding moiety such as an aliphatic group or a heteroatom. The heteroaryl groups used herein may optionally contain further substituents.

[0126] As used herein, “conjugated compound” means any atom, group of atoms, or bonded group of atoms suitable for use as a conjugated group. In certain embodiments, a conjugated compound may have or be conferred one or more properties, including, but not limited to, pharmacodynamic properties, pharmacokinetic properties, binding properties, absorption properties, cell distribution properties, cell uptake properties, charge properties, and / or clearance properties.

[0127] As used herein, unless otherwise indicated or modified, the term “double-stranded” refers to two distinct oligomeric compounds that hybridize with one another. Such double-stranded compounds may have one or more nucleosides or unhybridized nucleosides at one or both ends of one or both of their chains (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 a conjugated antisense compound comprising an antisense oligonucleotide and a conjugate.

[0129] a. A certain antisense oligonucleotide In certain embodiments, the present invention provides an antisense oligonucleotide. Such antisense oligonucleotides comprise linked nucleosides, each nucleoside comprising a sugar moiety and a nucleobase. The structure of such antisense oligonucleotides can be considered in terms of chemical features (e.g., modifications and modification patterns) as well as nucleobase sequences (e.g., the sequence of the antisense oligonucleotide, identity, and the sequence of the target nucleic acid).

[0130] i. A specific chemical characteristic In certain embodiments, the antisense oligonucleotide comprises one or more modifications. In certain such embodiments, the antisense oligonucleotide comprises one or more modified nucleosides and / or modified internucleoside linkages. In certain embodiments, the modified nucleoside comprises a modified sugar moiety and / or a modified nucleobase.

[0131] 1. A specific sugar portion In certain embodiments, the compounds of the present disclosure comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides can have desirable properties such as enhanced nuclease stability or increased binding affinity to a target nucleic acid compared to oligonucleotides comprising only nucleosides having 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 can comprise one or more substitutions corresponding to the substitution of the substituted sugar moiety.

[0132] In certain embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-crosslinked sugar substituents, 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'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position may be allyl, amino, azide, thio, O-allyl, or O-C1~C 10 Alkyl, O-C1~C 10 Selected from substituted alkyl groups, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C1-C 10 It is 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-crosslinked sugar substituents, e.g., a 2'-F-5'-methyl sugar moiety (for further 5',2'-bis-substituted sugar moieties and nucleosides, see, for example, PCT International Application WO2008 / 101157).

[0133] Nucleosides containing a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In certain embodiments, 2'-substituted nucleosides are halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O, S, or N(R) m )-alkyl; O, S, or N(R m )-alkenyl; O, S, or N(R m )-Alkynyl;O-Alkyrenyl-O-Alkyl,Alkynyl,Aralkyl,Aralkyl,O-Alkalyl,O-Aralkyl,O(CH2)2SCH3,O-(CH2)2-ON(R m )(R n ); or O-CH2-C(=O)-N(R m )(R n The formula includes a 2'-substituted selected from ), where each R m and Rn These are independently H, an amino protecting group, or substituted or unsubstituted C1-C1. 10 These are alkyl groups. These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl groups.

[0134] In certain embodiments, the 2'-substituted nucleoside is F, NH2, N3, OCF3, O-CH3, O(CH2)3NH2, CH2-CH=CH2, O-CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O-CH2-C(=O)-N(R m )(R n The formula includes a 2'-substituted selected from ), where each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C1. 10 It is alkyl.

[0135] In certain embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituent selected from F, OCF3, O-CH3, OCH2CH2OCH3, O(CH2)2SCH3, O-(CH2)2-ON(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2-C(=O)-N(H)CH3.

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

[0137] Certain modified sugar moieties include bridging sugar substituents that form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4'-furanose ring atom and the 2'-furanose ring atom. 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(CH2OCH3)-O-2';and their analogues (see, for example, U.S. Patent No. 7,399,845 issued July 15, 2008);4'-C(CH3)(CH3)-O-2' and its analogues (see, for example, 2009 See International Publication No. WO2009 / 006478, published on January 8); 4'-CH2-N(OCH3)-2' and its analogues (see, for example, International Publication No. WO2008 / 150729, published on December 11, 2008); 4'-CH2-ON(CH3)-2' (see, for example, US2004 / 0171570, published on 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 C1~C 12 It is alkyl); 4'-CH2-N(R)-O-2' (wherein R is H, C1~C) 12(which is an alkyl or a protecting group) (see U.S. Patent No. 7,427,672, issued September 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 its analogs (see PCT International Application No. WO2008 / 154401, published December 8, 2008), but are not limited thereto.

[0138] In certain embodiments, such a 4’-to-2’ bridge is 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 includes 1 to 4 linking groups independently selected from, wherein, x is 0, 1, or 2, n is 1, 2, 3, or 4, each R a and R b is 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 alicyclic radical, substituted C5-C7 alicyclic 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~C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12 Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution C5~C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1~C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl group or a protecting group.

[0139] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Examples of bicyclic nucleosides include (A) α-L-methyleneoxy(4'-CH2-O-2')BNA, (B) β-D-methyleneoxy(4'-CH2-O-2')BNA (also called locked nucleic acid or LNA), and (C) ethyleneoxy(4'-(CH2) This includes, but is not limited to, (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 called restricted ethyl or cEt), (G) methylene-thio(4'-CH2-S-2')BNA, (H) methylene-amino(4'-CH2-N(R)-2')BNA, (I) methyl carboncyclic(4'-CH2-CH(CH3)-2')BNA, and (J) propylene carboncyclic(4'-(CH2)3-2')BNA. [ka] [ka] In the formula, Bx is the nucleic acid base portion, and R is independently H, a protecting group, or C1-C 12 It is alkyl.

[0140] Further bicyclic sugar moieties are known in the art, e.g., 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., and WO2007 / 134181, US Patent Publication No. US2004 / 0171570, No. US2007 / 0287831, and No. US2008 / 0039618, US Patent No. 12 / 129,154, No. 60 / 989,574, No. 61 / 026,995, No. 61 / 026,998, No. 61 / 056,564, No. 61 / 086,231, No. 61 / 097,787, and No. 61 / 099,844, as well as PCT International Application Nos. PCT / US2008 / 064591, No. PCT / US2008 / 066154, and No. PCT / US2008 / 068922.

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

[0142] In certain embodiments, substituted sugar moieties include one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., a 5'-substitution and a 4'-2' bridged sugar) (see PCT International Application No. WO2007 / 134181, published November 22, 2007, where LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0143] In certain embodiments, the modified sugar moiety is a sugar substitute. In certain such embodiments, the oxygen atom of a naturally occurring sugar is substituted with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such a modified sugar moiety also includes the aforementioned crosslinking and / or non-crosslinking substituents. For example, certain sugar substitutes include substitutions at the 4'-sulfur atom and at the 2' position (see, for example, U.S. Patent Application No. US2005 / 0130923, published June 16, 2005) and / or at the 5' position. Further examples include carbocyclic bicyclic nucleosides having a 4'-2' bridge (see, for example, 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 substitute comprises a ring having more than five atoms. For example, in certain embodiments, the sugar substitute comprises a morpholino. Their use in morpholino compounds and oligomeric compounds has been reported in numerous patents and publications (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510, and U.S. Patents 5,698,685, 5,166,315, 5,185,444, and 5,034,506). As used herein, the term "morpholino" means a sugar substitute having the following structure: [ka]

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

[0146] As another example, in certain embodiments, the sugar substitute 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), fluoroHNA (F-HNA), and compounds having the following formula VI. [ka] In the formula, independently, for each of the at least one tetrahydropyrannucleoside analogs of formula VI, Bx is the nucleic acid base portion, T3 and T4 are, independently, internucleoside bonding groups that bond a tetrahydropyran nucleoside analog to an antisense compound, or one of T3 and T4 is an internucleoside bonding group that bonds a tetrahydropyran nucleoside analog to an antisense compound, and the other of T3 and T4 is H, a hydroxyl protecting group, a conjugated 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 of J1, J2, and J3 is independently H or C1-C6 alkyl.

[0147] In certain embodiments, a modified THP nucleoside of formula VI is provided, where 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 not H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, a THP nucleoside of formula VI is provided, where one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.

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

[0149] 2'-F-5'-methyl-substituted nucleosides (for other disclosed 5',2'-bis-substituted nucleosides, see PCT International Application WO2008 / 101157, published August 21, 2008), as well as substitution of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see US Patent Application US2005-, published June 16, 2005). Combinations of modifications are also provided, including, but are not limited to, those such as, or 5'-substitution of bicyclic nucleic acids (a 4'-CH2-O-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or 5'-vinyl group; see PCT international application WO2007 / 134181, published November 22, 2007). In addition to oligomerization and biochemical studies of carbocyclic and bicyclic nucleosides, their synthesis and preparation are also described (e.g., Srivastava et al., J.Am.Chem.Soc.2007, 129(26), 8362-8379).

[0150] In certain embodiments, the disclosure provides oligonucleotides comprising modified nucleosides. These modified nucleotides may comprise modified sugars, modified nucleic acid bases, and / or modified bonds. Specific modifications are selected such that the resulting oligonucleotide has desirable characteristics. In certain embodiments, the oligonucleotide comprises one or more RNA-like nucleosides. In certain embodiments, the oligonucleotide comprises one or more DNA-like nucleotides.

[0151] 2. A certain nucleic acid base modification In certain embodiments, the nucleoside of the Disclosure comprises one or more unmodified nucleic acid bases. In certain embodiments, the nucleoside of the Disclosure comprises one or more modified nucleic acid bases.

[0152] In certain embodiments, modified nucleic acid bases are selected from universal bases, hydrophobic bases, eclectic bases, size-enlarged bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines include, as defined herein, 2-aminopropyl adenine, 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, and 6-azo These include uracil, cytosine and thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, in particular 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, chaotic bases, size-expanded bases, and fluorinated bases. Furthermore, modified nucleic acid bases include tricyclic pyrimidines, such as phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), G-clamps, such as substituted phenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one).Modified nucleic acid bases may also include bases in which a purine or pyrimidine base is substituted with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Furthermore, nucleic acid bases are 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. This includes the bases disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and the bases disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, STand Lebleu, B., Eds., CRC Press, 1993, 273-288.

[0153] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and certain preparations of other modified nucleic acid bases include U.S. Patents No. 3,687,808, No. 4,845,205, No. 5,130,302, No. 5,134,066, No. 5,175,273, No. 5,367,066, No. 5,432,272, No. 5,457,187, No. 5,459,255, No. 5,484,908, No. 5,502,177, No. 5,525,711, and No. 5,552 This includes, but is not limited to, patents No. 540, 5587,469, 5594,121, 5596,091, 5614,617, 5645,985, 5681,941, 5750,692, 5763,588, 5830,653, and 6005,096, some of which are co-owned with this application, and each of them is incorporated herein by reference in whole.

[0154] 3. A specific nucleoside bond In certain embodiments, the disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides may be linked together using any internucleoside bond. Two main classes of internucleoside bonds are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiesters (PO), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (PS). Representative non-phosphorus-containing internucleoside bonds include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-), siloxanes (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified bonds can typically be used to alter, or increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester bonds. In certain embodiments, nucleoside bonds containing chiral atoms can be prepared as racemic mixtures or as distinct enantiomers. Typical chiral bonds include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing nucleoside bonds are well known to those skilled in the art.

[0155] The oligonucleotides described herein contain one or more chiral centers, and therefore, in terms of absolute stereochemistry, (R) or (S), in the case of sugar anomers, a or β, Or, in the case of amino acids, etc., they give rise to enantiomers, diastereomers, and other stereoisomer configurations 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 nucleoside interbonding includes, but is not limited to, phosphotriesters, methylphosphonates, 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'). Furthermore, neutral nucleoside interbonding includes siloxanes (dialkylsiloxanes), carboxylic acid esters, carboxamides, Nonionic bonds include sulfides, sulfonic acid esters, and amides (see, for example, *Carbohydrate Modifications in Antisense Research*; YSSanghvi and PDCook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Furthermore, neutral nucleoside bonds include nonionic bonds containing mixed N, O, S, and CH2 component moieties.

[0157] 4. A specific motif In certain embodiments, the antisense oligonucleotide comprises one or more modified nucleosides (e.g., nucleosides containing modified sugars and / or modified nucleic acid bases) and / or one or more intermodified nucleoside bonds. Such a pattern of modifications in the oligonucleotide is referred to herein as a motif. In certain embodiments, the sugar, nucleic acid base, and binding motif are independent of each other.

[0158] a. A certain sugar motif In certain embodiments, the oligonucleotide comprises one or more modified sugar moieties and / or naturally occurring sugar moieties arranged along the oligonucleotide or its region in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and / or other known sugar modifications.

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

[0160] i. A specific 5'-wing In a particular embodiment, the 5'-wing of the gapmer consists of 1 to 8 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 1 to 7 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 1 to 6 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 1 to 5 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 2 to 5 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 3 to 5 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 4 or 5 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 1 to 4 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of 1 to 3 linked nucleosides. In a particular embodiment, the 5'-wing of the gapmer consists of one or two linked nucleosides. In a particular embodiment, The 5'-wing of a gapmer consists of 2 to 4 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 2 or 3 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 3 or 4 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 1 nucleoside. In a particular embodiment, the 5'-wing of a gapmer consists of 2 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 3 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 4 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 5 linked nucleosides. In a particular embodiment, the 5'-wing of a gapmer consists of 6 linked nucleosides.

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

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

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

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

[0166] ii. A particular 3'-wing In a particular embodiment, the 3'-wing of the gapmer consists of 1 to 8 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 1 to 7 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 1 to 6 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 1 to 5 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 2 to 5 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 3 to 5 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 4 or 5 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 1 to 4 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of 1 to 3 linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of one or two linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of two to four linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of two or three linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of three or four linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of one nucleoside. In a particular embodiment, the 3'-wing of the gapmer consists of two linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of three linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of four linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of five linked nucleosides. In a particular embodiment, the 3'-wing of the gapmer consists of six linked nucleosides.

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

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

[0169] In certain embodiments, the 3'-wing of the gapmer contains at least one 2'-deoxynucleoside. In certain embodiments, each nucleoside of the 3'-wing of the gapmer is a 2'-deoxynucleoside. In certain embodiments, the 3'-wing of the gapmer contains at least one ribonucleoside. In certain embodiments, each nucleoside of the 3'-wing of the gapmer is a ribonucleoside. In certain embodiments, one, two or more, or each of the nucleosides of 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 restricted ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one restricted ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one restricted ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one restricted ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one restricted ethyl nucleoside and at least one 2'-deoxynucleoside.

[0172] In a particular embodiment, the 3'-wing of the gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In some 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 the 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 a particular embodiment, the gap of the gapmer consists of 6 to 20 linked nucleosides. In a particular embodiment, the gap of the gapmer consists of 6 to 15 linked nucleosides. In a particular embodiment, the gap of the gapmer consists of 6 to 12 linked nucleosides. In a particular embodiment, The gap of the gapmer consists of 6 to 10 linked nucleosides. In a particular embodiment, the gapmer consists of 6 to 9 linked nucleosides. In a particular embodiment, the gapmer consists of 6 to 8 linked nucleosides. In a particular embodiment, the gapmer consists of 6 or 7 linked nucleosides. In a particular embodiment, the gapmer consists of 7 to 10 linked nucleosides. In a particular embodiment, the gapmer consists of 7 to 9 linked nucleosides. In a particular embodiment, the gapmer consists of 7 or 8 linked nucleosides. In a particular embodiment, the gapmer consists of 8 to 10 linked nucleosides. In a particular embodiment, the gapmer consists of 8 or 9 linked nucleosides. In a particular embodiment, the gapmer consists of 6 linked nucleosides. In a particular embodiment, the gap of the gapmer consists of seven linked nucleosides. In a particular embodiment, the gap of the gapmer consists of eight linked nucleosides. In a particular embodiment, the gap of the gapmer consists of nine linked nucleosides. In a particular embodiment, the gap of the gapmer consists of ten linked nucleosides. In a particular embodiment, the gap of the gapmer consists of eleven linked nucleosides. In a particular embodiment, the gap of the gapmer consists of twelve linked nucleosides.

[0178] In certain embodiments, each nucleoside in the gap of the gapmer is a 2'-deoxynucleoside. In certain embodiments, the gap contains one or more modified nucleosides. In certain embodiments, each nucleoside in the gap of the gapmer is either a 2'-deoxynucleoside or a “DNA-like” modified nucleoside. In such embodiments, “DNA-like” means that the nucleoside has DNA-like characteristics so that the double strand containing the gapmer and RNA molecule can activate RNase H. For example, under certain conditions, 2'-(ara)-F has been shown to assist in the activation of RNase H and is therefore DNA-like. In certain embodiments, one or more nucleosides in the gap of the gapmer are neither 2'-deoxynucleosides nor DNA-like. In certain such embodiments, the gapmer still assists in the activation of RNase H (e.g., thanks to the number or placement of non-DNA nucleosides).

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

[0180] In certain embodiments, the gap contains one or more modified nucleosides. In certain embodiments, the gap contains one or more modified nucleosides selected from cEt, FHNA, LNA, and 2-thiothymidine. In certain embodiments, the gap contains one modified nucleoside. In certain embodiments, the gap contains a 5'-substituted sugar moiety selected from 5'-Me and 5'-(R)-Me. In certain embodiments, the gap contains two modified nucleosides. In certain embodiments, the gap contains three modified nucleosides. In certain embodiments, the gap contains four modified nucleosides. In certain embodiments The gap contains two or more modified nucleosides, each of which is identical. In a particular embodiment, the gap contains two or more modified nucleosides, each of which is different.

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

[0182] b. A specific nucleoside bond motif In certain embodiments, the oligonucleotide includes modified nucleoside bonds arranged along the oligonucleotide or a region thereof in a defined pattern or modified nucleoside bond motif. In certain embodiments, the oligonucleotide includes a region having alternating nucleoside bond motifs. In certain embodiments, the oligonucleotide of the Disclosure includes a region of uniformly modified nucleoside bonds. In certain such embodiments, the oligonucleotide includes a region uniformly linked by phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate nucleoside bonds. In certain embodiments, each nucleoside bond of the oligonucleotide is selected from phosphodiesters and phosphorothioates. In certain embodiments, each nucleoside bond of the oligonucleotide is selected from phosphodiesters and phosphorothioates, and at least one nucleoside bond is a phosphorothioate.

[0183] In certain embodiments, the oligonucleotide contains at least 6 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 7 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 8 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 9 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 10 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 11 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 12 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 13 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains at least 14 phosphorothioate nucleoside bonds.

[0184] In certain embodiments, the oligonucleotide comprises at least one block of at least six consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least seven consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least eight consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least nine consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one block of at least ten consecutive phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide comprises at least one 12 consecutive It includes at least one block of consecutive phosphorothioate nucleoside bonds. 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 at the 3' end of the oligonucleotide. In certain embodiments, the oligonucleotide contains fewer than 15 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 14 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 13 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 12 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 11 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 10 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 9 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 8 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 7 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 6 phosphorothioate nucleoside bonds. In certain embodiments, the oligonucleotide contains fewer than 5 phosphorothioate nucleoside bonds.

[0185] c. A specific nucleic acid base modification motif In certain embodiments, the oligonucleotide includes chemical modifications to nucleic acid bases arranged along the oligonucleotide or its region in a defined pattern or nucleic acid base modification motif. In certain such embodiments, the nucleic acid base modifications are arranged in gap motifs. In certain embodiments, the nucleic acid base modifications are arranged in alternating motifs. In certain embodiments, each nucleic acid base is modified. In certain embodiments, none of the nucleic acid bases are chemically modified.

[0186] In certain embodiments, the oligonucleotide contains a block of modified nucleic acid bases. In certain such embodiments, the block is located at the 3' end of the oligonucleotide. In certain embodiments, the block is located within the three nucleotides at 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 the three nucleotides at the 5' end of the oligonucleotide.

[0187] In certain embodiments, nucleic acid base modification is the function of a native base at a specific position on an oligonucleotide. For example, in certain embodiments, each purine or pyrimidine in the 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 the oligonucleotide are 5-methylcytosine moieties, or none are 5-methylcytosine moieties. In this specification, 5-methylcytosine is not a “modified nucleic acid base.” Therefore, unless otherwise indicated, unmodified nucleic acid bases include both cytosine residues with 5-methyl and cytosine residues lacking 5-methyl. In certain embodiments, the methylation state of all or some of the cytosine nucleic acid bases is specified.

[0189] In a particular embodiment, chemical modification of a nucleic acid base is performed on a particular conjugated group of nucleic acid. This includes binding to a base. In certain embodiments, each purine or pyrimidine in the oligonucleotide can be optionally modified to include a conjugated group.

[0190] d. A specific total length In certain embodiments, the disclosure provides oligonucleotides of a range 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 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 a particular embodiment, oligonucleotides are 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, 10-2 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 10 linked nucleosides. In embodiments where the number of nucleosides in the oligonucleotide of a compound is limited, regardless of a range or a specific number, the compound may still further contain 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 contain, for example, one or more conjugated groups, terminal groups, or other substituents.

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

[0192] 5. Chemical motifs of certain antisense oligonucleotides In certain embodiments, the chemical structural features of antisense oligonucleotides are characterized by their sugar motifs, nucleoside-linking motifs, nucleic acid base modification motifs, and overall length. In certain embodiments, each of these parameters is independent of the others. Thus, each nucleoside-link in an oligonucleotide having a gapmer sugar motif may be modified or unmodified, and may or may not follow the gapmer modification pattern of the sugar modification. Consequently, the nucleoside-links within the wing region of a sugar gapmer may be identical or different from those within the gap region. Similarly, such a sugar gapmer oligonucleotide may contain one or more modified nucleic acid bases independently of the gapmer pattern of the sugar modification. Those skilled in the art will recognize that such motifs can be combined to create a variety of oligonucleotides.

[0193] In certain embodiments, the selection of nucleoside-to-nucleoside bonding and nucleoside modification are not independent of each other.

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

[0195] In certain embodiments, the disclosure provides an antisense compound comprising an oligonucleotide that is completely complementary to a target nucleic acid over its entire length. In certain embodiments, the oligonucleotide is 99% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is 95% complementary to the target nucleic acid. In certain embodiments, such an oligonucleotide is 90% complementary to the 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 compound is fully complementary to the target nucleic acid and includes a region that is at least 80% complementary to the target nucleic acid throughout the entire length of the oligonucleotide. In certain such embodiments, the fully complementary region is 6 to 14 nucleic acid bases long.

[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 1 to 4 terminal nucleosides. In certain embodiments, the terminal nucleosides are 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 oligonucleotides with a target nucleic acid, where this hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupation, for example, by simultaneous inhibition or stimulation of cellular mechanisms involving translation, transcription, or splicing of the target nucleic acid.

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

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

[0201] ii. A certain detachable portion In certain embodiments, the cleavable portion is a cleavable bond. In certain embodiments, the cleavable portion includes a cleavable bond. In certain embodiments, the conjugated group includes a cleavable portion. In certain such embodiments, the cleavable portion binds to an antisense oligonucleotide. In certain such embodiments, the cleavable portion binds directly to a cellular target site. In certain such embodiments, the cleavable portion binds to a conjugated linker. In certain embodiments, the cleavable portion includes a phosphate or phosphodiester. In certain embodiments, the cleavable portion is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog is pre- The cleavable portion comprises a substituted purine, a pyrimidine, or an optionally protected heterocyclic base selected from substituted pyrimidines. In certain embodiments, the cleavable portion 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 portion is a 2'-deoxynucleoside bonded 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 portion is 2'-deoxyadenosine bonded to the 3' position of the antisense oligonucleotide by a phosphodiester bond and to the linker by a phosphodiester or phosphorothioate bond. In a particular embodiment, the cleavable portion is 2'-deoxyadenosine, which is bonded 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 portion is bonded to the 3' position of the antisense oligonucleotide. In certain embodiments, the cleavable portion is bonded to the 5' position of the antisense oligonucleotide. In certain embodiments, the cleavable portion is bonded to the 2' position of the antisense oligonucleotide. In certain embodiments, the cleavable portion is bonded to the antisense oligonucleotide by a phosphodiester bond. In certain embodiments, the cleavable portion is bonded to the linker by either a phosphodiester bond or a phosphorothioate bond. In certain embodiments, the cleavable portion is bonded to the linker by a phosphodiester bond. In certain embodiments, the conjugated group does not contain a cleavable portion.

[0203] In certain embodiments, the cleavable portion is cleaved only after the complex is administered to an animal, after it has been taken up by the target cell. The cleavable portion is cleaved within the cell, thereby releasing an active antisense oligonucleotide. Although we do not wish to be bound by theory, it is conceivable that the cleavable portion is cleaved within the cell by one or more nucleases. In certain embodiments, one or more nucleases cleave the phosphodiester bond between the cleavable portion and the linker. In certain embodiments, the cleavable portion has a structure selected from the following: [ka] In the formula, each of Bx, Bx1, Bx2, and Bx3 is independently a heterocyclic base moiety. In a particular embodiment, the cleavable moiety has a structure selected from the following: [ka]

[0204] iii. A specific linker In certain embodiments, the conjugated group includes a linker. In certain such embodiments, the linker is covalently bonded to a cleavable portion. In certain such embodiments, the linker is covalently bonded to an antisense oligonucleotide. In certain embodiments, the linker is covalently bonded to a cellular target portion. In certain embodiments, the linker further includes a covalent bond to a solid support. In certain embodiments, the linker further includes a covalent bond to a protein binding portion. In certain embodiments, the linker further includes a covalent bond to a solid support and also further includes a covalent bond to a protein binding portion. In certain embodiments, the linker includes multiple positions for tether ligand binding. In certain embodiments, the linker includes multiple positions for tether ligand binding and is not bonded to a branched group. In certain embodiments, the linker further includes one or more cleavable bonds. In certain embodiments, the conjugated group does not include a linker.

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

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

[0207] In certain embodiments, the linker includes a protein-binding moiety. In certain embodiments, the protein-binding moiety may be, for example, cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or Lipids include, but are not limited to, phenoxazine, vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomal lysants, steroids (e.g., ubaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-derived lithocholic acid), or 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 a particular embodiment, the linker has a structure selected from the following: [ka] In the formula, each n is independently between 1 and 20, and p is between 1 and 6.

[0209] In a particular embodiment, the linker has a structure selected from the following: [ka] In the formula, each n is independently between 1 and 20.

[0210] In a particular embodiment, the linker has a structure selected from the following: [ka] In the formula, n is between 1 and 20.

[0211] In a particular embodiment, the linker has a structure selected from the following: [ka] In the formula, each L is independently either a phosphorus-bonding group or a neutral-bonding group. Each n is independently between 1 and 20.

[0212] In a particular embodiment, the linker has a structure selected from the following: [ka] [ka]

[0213] In a particular embodiment, the linker has a structure selected from the following: [ka]

[0214] In a particular embodiment, the linker has a structure selected from the following: [ka]

[0215] In a particular embodiment, the linker has a structure selected from the following: [ka] In the formula, n is between 1 and 20.

[0216] In a particular embodiment, the linker has a structure selected from the following: [ka]

[0217] In a particular embodiment, the linker has a structure selected from the following: [ka]

[0218] In a particular embodiment, the linker has a structure selected from the following: [ka]

[0219] In a particular embodiment, the conjugated linker has the following structure: [ka]

[0220] In a particular embodiment, the conjugated linker has the following structure: [ka]

[0221] In a particular embodiment, the linker has a structure selected from the following: [ka]

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

[0223] iv. A specific cell target region In certain embodiments, the conjugated group comprises a cell-targeting moiety. In certain embodiments, such a cell-targeting moiety increases the cellular uptake of the antisense compound. In certain embodiments, the cell-targeting moiety comprises a branched group, one or more tethers, and one or more ligands. In certain embodiments, the cell-targeting moiety comprises a branched group, one or more tethers, one or more ligands, and one or more cleavable bonds.

[0224] 1. A specific branching point In certain embodiments, the conjugated group comprises a target moiety containing a branched group and at least two tether ligands. In certain embodiments, the branched group binds a conjugated linker. In certain embodiments, the branched group binds a cleavable moiety. In certain embodiments, the branched group binds an antisense oligonucleotide. In certain embodiments, the branched group is covalently bonded to each of the linker and the tether ligand. In certain embodiments, the branched group comprises a branched aliphatic group containing a group selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the branched group contains a group selected from alkyl, amide, and ether groups. In certain embodiments, the branched group contains a group selected from alkyl and ether groups. In certain embodiments, the branched group comprises a monocyclic or polycyclic ring system. In certain embodiments, the branched group contains one or more cleavable bonds. In certain embodiments, the conjugated group does not contain a branched group.

[0225] In a particular embodiment, the branching base has a structure selected from the following: [ka] [ka] In the formula, each n is independently between 1 and 20. j is 1 to 3, m is between 2 and 6.

[0226] In a particular embodiment, the branching base has a structure selected from the following: [ka] Each n is independently between 1 and 20. m is between 2 and 6.

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

[0228] In a particular embodiment, the branching base has a structure selected from the following: [ka] In the formula, each A1 is independently O, S, C=O, or NH. Each n is independently between 1 and 20.

[0229] In a particular embodiment, the branching base has a structure selected from the following: [ka] In the formula, each A1 is independently O, S, C=O, or NH. Each n is independently between 1 and 20.

[0230] In a particular embodiment, the branching base has a structure selected from the following: [ka] In the formula, A1 is O, S, C=O, or NH. Each n is independently between 1 and 20.

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

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

[0233] In a particular embodiment, 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 branched group. In certain embodiments, the conjugated group comprises one or more tethers covalently bonded to the bonding 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 a particular embodiment, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiesters in any combination. In a particular embodiment, each tether is at least one Contains a neutron bond group or a neutral bond group.

[0235] In certain embodiments, the tether includes one or more cleavable bonds. In certain embodiments, the tether is bonded to the branched group via either an amide group or an ether group. In certain embodiments, the tether is bonded to the branched group via a phosphodiester group. In certain embodiments, the tether is bonded to the branched group via a phosphorus-binding group or a neutral-binding group. In certain embodiments, the tether is bonded to the branched group via an ether group. In certain embodiments, the tether is bonded to the ligand via either an amide group or an ether group. In certain embodiments, the tether is bonded to the ligand via an ether group. In certain embodiments, the tether is bonded to the ligand via either an amide group or an ether group. In certain embodiments, the tether is bonded to the ligand via an ether group.

[0236] In a particular embodiment, each tether has a chain length of about 8 to about 20 between the ligand and the branching group. In a particular embodiment, each tether group has a chain length of about 10 to about 18 between the ligand and the branching group. In a particular embodiment, each tether group has a chain length of about 13.

[0237] In a particular embodiment, the tether has a structure selected from the following: [ka] In the formula, each n is independently between 1 and 20. Each p is between 1 and approximately 6.

[0238] In a particular embodiment, the tether has a structure selected from the following: [ka]

[0239] In a particular embodiment, the tether has a structure selected from the following: [ka] In the formula, each n is independently between 1 and 20.

[0240] In a particular embodiment, the tether has a structure selected from the following: [ka] In the formula, L is either a phosphorus-bonding group or a neutral-bonding 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 between 0 and 20, and at least one m1 is greater than 0 for each tether.

[0241] In a particular embodiment, the tether has a structure selected from the following: [ka]

[0242] In a particular embodiment, the tether has a structure selected from the following: [ka] In the formula, Z2 is either H or CH3. Each m1 is independently between 0 and 20, and at least one m1 is greater than 0 for each tether.

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

[0244] In certain embodiments, the tether includes a phosphorus-binding group. In certain embodiments, the tether does not include any amide bonds. In certain embodiments, the tether includes a phosphorus-binding group but does not include any amide bonds.

[0245] 3. A specific ligand In certain embodiments, the disclosure provides ligands to which each ligand is covalently bound to a tether. In certain embodiments, each ligand is selected to have affinity for at least one receptor in target cells. In certain embodiments, a ligand is selected to have affinity for at least one receptor on the surface of mammalian liver cells. 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-acetylgalactoseamine, mannose, glucose, glucosamine, and fucose. In certain embodiments, each ligand is N-acetylgalactoseamine (GalNAc). In certain embodiments, the target moiety comprises 2 to 6 ligands. In certain embodiments, the target moiety comprises 3 ligands. In certain embodiments, the target moiety comprises 3 N-acetylgalactoseamine ligands.

[0246] In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, modified carbohydrate, polyhydric carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or 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-formamide-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfamino-D-glucopyranose, and N-sulfo-D-glucosamine, and N-glycoyl-α-neuraminic acid. For example, the thiosaccharide can 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-glucopyranose, 4-thio-β-D-galactopyranoside, 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, which is 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, which includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, both β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose may be used synonymously. Therefore, in structures in which one form is shown, these structures are intended to also include the other forms. For example, α-form: 2-(acetylamino)-2-deoxy- Where the structure of D-galactopyranose is shown, this structure is intended to also include other forms. In certain embodiments, and in certain preferred embodiments, the β-form: 2-(acetylamino)-2-deoxy-D-galactopyranose is a preferred embodiment. [ka] [ka] [ka]

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

[0249] In a particular embodiment, one or more ligands have a structure selected from the following: To possess. [ka]

[0250] In a particular embodiment, one or more ligands have a structure selected from the following: [ka]

[0251] In a particular embodiment, one or more ligands have a structure selected from the following: [ka]

[0252] i. A specific conjugate In a particular embodiment, the conjugated group includes the structural features described above. In a particular such embodiment, the conjugated group has the following structure: [ka] In the formula, each n is independently between 1 and 20.

[0253] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0254] In a particular such embodiment, the conjugated group has the following structure: [ka] In the formula, each n is independently between 1 and 20. Z is H or a bonded solid support. Q is an antisense compound, X is either O or S, Bx is the heterocyclic base portion.

[0255] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0256] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0257] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0258] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0259] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0260] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0261] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0262] In a particular such embodiment, the conjugated group has the following structure: [ka]

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

[0264] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0265] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0266] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0267] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0268] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0269] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0270] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0271] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0272] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0273] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0274] In a particular such embodiment, the conjugated group has the following structure: [ka]

[0275] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of 6 to 11 consecutively bonded atoms.

[0276] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of 10 consecutively bonded atoms.

[0277] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of 4 to 11 consecutively bonded atoms, the tether containing exactly one amide bond.

[0278] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, Y and Z are C1 to C 12 The group is independently selected from substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, or from groups comprising ethers, ketones, amides, esters, carbamates, amines, piperidines, phosphates, phosphodiesters, phosphorothioates, triazoles, pyrrolidines, disulfides, or thioethers.

[0279] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, Y and Z are C1 to C 12The group is independently selected from substituted or unsubstituted alkyl groups, or from groups comprising exactly one ether or exactly two ethers, amides, amines, piperidines, phosphates, phosphodiesters, or phosphorothioates.

[0280] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure , [ka] In the formula, Y and Z are C1 to C 12 Selected independently of substituted or unsubstituted alkyl groups.

[0281] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0282] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.

[0283] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of 4 to 13 consecutively bonded atoms, and X does not contain an ether group.

[0284] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of eight consecutively bonded atoms, and X does not contain an ether group.

[0285] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of 4 to 13 consecutively bonded atoms, the tether contains exactly one amide bond, and X does not contain an ether group.

[0286] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, X is a substituted or unsubstituted tether of 4 to 13 consecutively bonded atoms, the tether being an amide bond and substituted or unsubstituted C2-C 11 It consists of alkyl groups.

[0287] In a particular embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, Y is C1~C 12 The group is selected from substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, or from groups including ethers, ketones, amides, esters, carbamates, amines, piperidines, phosphates, phosphodiesters, phosphorothioates, triazoles, pyrrolidines, disulfides, or thioethers.

[0288] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, Y is C1~C 12The group is selected from substituted or unsubstituted alkyl groups, or from groups including ethers, amines, piperidines, phosphates, phosphodiesters, or phosphorothioates.

[0289] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, Y is C1~C 12 Selected from substituted or unsubstituted alkyl groups.

[0290] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0291] In a particular such embodiment, the cell-targeting portion of the conjugated group has the following structure: [ka] In the formula, n is 4, 5, 6, 7, or 8.

[0292] b. Certain conjugated antisense compounds In certain embodiments, the conjugate is bonded to the nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside. In certain embodiments, the conjugated antisense compound has the following structure: [ka] During the ceremony, A is an antisense oligonucleotide, B is a part that can be cut, C is a conjugate linker, D is a branching base, Each E is a tether, Each F is an ligand, q is an integer between 1 and 5.

[0293] In a particular embodiment, the conjugated antisense compound has the following structure: [ka] During the ceremony, A is an antisense oligonucleotide, C is a conjugate linker, D is a branching base, Each E is a tether, Each F is an ligand, q is an integer between 1 and 5.

[0294] In a particular such embodiment, the conjugated linker includes at least one severable bond.

[0295] In a particular such embodiment, the branching group includes at least one cleavable bond.

[0296] In a particular embodiment, each tether includes at least one detachable bond.

[0297] In a particular embodiment, the conjugate is bound to the nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside.

[0298] In a particular embodiment, the conjugated antisense compound has the following structure: [ka] During the ceremony, A is an antisense oligonucleotide, B is a part that can be cut, C is a conjugate linker, Each E is a tether, Each F is an ligand, q is an integer between 1 and 5.

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

[0300] In a particular embodiment, the conjugated antisense compound has the following structure: [ka] During the ceremony, A is an antisense oligonucleotide, B is a part that can be cut, D is a branching base, Each E is a tether, Each F is an ligand, q is an integer between 1 and 5.

[0301] In a particular embodiment, the conjugated antisense compound has the following structure: [ka] During the ceremony, A is an antisense oligonucleotide, D is a branching base, Each E is a tether, Each F is an ligand, q is an integer between 1 and 5.

[0302] In a particular such embodiment, the conjugated linker includes at least one severable bond.

[0303] In a particular embodiment, each tether includes at least one detachable bond.

[0304] In a particular embodiment, the conjugated antisense compound has a structure selected from the following: [ka]

[0305] In a particular embodiment, the conjugated antisense compound has a structure selected from the following: [ka]

[0306] In a particular embodiment, the conjugated antisense compound has a structure selected from the following: [ka]

[0307] Representative U.S. patents, U.S. patent application publications, and international patent application publications teaching specific preparations of the aforementioned conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties, and other modifications include, but are not limited to, U.S. Patents 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 WO2013 / 033230, and WO2012 / 037254, each of which is incorporated herein by reference in its entirety.

[0308] Representative publications that teach specific preparations of the aforementioned 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, and LEE et al., “New an 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 This includes, but is not limited to, "Asialoglycoprotein Receptor" Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated herein by reference in whole.

[0309] In certain embodiments, the conjugated antisense compound comprises an RNase H-based oligonucleotide (such as a gapmer) or a splice-modified oligonucleotide (such as a fully modified oligonucleotide), and any conjugated group containing at least one, two, or three GalNAc groups. In certain embodiments, the conjugated antisense compound is as follows (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.,Geneberg 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. 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, 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 Applications US2011 / 0097264, US2011 / 0097265, US2013 / 0004427, US2005 / 0164235, US2006 / 0148740, US2008 / 0281044, US2010 / 0240730, US2003 / 0119724, US2006 / 0183886 No. US2008 / 0206869, No. US2011 / 0269814, No. US2009 / 0286973, No. US2011 / 0207799, No. US2012 / 0136042, No. US2012 / 0165393, No. US2008 / 0281041, No. US2009 / 0203135, No. US2012 / 0035115, No. US2012 / 009, This includes any conjugated group found in any of the following publications: 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 in whole by reference. C. Specific uses and characteristics

[0310] In certain embodiments, the conjugated antisense compound exhibits a potent reduction of target RNA in vivo. In certain embodiments, the unconjugated antisense compound accumulates in the kidney. In certain embodiments, the conjugated antisense compound accumulates in the liver. In certain embodiments, the conjugated antisense compound exhibits good tolerability. Such properties make the conjugated antisense compound particularly useful for inhibiting many target RNAs, including but not limited to target RNAs involved in metabolic, cardiovascular, and other diseases, disorders, or conditions. Accordingly, a method for treating such diseases, disorders, or conditions is provided herein by contacting liver tissue with a conjugated antisense compound that targets RNA associated with such diseases, disorders, or conditions. Accordingly, a method for improving any of the various metabolic, cardiovascular, and other diseases, disorders, or conditions using the conjugated antisense compounds of the present invention is also provided.

[0311] In certain embodiments, a conjugated antisense compound is more potent than its unconjugated counterpart at specific tissue concentrations. Without being constrained by any theory or mechanism, in certain embodiments, the conjugate may allow the conjugated antisense compound to enter cells more efficiently or more productively. For example, in certain embodiments, a conjugated antisense compound may exhibit higher targeted reduction compared to its unconjugated counterpart, and both the conjugated antisense compound and its unconjugated counterpart are present in tissues at the same concentrations. For example, in certain embodiments, a conjugated antisense compound may exhibit higher targeted reduction compared to its unconjugated counterpart, and both the conjugated antisense compound and its unconjugated counterpart are present in the liver at the same concentrations.

[0312] Productive and unproductive uptake of oligonucleotides has been discussed previously (e.g., Geary, RS, 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. Vincent, et al. (2011). “Mechanisms of single-stranded phosphorothioate See "Modified antisense oligonucleotide accumulation in hepatocytes." Nucleic Acids Res. 39(11):4795-807). The conjugated groups described herein may improve productive uptake.

[0313] In certain embodiments, the conjugated groups described herein may further improve strength by increasing the affinity of a conjugated antisense compound to a particular type of cell or tissue. In certain embodiments, the conjugated groups described herein may further improve strength by increasing the recognition of a conjugated antisense compound by one or more cell surface receptors. In certain embodiments, the conjugated groups described herein may further improve strength by increasing the recognition of a conjugated antisense compound by one or more cell surface receptors. Strength can be further improved by promoting endocytosis of the anthisense compound.

[0314] In certain embodiments, the cleavable portion may further improve strength 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] Phosphothioate bonds have previously been incorporated into antisense oligonucleotides. Such phosphorothioate bonds improve the stability of oligonucleotides by making them resistant to nucleases. Furthermore, phosphorothioate bonds can also bind to certain proteins, leading to the accumulation of antisense oligonucleotides in the liver. Oligonucleotides with fewer phosphorothioate bonds accumulate less in the liver and more in the kidneys (e.g., Geary, R., “Pharmacokinetic”). See "Properties of 2'-O-(2-Methoxyethyl)-Modified Oligonucleotide Analogs in Rats," Journal of Pharmacology and Experimental Therapeutics, Vol.296, No.3, 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 nucleoside bonds and more phosphodiester nucleoside bonds accumulate less in the liver and more in the kidneys. When treating liver disease, this is undesirable for several reasons, including (1) the drug does not reach the desired site of action (liver), (2) the drug is excreted in the urine, and (3) the kidneys are exposed to relatively high concentrations of the drug, which can be renally toxic. Therefore, in the case of liver disease, phosphorothioate bonds offer significant benefits.

[0316] However, in certain embodiments, administration of oligonucleotides uniformly linked by phosphorothioate nucleoside linkages induces one or more pro-inflammatory responses (see, for example, J Lab Clin Med. 1996 Sep;128(3):329-38. “Amplification of antibody production by phosphorothioate oligodeoxynucleotides”. Branda et al., and also see, for example, 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 nucleoside linkages are phosphorothioate nucleoside linkages induces one or more pro-inflammatory responses.

[0317] In certain embodiments, the degree of pro-inflammatory effect may depend on several variables (e.g., skeletal modifications, off-target effects, nucleic acid base modifications, and / or nucleoside modifications) (e.g., Toxicologic Properties in Antisense). See a 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 variable compounds. For example, the degree of pro-inflammatory effect of a given oligonucleotide can be reduced by replacing any number of phosphorothioate nucleoside bonds with phosphodiester nucleoside bonds. This can be mitigated by reducing the total number of phosphorothioate nucleoside bonds.

[0318] In certain embodiments, it is desirable to reduce the number of phosphorothioate bonds, which can be done without losing stability or altering the distribution from the liver to the kidney. For example, in certain embodiments, the number of phosphorothioate bonds can be reduced by replacing them with phosphodiester bonds. In such embodiments, antisense compounds having fewer phosphorothioate bonds and more phosphodiester bonds induce a lower pro-inflammatory response or no response at all. Antisense compounds having fewer phosphorothioate bonds and more phosphodiester bonds induce a lower pro-inflammatory response, but they do not accumulate in the liver and may be less effective at the same or similar doses compared to antisense compounds with more phosphorothioate bonds. Therefore, in certain embodiments, it is desirable to design antisense compounds having multiple phosphodiester bonds and multiple phosphorothioate bonds, while also having stability and good distribution to the liver.

[0319] In certain embodiments, even when some of the phosphorothioate bonds are replaced with less pro-inflammatory phosphodiester nucleoside bonds, conjugated antisense compounds accumulate more in the liver and less in the kidneys compared to their unconjugated counterparts. In certain embodiments, even when some of the phosphorothioate bonds are replaced with less pro-inflammatory phosphodiester nucleoside bonds, 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 conjugated compounds makes it possible to design antisense drugs that are more potent and have better resistance. In fact, in certain embodiments, conjugated antisense compounds have a higher therapeutic index than their unconjugated counterparts. This allows conjugated antisense compounds to be administered at higher absolute doses because they have a lower risk of pro-inflammatory responses and a lower risk of nephrotoxicity. These higher doses allow for lower-frequency administration because elimination (metabolism) is expected to be similar. Furthermore, as mentioned above, because the compound is more potent, it can be concentrated at a lower level before the next dose without losing therapeutic activity, allowing for a longer interval between doses.

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

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

[0322] In certain embodiments, antisense compounds containing one or more conjugated groups as described herein have increased activity and / or strength and / or tolerance compared to parent antisense compounds lacking such one or more conjugated groups. Therefore, in certain embodiments, the conjugation of such conjugated groups to oligonucleotides is desirable. Such conjugated groups may be conjugated to the 5' and / or 3' ends of the oligonucleotide. In certain cases, conjugation at the 5' end is synthetically desirable. Typically, oligonucleotides are synthesized using techniques well known in the art by the conjugation of a 3'-terminal nucleoside to a solid support and the sequential coupling of nucleosides from 3' to 5'. Therefore, if the conjugated group is desired at the 3' end, (1) the conjugated group can be conjugated to the 3'-terminal nucleoside and the conjugated nucleoside can be conjugated to a solid support for subsequent preparation of the oligonucleotide, or (2) after synthesis, the conjugated group can be conjugated to the 3'-terminal nucleoside of the complete oligonucleotide. Neither of these means is very efficient, and therefore, both are costly. Specifically, the binding of conjugated nucleosides to solid supports, as demonstrated in the examples herein, is an inefficient process. In certain embodiments, binding a conjugated group to a 5'-terminated nucleoside is synthetically easier than binding at the 3' terminus. Using a well-defined standard reaction, a non-conjugated 3'-terminated nucleoside can be bound to a solid support to prepare this oligonucleotide. Only then is it necessary to bind the 5'-nucleoside containing the conjugated group in the final coupling step. In certain embodiments, this is more efficient than the direct binding of a conjugated nucleoside to a solid support, which is typically performed to prepare 3'-conjugated oligonucleotides. The examples herein demonstrate binding at the 5' terminus. In addition, certain conjugated groups offer synthetic advantages. For example, certain conjugated groups containing a phosphorus-binding group are synthetically simpler and more efficiently prepared than other conjugated groups containing previously reported conjugated groups (e.g., International Publication No. WO / 2012 / 037254).

[0323] In certain embodiments, conjugated antisense compounds are administered to a subject. In such embodiments, antisense compounds containing one or more conjugated groups as described herein have increased activity and / or strength and / or tolerance compared to parent antisense compounds lacking such one or more conjugated groups. Without being constrained by a mechanism, the conjugated groups are thought to aid in distribution, delivery, and / or uptake to target cells or tissues. In certain embodiments, it is desirable that upon entering target cells or tissues, all or some of the conjugated groups are cleaved to release the active oligonucleotide. In certain embodiments, it is not necessary for all conjugated groups to be cleaved from the oligonucleotide. For example, in Example 20, conjugated oligonucleotides were administered to mice, and several different chemical species containing different portions of the conjugated groups remaining in the oligonucleotide were detected (Table 10a). This conjugated antisense compound showed good strength (Table 10). Therefore, in certain embodiments, such metabolite profiles of multiple partial cleavages of conjugated groups are desirable. The yl does not impede activity / strength. Nevertheless, in certain embodiments, it is desirable that the prodrug (conjugated oligonucleotide) produce a single active compound. In certain cases, if multiple forms of the active compound are found, it may be necessary to determine the relative amount and activity of each form. In certain embodiments, if 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 that such a single active species is an antisense oligonucleotide lacking any portion of the conjugated group. In certain embodiments, the 5'-terminal conjugated group is likely to result in complete metabolism of the conjugated group. Without being constrained by mechanism, an endogenous enzyme involved in the metabolism of the 5' terminus (e.g., 5' nuclease) may be more active / efficient than the 3' equivalent. In certain embodiments, these particular conjugated groups are more compliant with metabolism to a single active species. In certain embodiments, certain conjugated groups are more compliant with 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 the 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 codes for a protein. In certain embodiments, the target nucleic acid is selected from mRNA, premRNA, 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 mimetic, which typically binds to multiple targets.

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

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

[0327] One type of antisense mechanism involving the degradation of target RNA is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA strand in RNA:DNA double strands. It is known in the art that "DNA-like" single-stranded antisense compounds induce RNase H activity in mammalian cells. Therefore, RNase H Activation of this molecule leads to cleavage of RNA targets, thereby significantly improving the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

[0328] Antisense mechanisms include, but are not limited to, RNAi mechanisms that utilize the RISC pathway. Such RNAi mechanisms include, but are not limited to, siRNA, ssRNA, and microRNA mechanisms. Such mechanisms include the creation of microRNA mimes and / or anti-microRNAs.

[0329] Antisense mechanisms include, but are not limited to, mechanisms that hybridize or mimic microRNAs or non-coding RNAs other than mRNA. Such non-coding RNAs include, but are not limited to, promoter-directed RNAs that result in the transcription or translation of one or more nucleic acids, as well as short and long RNAs.

[0330] In certain embodiments, the oligonucleotide containing the conjugate described herein is an RNAi compound. In certain embodiments, the oligomeric oligonucleotide containing the conjugate described herein is an ssRNA compound. In certain embodiments, the oligonucleotide containing the conjugate described herein is paired with a second oligomeric compound to form an siRNA. In certain such embodiments, the second oligomeric compound also includes the conjugate. In certain embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In certain embodiments, the oligonucleotide containing the conjugate described herein is the antisense strand in the siRNA compound. In certain embodiments, the oligonucleotide containing the conjugate described herein is the sense strand in the siRNA compound. In embodiments where the conjugated oligomeric compound is a double-stranded siRnA, the conjugate may be present on the sense strand, on the antisense strand, or on both the sense and antisense strands. 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, modification of the target nucleic acid yields clinical benefits. Certain target nucleic acids include, but are not limited to, those illustrated in Table 1. [Table 1]

[0332] The targeting process typically involves determining at least one target region, segment, or site within the target nucleic acid so that an antisense interaction occurs and the desired effect is consequently achieved.

[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 encompass the 3'UTR, 5'UTR, exon, intron, coding region, translation initiation region, translation termination region, or other defined nucleic acid region or target segment.

[0334] In a particular embodiment, the target segment is at least about eight nucleic acid bases of the target region targeted by the conjugated antisense compound. The target segment may include a DNA or RNA sequence containing at least eight consecutive nucleic acid bases from one 5' end of the target segment (the remaining nucleic acid bases begin immediately upstream of the 5' end of the target segment). The target segment is also represented by a DNA or RNA sequence containing at least eight consecutive nucleic acid bases from one of the 3' ends of the target segment (the remaining nucleic acid bases are a continuous sequence of identical DNA or RNA starting immediately downstream of the 3' end of the target segment and continuing until the DNA or RNA contains approximately eight to approximately 30 nucleic acid bases). The target segment is also represented by a DNA or RNA sequence containing at least eight consecutive nucleic acid bases from an internal portion of the target segment sequence, which may extend in either direction or both directions until the conjugated antisense compound contains approximately eight to approximately 30 nucleic acid bases.

[0335] In certain embodiments, antisense compounds targeting nucleic acids listed in Table 1 may be modified as described herein. In certain embodiments, the antisense compound may have a modified sugar moiety, an unmodified sugar moiety, or a mixture of modified and unmodified sugar moieties as described herein. In certain embodiments, the antisense compound may have a modified nucleoside bond, an unmodified nucleoside bond, or a mixture of modified and unmodified nucleoside bonds as described herein. In certain embodiments, the antisense compound may have a modified nucleic acid base, an unmodified nucleic acid base, or a mixture of modified and unmodified nucleic acid bases as described herein. In certain embodiments, the antisense compound may have a motif as described herein.

[0336] In a particular embodiment, antisense compounds targeting nucleic acids listed in Table 1 may be conjugated as described herein.

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

[0338] HBV, a virus, is a double-stranded liver-targeting virus that infects only humans and non-human primates. Viral replication primarily occurs in the liver, but to a lesser extent, also in the kidneys, pancreas, bone marrow, and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64:2000; 51-68). Viral and immunomarkers 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 may be high during the incubation period, but levels of HBV DNA and HBeAg begin to decline at the onset of the disease and may 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 and correlates with active viral replication, and consequently, high viral load and high infectivity (Hepatitis B e 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 associated with recovery and immunity in previously infected individuals. It demonstrates infectious power.

[0339] The American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of Liver Diseases (EASL) currently recommend therapies for chronic HBV infection, including interferon-alpha (INFα), pegylated interferon-alpha-2a (Peg-IFN2a), entecavir, and tenofovir. While entecavir and tenofovir, which are nucleoside and nucleic acid base therapies, effectively reduce viral load, their HBeAg seroconversion rates and HBsAg loss rates are even lower than those achieved with IFNα therapy. Other similar therapies, including lamivudine (3TC), terbivudine (LdT), and adefovir, are also used, but in general, with nucleoside / nucleo acid base therapies, resistance development limits treatment efficacy.

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

[0341] Antisense compounds targeting HBV have been previously disclosed in International Publications WO2011 / 047312, WO2012 / 145674, and WO2012 / 145697, respectively, each incorporated herein by reference in its entirety. Clinical studies are planned to evaluate the effects of HBV-targeting antisense compounds in patients. However, there remains a need to provide patients with more viable treatment options.

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

[0343] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 3.

[0344] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 4.

[0345] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 5.

[0346] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 contains at least eight consecutive nucleic acid base sequences of SEQ ID NO: 6. In a particular embodiment, the sequence number The conjugated antisense compound targeting compound 1 contains the nucleic acid sequence of SEQ ID NO: 6.

[0347] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 7.

[0348] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 8.

[0349] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 9.

[0350] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 10.

[0351] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 1 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 11. [Table 2]

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

[0353] In certain embodiments, the compound comprises or consists of ISIS 509934 and a conjugated group. ISIS 509934 has the formula: mCes mCes Ae A modified oligonucleotide having s Aes Tes Tds Tds Ads Tds Gds mCds mCds Tds Ads mCds Aes Ges mCes mCes Te, in which, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0354] In certain embodiments, the compound comprises or consists of ISIS 510100 and a conjugated 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, in which, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0355] In certain embodiments, the compound comprises or consists of ISIS 552023 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0356] In certain embodiments, the compound comprises or consists of ISIS 552024 and a conjugated group. ISIS 552024 has the formula: Ges Tes Ges Aes Aes Ges mCds Gds Ads Ads Gds Tds Gds A modified oligonucleotide having mCds Ads mCds Aes mCes Ges Ge, in which, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0357] In a particular embodiment, the compound comprises ISIS 552032 and a conjugated group. or consisting of them. ISIS 552032 is formula: Ges Tes Ges mCes Aes Ges Ads Gds Gds Tds Gds Ads Ads A modified oligonucleotide having Gds mCds Gds Aes Aes Ges Te, in which, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0358] In certain embodiments, the compound comprises or consists of ISIS 552859 and a conjugated 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, in which, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, k is a cEt-modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0359] In certain embodiments, the compound comprises or consists of ISIS 552925 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, k is a cEt-modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond. s represents a phosphorothioate nucleoside bond.

[0360] In certain embodiments, the compound comprises or consists of ISIS 577119 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, k is a cEt-modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

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

[0362] In a particular embodiment, the compound comprises or consists of ISIS 712408 having the following chemical structure. [ka]

[0363] In certain embodiments, the compound comprises or consists of ISIS 695324 having the following chemical structure. [ka]

[0364] In certain embodiments, the compound includes or consists of the chemical modifications represented by SEQ ID NO: 3, 5'-GalNAc, and the following chemical structures: [ka] In the formula, R 1 However, it is -OCH2CH2OCH3(MOE), and R 2 However, is it H or R 1 and R 2 Either they come together to form a bridge, and there, R 1 However, it is -O- and R 2 However, R is selected from -CH2-, -CH(CH3)-, or -CH2CH2-, and the resulting bridge is selected from -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-. 1 and R 2 They are directly connected, R of each ring on the same ring 3 and R 4 For each pair, independently for each ring, R 3 However, H and -OCH2CH2OCH3 are selected, and R 4 However, is it H or R 3 and R 4 Either they come together to form a bridge, and there, R 3 However, it is -O- and R 4 However, R is selected from -CH2-, -CH(CH3)-, or -CH2CH2-, and the resulting bridge is selected from -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-. 3 and R 4 They are directly connected, R 5 However, it is selected from H and -CH3, Z, S - and O - Selected from.

[0365] In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2012 / 145697, which is incorporated herein in whole by reference, and a conjugated group described herein. In certain embodiments, the compound comprises SEQ ID NOs. 5-310, 321, disclosed in International Publication No. WO2012 / 145697. The compound comprises an antisense oligonucleotide having one of the nucleic acid base sequences ~802, 804~1272, 1288~1350, 1364~1372, 1375, 1376, and 1379, and a conjugated group as described herein. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2011 / 047312, which is incorporated herein in whole by reference, and a conjugated group as described herein. In certain embodiments, the compound comprises an antisense oligonucleotide having one of the nucleic acid base sequences of SEQ ID NOs. 14~22, disclosed in International Publication No. WO2011 / 047312, and a conjugated group as described herein. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2012 / 145674, which is incorporated herein in whole by reference, and a conjugated group as described herein. In certain embodiments, the compound comprises an antisense oligonucleotide having one of the nucleic acid sequences of SEQ ID NOs. 18-35 disclosed in International Publication No. WO2012 / 145674. In certain embodiments, the compound comprises a double-stranded oligonucleotide disclosed in International Publication No. WO2013 / 159109, which is incorporated herein by reference in whole, and a conjugated group described herein. In certain embodiments, the compound comprises a double-stranded oligonucleotide in which one strand has one of the nucleic acid sequences of SEQ ID NOs. 30-125 disclosed in International Publication No. WO2013 / 159109. All of the nucleic acid sequences of the referenced SEQ ID NOs are incorporated herein by reference.

[0366] HBV treatment index In certain embodiments, the present invention provides a method for regulating HBV expression in a target using a conjugated antisense compound that targets HBV nucleic acid. In certain embodiments, HBV expression is reduced.

[0367] In certain embodiments, the present invention provides a method for treating a subject using a conjugated antisense compound that targets HBV nucleic acid in a pharmaceutical composition. In certain embodiments, the subject has an HBV-associated condition. In certain embodiments, an HBV-associated condition includes, but is not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, hepatitis, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. In certain embodiments, an HBV-associated condition may have symptoms that include, in conjunction with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or antibodies specific to hepatitis B virus antigen, one 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-colored urine. In certain embodiments, the subject is at risk of an HBV-associated condition. This includes subjects who have one or more risk factors for developing an HBV-related condition, including sexual exposure to an individual infected with the hepatitis B virus, cohabitation with an individual with lifelong hepatitis B virus infection, exposure to human blood infected with the hepatitis B virus, infusion of illicit drugs, contact with a person with hemophilia, and visits to areas where hepatitis B is common. In certain embodiments, subjects are identified as requiring treatment for an HBV-related condition.

[0368] Certain embodiments provide a method for reducing HBV DNA and / or HBV antigen levels in an animal infected with HBV, comprising administering 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 a particular embodiment, the present invention relates to conjugated antisense targeting HBV nucleic acid. This invention provides a method for preparing pharmaceuticals using a compound.

[0370] In certain embodiments, the present invention provides a conjugated antisense compound or a pharmaceutically acceptable salt thereof that targets HBV nucleic acid for therapeutic use.

[0371] Certain embodiments provide conjugated antisense compounds targeting HBV nucleic acid for use in the treatment of HBV-related conditions. HBV-related conditions include, but are not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, hepatitis, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia.

[0372] Certain embodiments provide a conjugated antisense compound targeting HBV nucleic acid for use in reducing HBV DNA and / or HBV antigen levels in animals infected with HBV, comprising administering the HBV nucleic acid-targeting conjugated antisense compound to the animals. 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 methods and uses described above. For example, in a particular embodiment, the conjugated antisense compounds targeting HBV nucleic acid in the methods and uses described above include: a conjugated antisense compound targeting SEQ ID NO: 1 containing at least eight consecutive nucleic acid base sequences from SEQ ID NOs: 3 to 11; a conjugated antisense compound targeting SEQ ID NO: 1 containing any nucleic acid base sequences from SEQ ID NOs: 3 to 11; ISIS 505358, ISIS 509934, ISIS 510100, ISIS 552023, ISIS 552024, ISIS 552032, ISIS 552859, ISIS 552925, or ISIS Compounds comprising or consisting of 577119 and a conjugated group; and compounds comprising an antisense oligonucleotide disclosed in International Publication No. WO2012 / 145697, which is incorporated herein in whole by reference, and a conjugated group; compounds comprising an antisense oligonucleotide having any of the nucleic acid base sequences of SEQ ID NOs. 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379 disclosed in International Publication No. WO2012 / 145697, and a conjugated group described herein; International Publication No. Compounds comprising an antisense oligonucleotide having one of the nucleic acid base sequences among Sequence ID Nos. 14-22 disclosed in WO2011 / 047312 and a conjugated group described herein; compounds comprising an antisense oligonucleotide having one of the nucleic acid base sequences among Sequence ID Nos. 18-35 disclosed in International Publication No. WO2012 / 145674; or compounds comprising a double-stranded oligonucleotide in which one strand has one of the nucleic acid base sequences among Sequence ID Nos. 30-125 disclosed in International Publication No. WO2013 / 159109, may include, but are not limited to, these.

[0374] 2. Transthyretin (TTR) TTR (also known as prealbumin, hyperthyroxinemia, prealbumin abnormality, thyroxine; senile systemic amyloidosis, amyloid polyneuropathy, amyloidosis I, PALB; transthyretin disorder, HST2651; TBPA; prealbumin disorder, normal thyroid function, 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 misfoldings in the protein lead to amyloid fibril deposition, resulting in senile systemic amyloidosis (SSA), familial amyloid polyneuropathy. It is associated with disorders such as familial amyloid cardiopathy (FAP) and familial amyloid heart disease (FAC).

[0375] TTR is primarily synthesized by the liver and 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 bloodstream, transthyretin originating from the choroid plexus is destined to become 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 as rare and endemic as previously thought, but can affect up to 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 deposition in FAP patients (Costa et al, Proc. Natl. Acad. Sci. USA 1978, 75:4499-4503), and later, substitution of methionine with valine at the 30th position 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 tissue, particularly in the peripheral nervous system (Sousa and Saraiva, Prog. Neurobiol. 2003, 71:385-400). Following TTR deposition, axonal degeneration occurs, beginning with small-diameter unmyelinated and myelinated fibers, and ultimately leading to neuronal loss at ganglion sites.

[0378] Antisense compounds targeting TTR are previously disclosed in U.S. Patent No. US2005 / 0244869, International Publication No. WO2010 / 017509, and International Publication No. WO2011 / 139917, each of which is incorporated herein by reference in whole. TTR-targeting antisense oligonucleotide, ISIS-TTR Rx Currently, this is being tested in Phase II / III clinical trials to evaluate its efficacy in treating patients with familial amyloid polyneuropathy. However, there is still a need to provide patients with more viable treatment options.

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

[0380] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 comprises at least one of the SEQ ID NOs: 12-19, which is a sequence of at least eight consecutive nucleic acid bases.

[0381] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 12.

[0382] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 13.

[0383] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 14.

[0384] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 15.

[0385] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 16 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 78.

[0386] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 17.

[0387] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 18.

[0388] In a particular embodiment, the conjugated antisense compound targeting SEQ ID NO: 2 includes at least eight consecutive nucleic acid base sequences of SEQ ID NO: 19. [Table 3]

[0389] In certain embodiments, the compound comprises or consists of ISIS 420915 and a conjugated 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, in which, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0390] In certain embodiments, the compound comprises or consists of ISIS 304299 and a conjugated 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 in the formula, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0391] In certain embodiments, the compound comprises or consists of ISIS 420921 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0392] In certain embodiments, the compound comprises or consists of ISIS 420922 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0393] In certain embodiments, the compound comprises or consists of ISIS 420950 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0394] In certain embodiments, the compound comprises or consists of ISIS 420955 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0395] In certain embodiments, the compound comprises or consists of ISIS 420957 and a conjugated 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, where, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

[0396] In certain embodiments, the compound comprises or consists of ISIS 420959 and a conjugated 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, in the formula, A is adenine, mC is 5'-methylcytosine, G stands for guanine, T is thymin, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, s represents a phosphorothioate nucleoside bond.

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

[0398] In a particular embodiment, the compound comprises or consists of ISIS 682877 having the following chemical structure. [ka]

[0399] In a particular embodiment, the compound comprises or consists of ISIS 682884 having the following chemical structure. [ka]

[0400] In a particular embodiment, the compound includes or consists of the chemical modifications represented by SEQ ID NO: 12, 5'-GalNAc, and the following chemical structures: [ka] In the formula, R 1However, it is -OCH2CH2OCH3(MOE), and R 2 However, is it H or R 1 and R 2 Either they come together to form a bridge, and there, R 1 However, it is -O- and R 2 However, R is selected from -CH2-, -CH(CH3)-, or -CH2CH2-, and the resulting bridge is selected from -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-. 1 and R 2 They are directly connected, R on the same ring 3 and R 4 For each pair, independently for each ring, R 3 However, H and -OCH2CH2OCH3 are selected, and R 4 However, is it H or R 3 and R 4 Either they come together to form a bridge, and there, R 3 However, it is -O- and R 4 However, R is selected from -CH2-, -CH(CH3)-, or -CH2CH2-, and the resulting bridge is selected from -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-. 3 and R 4 They are directly connected, R 5 However, it is selected from H and -CH3, Z, S - and O - 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, which is incorporated in whole herein by reference, and a conjugated group. In certain embodiments, the compound comprises an antisense oligonucleotide disclosed in International Publication No. WO2011 / 139917, sequence number The compound comprises an antisense oligonucleotide having one of the nucleic acid base sequences from sequences 8-160 or 170-177, and a conjugated group described herein. In a particular embodiment, the compound comprises an antisense oligonucleotide having one of the nucleic acid base sequences from sequence numbers 12-89 disclosed in U.S. Patent No. 8,101,743, and a conjugated group described herein. In a particular embodiment, the compound comprises an antisense oligonucleotide having a nucleic acid base sequence complementary to one of the preferred target segments from sequence numbers 90-133 disclosed in U.S. Patent No. 8,101,743, and a conjugated group described herein. All nucleic acid base sequences from the aforementioned referenced sequence numbers are incorporated herein by reference.

[0402] TTR treatment index In a particular embodiment, the present invention provides a method for regulating TTR expression in a target using a conjugated antisense compound that targets TTR nucleic acid. In a particular embodiment, TTR expression is reduced.

[0403] In certain embodiments, the present invention provides a method for treating a subject using a conjugated antisense compound that targets 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," "transthyretin amyloidosis," or "transthyretin amyloid disease" as used herein are any lesion or disease associated with a dysfunction or abnormal regulation of transthyretin resulting in the formation of transthyretin-containing amyloid fibrils. Transthyretin amyloidosis includes, but is not limited to, hereditary TTR amyloidosis, leptomeningeal amyloidosis, familial polymyloid neuropathy (FAP), familial amyloid cardiomyopathy, familial leptomeningeal amyloidosis, senile cardiac amyloidosis, or senile systemic amyloidosis.

[0404] In a particular embodiment, the present invention provides a method for preparing a drug using a conjugated antisense compound that targets TTR nucleic acids.

[0405] In a particular embodiment, the present invention provides a conjugated antisense compound or a pharmaceutically acceptable salt thereof that targets a TTR nucleic acid for therapeutic use.

[0406] In certain embodiments, conjugated antisense compounds targeting TTR nucleic acids are provided for use in the treatment of transthyretin-related diseases, disorders, or conditions or symptoms 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 methods and uses described above. For example, in a particular embodiment, the conjugated antisense compounds targeting the TTR nucleic acid in the methods and uses described above include: a conjugated antisense compound targeting SEQ ID NO: 2, which contains at least one of the SEQ ID NOs from 12 to 19; a conjugated antisense compound targeting SEQ ID NO: 2, which contains one of the SEQ ID NOs from 12 to 19; ISIS 420915, ISIS 304299, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, or ISIS Compounds comprising or consisting of 420959 and a conjugated group; antisense oligonucleotides disclosed in International Publication No. WO2011 / 139917 or U.S. Patent No. US8,101,743, which are incorporated herein in whole by reference, and a conjugated group Compounds comprising: an antisense oligonucleotide having one of the nucleic acid base sequences among Sequence IDs 8-160 and 170-177 disclosed in International Publication No. WO2011 / 139917, and a conjugated group described herein; an antisense oligonucleotide having one of the nucleic acid base sequences among Sequence IDs 12-89 disclosed in U.S. Patent No. US8,101,743, and a conjugated group described herein; or a compound comprising an antisense oligonucleotide having a nucleic acid base sequence complementary to one of the preferred target segments among Sequence IDs 90-133 disclosed in U.S. Patent No. US8,101,743, and a conjugated group described herein. All nucleic acid base sequences among the aforementioned referenced Sequence IDs are incorporated herein by reference. E. A specific pharmaceutical composition

[0408] In certain embodiments, the disclosure provides a pharmaceutical composition comprising one or more antisense compounds. In certain embodiments, such a pharmaceutical composition comprises 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 a pharmaceutical composition consists 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, the antisense compound may be mixed with pharmaceutically acceptable active and / or inactive substances to prepare a pharmaceutical composition or formulation. The composition and the method for formulating the pharmaceutical composition depend on several criteria, including, but not limited to, the route of administration, the severity of the disease, or the dose administered.

[0410] A pharmaceutical composition containing an antisense compound includes any pharmaceutically acceptable salt, ester, or salt of such ester. In certain embodiments, a pharmaceutical composition containing an antisense compound includes one or more oligonucleotides that, upon administration to an animal, including a human, can (directly or indirectly) provide a biologically active metabolite or its residue. Thus, for example, this disclosure covers pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents of antisense compounds. Preferred pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0411] The prodrug may involve the incorporation of additional nucleosides at one or both ends of an oligonucleotide that is cleaved in the body by an endogenous nuclease to form an active antisense oligonucleotide.

[0412] Lipid moieties are used in nucleic acid therapy in various ways. In certain such methods, nucleic acids are introduced into pre-formed liposomes or lipoplexes made from a mixture of cationic and neutral lipids. In certain methods, DNA complexes with mono- or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of a drug to specific cells or tissues. In certain embodiments, lipid moieties increase the distribution of a drug to adipose tissue. They are selected to increase the distribution of the drug to muscle tissue. In a particular embodiment, the lipid portion is selected to increase the distribution of the drug 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 excipients are selected from water, saline solution, 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 that include a hydrophobic compound. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0415] In certain embodiments, the pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver one or more drugs of the disclosure to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[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 with hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which is a solution of anhydrous ethanol containing 3 w / v% benzyl alcohol, 8 w / v% the nonpolar surfactant Polysorbate 80™, and 65 w / v% polyethylene glycol 300. The proportions of such cosolvent systems can be varied considerably without significantly altering their solubility and toxic properties. Furthermore, the identity of the cosolvent components can be varied; for example, other surfactants may be used instead of Polysorbate 80™, the fraction size of polyethylene glycol may be varied, other biocompatible polymers may replace polyethylene glycol, such as polyvinylpyrrolidone, and other sugars or polysaccharides may 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 oral administration.

[0418] In certain embodiments, the pharmaceutical composition is prepared for administration by infusion (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution, e.g., water or a physiologically compatible buffer, e.g., Hanks' solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other components (e.g., components that aid in dissolution or act as preservatives) are included. In certain embodiments, the infusionable suspension is prepared using a suitable liquid carrier, suspending agent, etc. Certain pharmaceutical compositions for infusion are provided in unit dosage forms, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for infusion are suspensions, solutions, or emulsions in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Certain solvents suitable for use in pharmaceutical compositions for injection 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 are carboxymethyl The suspension may contain substances that increase viscosity, such as sodium cellulose, sorbitol, or dextran. Optionally, such suspension may also contain agents that increase the solubility of suitable stabilizers or agents that enable the preparation of highly concentrated solutions.

[0419] In certain embodiments, pharmaceutical compositions are prepared for transmucosal administration. In certain such embodiments, a suitable penetrating agent is used in the formulation for the penetration barrier. Such penetrating agents are generally known in the art.

[0420] In certain embodiments, the pharmaceutical composition provided herein contains a therapeutically effective amount of oligonucleotide. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate, or improve the symptoms of a disease, or to prolong the survival of the subject being treated. Determining the 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 more biologically, pharmacokinetically, or therapeutically active form of the oligonucleotide. In certain embodiments, the prodrug is useful because it is easier to administer than the corresponding active form. For example, in certain cases, the prodrug is more biologically available than the corresponding active form (e.g., by oral administration). 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 a prodrug has excellent permeability across cell membranes where water 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) bonded to an acid group. In certain such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0422] In certain embodiments, this disclosure provides compositions and methods for reducing the amount or activity of a target nucleic acid within a cell. In certain embodiments, the cell is located within 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 Disclosure provides a method for administering a pharmaceutical composition comprising the oligonucleotides of the Disclosure to an animal. Preferred routes of administration include, but are not limited to, oral, rectal, mucosal, intestinal, enteral, topical, suppository, inhalation, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous) administration. In certain embodiments, a pharmaceutical intrathecal agent is administered to achieve local exposure rather than systemic exposure. For example, the pharmaceutical composition may be injected directly into a desired affected area (e.g., the liver).

[0424] Non-exclusive disclosure and incorporation by reference While certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, the following examples are intended solely to illustrate the compounds described herein and are not intended to limit them. Each of the references, GenBank acceptance numbers, etc., listed in this application is incorporated herein by reference in their entirety. Born.

[0425] In this specification, certain compounds, compositions, and methods are said to "exactly contain" or "exactly contain" a certain number of certain elements or features. Such descriptions indicate that a compound, composition, or method may contain other elements, but the number of specific elements or features is specified. For example, a "conjugate containing exactly one GalNAc" is a conjugate containing only one GalNAc, but it may also contain other elements in addition to that one GalNAc.

[0426] While the sequence listings attached to this application may identify each sequence as either "RNA" or "DNA" as needed, in practice, these sequences may be modified with any combination of chemical modifications. Those skilled in the art will readily recognize that such designations of "RNA" or "DNA" to describe modified oligonucleotides are arbitrary in certain cases. For example, an oligonucleotide containing a nucleoside with a 2'-OH sugar moiety and a thymine base may be described as DNA with a modified sugar (2'-OH in the case of the natural 2'-H of DNA) or RNA with a modified base (thymine (methylated uracil) in the case of the natural uracil of RNA). Therefore, nucleic acid sequences provided herein, including but not limited to those in the sequence listings, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to those containing modified nucleic acid bases. Further examples, but not limited to, include oligonucleotides having the nucleic acid base sequence "ATCGATCG", RNA bases whether modified or unmodified, for example, oligonucleotides having the sequence "AUCGAUCG", and oligonucleotides having several DNA bases and several RNA bases such as "AUCGATCG", and "AT me This includes, but is not limited to, any oligonucleotide having such a nucleic acid base sequence, including, oligonucleotides having other modified bases such as "CGAUCG", in the formula, me C represents a cytosine base containing a methyl group at position 5. [Examples]

[0427] The following examples illustrate, but do not limit, certain embodiments of the present disclosure. Furthermore, where specific embodiments are provided, the inventors intend for those specific embodiments to be generally applicable. For example, the disclosure of an oligonucleotide having a particular motif provides reasonable support for further oligonucleotides having that motif or a similar motif. Similarly, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered preferred unless otherwise indicated.

[0428] Example 1: General method for preparing phosphoramidites (compounds 1, 1a, and 2) [ka] Compounds 1, 1a, and 2 were prepared according to the procedures well known in the art as described herein (Seth et al., Bioorg. Med. Chem., 2011, 21 See also (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 U.S. Patent 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 a conjugated ASO (compound 29) containing GalNAc3-1 at the 3' end. [ka] [ka] Protected GalNAc3-1 has the following structure: [ka]

[0437] GalNAc3 cluster portion of the conjugated group GalNAc3-1 (GalNAc3-1 a By combining ) with any cleavable portion, various conjugated groups can be obtained. GalNAc3-1 a It has the following formula: [ka]

[0438] Solid support-bound protection GalNAc3-1 (compound 25) was prepared according to the procedure illustrated in Example 7. Oligomer compound 29 containing GalNAc3-1 at the 3' end was prepared using a standard procedure in automated DNA / RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite construction blocks (compounds 1 and 1a) were prepared according to the procedure illustrated in Example 1. The exemplified phosphoramidites are representative and not intended to be limiting, as oligomer compounds with predetermined sequences and compositions can be prepared using other phosphoramidite construction blocks. The gapped oligomer compounds described herein can be prepared by adjusting the order and amount of phosphoramidites added to the solid support. Such gapped oligomer compounds may have predetermined compositions and nucleotide sequences indicated by any given target.

[0439] Example 10: General preparation of a conjugated ASO (compound 34) containing GalNAc3-1 at the 5' end. [ka] [ka] Unylinker(trademark) 30 is a commercially available product. It has GalNAc3- at its 5' end. Oligomer compound 34 containing one cluster was prepared using a standard procedure in automated DNA / RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite construction blocks (compounds 1 and 1a) were prepared according to the procedure illustrated in Example 1. The phosphoramidites illustrated are representative and not intended to be limiting, as oligomer compounds with predetermined sequences and compositions can be prepared using other phosphoramidite construction blocks. The gapped oligomer compounds described herein can be prepared by adjusting the order and amount of phosphoramidites added to the solid support. Such gapped oligomer compounds may have predetermined compositions and nucleotide sequences indicated 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 preparing conjugated ASOs containing GalNAc3-1 at the 3' position by solid-phase technique (preparation of ISIS 647535, 647536, and 651900) Unless otherwise specified, all reagents and solutions used in the synthesis of oligomeric compounds shall be purchased from commercial sources. Standard phosphoramidite construction blocks and solid supports shall be purchased, for example, T, A, G, and m It is used for the incorporation of nucleoside residues, including C residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2'-deoxyribonucleoside and 2'-MOE.

[0449] ASO synthesis was performed using a phosphoramidite coupling method on a VIMAD solid support (110 μmol / g, Guzaev et al., 2003) packed with GalNAc3-1 in a column, on an ABI 394 synthesizer (1-2 μmol scale) or a GE Healthcare Bioscience AeKTA oligo-pilot synthesizer (40-200 μmol scale). For this coupling step, phosphoramidite in a quantity exceeding four times the packing ratio of the solid support was delivered, and phosphoramidite condensation was performed for 10 minutes. 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. During the coupling step, 4,5-dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as the activator. The phosphorothioate bond was introduced by sulfidation with a 0.1 M solution of 1:1 pyridine / CH3CN xanthan hydride with a contact time of 3 minutes. A 20% solution of tert-butyl hydroperoxide in H3CN was used as an oxidizing agent, and a phosphodiester nucleoside bond was provided after a contact time of 12 minutes.

[0450] After the desired sequence was constructed, the cyanoethyl phosphate protecting group was deprotected with a 1:1 (v / v) mixture of triethylamine and acetonitrile with a contact time of 45 minutes. The solid support-bound ASO was suspended in aqueous ammonia (28-30% by weight) and heated at 55°C for 6 hours.

[0451] Subsequently, the unbound ASO was filtered and ammonia was removed by boiling. The residue was purified by high-pressure liquid chromatography on a strong anion exchange column (GE Healthcare). Bioscience, Source 30Q, 30 μm, 2.54 × 8 cm, A = 100 mM ammonium acetate in 30% CH3CN aqueous solution, B = 1.5 M NaBr in A, 0–40% B after 60 mins, flow rate 14 mL / min-1, λ = 260 nm). The residue was desalted by HPLC on a reversed-phase column to obtain the desired ASO with an isolation yield of 15–30% based on the initial packing onto the solid support. The ASO was characterized by ion-pair HPLC coupling MS analysis using an Agilent 1100 MSD system.

[0452] Conjugate-free antisense oligonucleotides were synthesized using a standard oligonucleotide synthesis procedure 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 had the same nucleic acid base sequence; ISIS 304801 was a 5-10-5 MOE gapmer with all phosphorothioate bonds, ISIS 647535 was identical to ISIS 304801 except that GalNAc3-1 was conjugated at its 3' end, and ISIS 647536 was identical to ISIS 647535 except that a certain nucleoside bond in the compound was a phosphodiester bond. As further summarized in Table 4, two distinct antisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a 2-10-2 cEt gapmer with all phosphorothioate nucleoside bonds, and ISIS 651900 is ISIS, except that it includes 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. "d" indicates (e.g., cEt), "s" indicates a phosphorothioate nucleoside bond (PS), "o" indicates a phosphodiester nucleoside bond (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. "GalNAc3-1" indicates a conjugated group having the structure shown earlier in Example 9. GalNAc3-1 contains cleavable adenosine that links ASO to the rest of the conjugate, and "GalNAc3-1 a Please note that it is specified as "A". The table above uses this nomenclature to show the complete nucleic acid base sequence including adenosine, which is part of the conjugate. Therefore, in the table above, "A do Sequences ending in "GalNAc3-1" can also be listed, omitting the ". The convention of using the subscript "a" to indicate a cleavable nucleoside or a portion of a conjugated group lacking a cleavable moiety is used throughout these embodiments. This portion of a conjugated group lacking a cleavable moiety is referred to herein as a "cluster," "conjugated cluster," or "GalNAc3 cluster." In certain cases, this is convenient for describing a conjugated 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 targeting human ApoC III and described above, were tested separately in dose-dependent studies to evaluate their ability to inhibit human ApoC III in human ApoC III transgenic mice.

[0456] process Human ApoCIII transgenic mice were maintained in a 12-hour light-dark cycle and continuously fed the Teklad experimental diet. The animals were acclimatized in the research facility for at least 7 days prior to the start of the experiment. ASO was prepared in PBS and sterilized by filtration through a 0.2 micron filter. ASO was dissolved in 0.9% PBS for injection.

[0457] Human ApoC III transgenic mice were intraperitoneally injected with ISIS 304801 or 647535 at concentrations of 0.08, 0.25, 0.75, 2.25, or 6.75 μmol / kg, or with PBS as a control, once a week for two weeks. Each treatment group consisted of four animals. Blood was collected from each mouse 48 hours after the final dose, 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) according to standard protocols. ApoC III mRNA levels in mouse liver were determined using [a specific method]. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen) before normalization to a PBS-treated control. The following results are presented as the mean percentage of ApoC III mRNA levels for each treatment group normalized to a PBS-treated control, and are denoted as "%PBS". The half-effect concentration (ED) of each ASO was determined. 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) We determined the plasma ApoC III protein analysis using the procedure reported by Graham et al (Circulation Research), which was published online prior to its publication on March 29, 2013.

[0461] Approximately 100 μL of plasma isolated from mice was analyzed without dilution 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 followed as described by the supplier.

[0462] As shown in Table 6 below, 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 analyzed using the Bligh and Dyer method (Bligh, E. and Dyer, W. J. Can. J. Biochem. Physiol. 37:911-917, 1959). Extraction was performed using (37, 911-917, 1959), and measurement was performed using a Beckmann-Coulter clinical analyzer and commercially available reagents.

[0464] Triglyceride levels were measured relative to mice injected with PBS and 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 fractions of cholesterol (HDL and LDL). The results are presented in Tables 8 and 9. As illustrated, both antisense compounds lowered total cholesterol levels, decreased 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 decrease in LDL levels are the beneficial cardiovascular effects of antisense inhibition of ApoC III. [Table 8] [Table 9]

[0466] Pharmacokinetic analysis (PK) The PK of ASO was also evaluated. Liver and kidney samples were cleaved and extracted using a standard protocol. Samples were analyzed using MSD1 with 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 compounds were similar to those of these two antisense compounds. Therefore, although 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 in significantly higher concentrations within the liver. In fact, the calculated EC 50 (Provided in Table 10) supports the idea that the observed increase in the strength of the conjugated compound is not solely attributable to the increase in accumulation. This result suggests that the conjugate improved the strength through mechanisms other than hepatic accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.

[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. For therapeutic targets where renal activity is not required, renal exposure carries the risk of nephrotoxicity and does not yield commensurate benefits. Furthermore, high concentrations in the kidney typically result in the loss of the compound into the urine, leading to more rapid clearance. Therefore, renal accumulation is undesirable for non-renal targets. 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 main metabolite of ISIS 647535 is full-length ASO lacking the total conjugate (i.e., ISIS 304801), which originates from cleavage at cleavage site A shown below. Further metabolites originating from other cleavage sites were also observed. These results suggest that the introduction of other cleavable bonds between GalNAc3-1 sugar and ASO, such as esters, peptides, disulfides, phosphoramidates, or acylhydrazones, which can be cleaved by intracellular enzymes, under the reducing environment of the cytosol, or adapted to the acidic pH in 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 targeting human ApoC III and listed in Table 4, were further evaluated in a single-dose study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

[0470] process Human ApoCIII transgenic mice were kept in a 12-hour light-dark cycle, and Tek The animals were continuously fed the lad experimental diet. Before the start of the experiment, the animals were acclimatized in the research facility for at least 7 days. ASO was prepared in PBS and sterilized by filtration through a 0.2 micron filter. ASO was dissolved in 0.9% PBS for injection.

[0471] Human ApoC III transgenic mice were intraperitoneally infused once with ISIS 304801, 647535, or 647536 (as described above), or a PBS-treated control, at the doses shown below. The treatment group consisted of 3 animals, and the control group consisted of 4 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 evaluated 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-infused mice using a standard protocol. ALT and AST levels indicated good tolerance to the antisense compound at all doses.

[0473] These results demonstrate improved strength in antisense compounds containing the 3'-terminal GalNAc3-1 conjugate (ISIS 647535 and 647536) compared to antisense compounds lacking the GalNAc3-1 conjugate (ISIS 304801). Furthermore, ISIS 647536, containing the GalNAc3-1 conjugate and several phosphodiester bonds, is as potent as ISIS 647535, which contains the same conjugate, and all nucleoside-to-nucleoside bonds within the ASO are phosphorothioate bonds. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0474] These results support the improvement of the strength of antisense compounds by GalNAc3-1 conjugates. These results also show equivalent strength for GalNAc3-1 conjugated antisense compounds, and these antisense oligonucleotides have mixed conjugations (ISIS 647536 with six phosphodiester bonds) and a complete phosphorothioate version of the same antisense compound (ISIS 647535).

[0475] Phosphothioate binding provides several properties to antisense compounds. For example, they resist nuclease digestion, bind to proteins, and lead to the accumulation of compounds in the liver rather than the kidneys / urine. These are particularly desirable properties when treating signs in the liver. However, phosphorothioate binding is also associated with inflammatory responses. Therefore, while reducing the number of phosphorothioate bonds in the compound is expected to reduce the risk of inflammation, it also lowers the concentration of the compound in the liver, increases its concentration in the kidney and urine, reduces its stability in the presence of nucleases, and lowers its overall potency. These results indicate that GalNAc3-1 conjugated antisense compounds in which certain phosphorothioate bonds are replaced with phosphodiester bonds are as potent against liver targets as their counterparts with complete phosphorothioate bonds. Such compounds are expected to be less pro-inflammatory (see Example 24, which illustrates experiments showing that a reduction in PS results in a reduction in inflammatory effects).

[0476] Example 22: Effects of GalNAc3-1-conjugated ASO targeting SRB-1 in vivo ISIS 440762 and 651900, both targeting SRB-1 and listed in Table 4, were evaluated in dose-dependent studies for their ability to inhibit SRB-1 in Balb / c mice.

[0477] process Six-week-old male Balb / c mice (Jackson Laboratory, Bar Harbor, ME) were subcutaneously injected once with either ISIS 440762, 651900, or a PBS-treated control at the doses shown below. Each treatment group consisted of four animals. Mice were sacrificed 48 hours after the final dose, and SRB-1 mRNA levels in the liver were determined according to a standard protocol using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR). SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen) before normalization to the PBS-treated control. The results below are presented as the mean percentage of SRB-1 mRNA levels for each treatment group normalized to the PBS-treated control and are denoted as "%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 far more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 440762). These results demonstrate the observed potency benefit of the GalNAc3-1 conjugate using antisense oligonucleotides complementary to different targets and possessing different chemically modified nucleosides, in this case the modified nucleoside containing a restricted ethyl sugar moiety (bicyclic sugar moiety). [Table 16]

[0479] Example 23: Human peripheral blood mononuclear cell (hPBMC) assay protocol hPBMC assays were performed using the BD Vautainer CPT tube method. Whole blood samples were obtained from volunteer donors who gave informed consent at the US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) and collected in 4 to 15 BD Vacutainer CPT 8mL tubes (VWR catalog number BD362753). The approximate starting total whole blood volume in each donor's CPT tube was recorded using the PBMC assay datasheet.

[0480] Blood samples were remixed immediately before centrifugation by gentle inversion of the tubes 8-10 times. CPT tubes were centrifuged in a horizontal (swing-out) rotor at 1500-1800 RCF with the brake off for 30 minutes at room temperature (18-25°C) (2700 RPM, Beckman Allegra 6R). Cells were harvested from the buffy coat interface (between Ficoll and the polymer gel layer) and transferred to 50 mL sterile conical tubes, with a maximum of 5 CPT tubes / 50 mL conical tubes / donor pooled. Cells were then sterilized in PBS (Ca ++ Mg ++Washed twice with a solution that does not contain (GIBCO). Filled the tube to a maximum of 50 mL and mixed by inverting several times. Then, the sample was centrifuged at 330 × g (1215 RPM, Beckman Allegra 6R) at room temperature for 15 minutes, and aspirated as much supernatant as possible without disturbing the pellet. Gently rotated the tube to remove the cell pellet and resuspended the cells in RPMI + 10% FBS + pen / strep (approximately 1 mL / 10 mL of starting whole blood volume). Pipetteed 60 μL of the sample into a sample vial (Beckman Coulter) containing 600 μL of VersaLyse reagent (Beckman Coulter, catalog no. A09777) and gently vortexed for 10-15 seconds. Incubated the sample at room temperature for 10 minutes and mixed again before coefficient. Cell suspensions were counted using a Vicell XR cell viability analyzer (Beckman Coulter) with PBMC cell types (1:11 dilution factor was saved for other parameters). Live cells / mL and viability were recorded. Cell suspensions were incubated in RPMI + 10% FBS + pen / strep at a rate of 1 × 10⁶. 7 Diluted to raw PBMC / mL.

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

[0482] Example 24: Evaluation of the pro-inflammatory effect of GalNAc3-1-coupled ASO in an hPBMC assay. The antisense oligonucleotides (ASOs) listed in Table 17 were evaluated for their pro-inflammatory effects in an hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high-response to 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] The IL-6 level was used as the primary readout. EC was performed using the standard procedure. 50 and E max The calculation was performed. The result was E from two donors. max / EC 50 It is expressed as the average ratio, "E max / EC 50 This is displayed as follows: A lower percentage indicates a relative decrease in the pro-inflammatory response, while a higher percentage indicates a relative increase in the pro-inflammatory response.

[0484] Regarding the test compounds, the compound with the lowest pro-inflammatory activity was PS / PO-bound ASO(ISI (S 616468) was found. The GalNAc3-1 conjugated ASO (ISIS 647535) was slightly less pro-inflammatory than its unconjugated counterpart (ISIS 304801). These results suggest that the incorporation of several PO bonds reduces the pro-inflammatory response, and that the addition of the GalNAc3-1 conjugate does not increase the pro-inflammatory properties of the compound but can reduce the pro-inflammatory response. Therefore, it is expected that antisense compounds containing both mixed PS / PO bonds and the GalNAc3-1 conjugate will result in a lower pro-inflammatory response compared to fully PS-bonded antisense compounds (with or without the GalNAc3-1 conjugate). These results suggest that GalNAc 3- This study demonstrates that conjugated antisense compounds, particularly those with reduced PS content, exhibit lower pro-inflammatory activity.

[0485] In summary, these results suggest that GalNAc3-1 conjugated compounds, particularly those with reduced PS content, can be administered at higher doses than their counterparts, complete PS antisense compounds lacking the GalNAc3-1 conjugator. Since the half-lives of these compounds are not expected to be substantially different, such higher doses would result in lower dosing frequency. In practice, the frequency of such dosing would be even lower, as GalNAc3-1 conjugated compounds are more potent (see Examples 20-22) and re-dosing is required when the concentration of the compound falls below the desired level, and such desired level 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 nucleoside bond (PS), "o" indicates a phosphodiester nucleoside bond (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. do’ -GalNAc3-1 a This indicates a conjugate having the structure GalNAc3-1 shown in Example 9, which is bound to the 3' end of an antisense oligonucleotide, as shown. [Table 18]

[0487] Example 25: Effects of GalNAc3-1-coupled modification 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 approximately 16 hours of treatment, RNA was isolated from the cells, mRNA levels were measured by quantitative real-time PCR, and hApoC III mRNA levels were adjusted according to the total RNA content measured by RIBOGREEN.

[0488] Using standard methods, IC 50 The values ​​were calculated and the results are presented in Table 19. As illustrated, comparable intensity was observed in cells treated with ISIS 647535 compared to the control ISIS 304801. [Table 19]

[0489] In this experiment, the significant strength benefit of GalNAc3-1 coupling observed in vivo was not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro showed increased strength of oligonucleotides containing various GalNAc conjugates compared to oligonucleotides lacking GalNAc conjugates (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 infused with either ISIS 304801 or ISIS 616468 (both mentioned above) or a PBS-treated control at a dose of 25 mg / kg once weekly for two weeks. The treatment group 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). The data from these analyses are presented in Table 20 below.

[0492] These results show a decrease in the strength of the antisense compound (ISIS 616468) with PO / PS in the wings compared to pure 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 identified by structural analysis.

[0495] Since phosphoramidites having the predetermined composition can be prepared using other single-protected substituted or unsubstituted alkyldiols, including but not limited to those presented herein, compound 57 is representative and not intended to be limiting. .

[0496] Example 29: Preparation of Compound 63 [ka] Compounds 61 and 62 were prepared using the same procedure as 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 may be prepared using a procedure similar to that reported in the scientific literature and patent documents of Kim et al. (Synlett, 2003, 12, 1838-1840, and Kim et al.'s published PCT international application WO2004063208).

[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 was prepared using a procedure similar to that reported in the published PCT international application WO2009003009 by Or et al. The protecting group used in Compound 65 is representative and not intended to be limiting, as other protecting groups, including but not limited to those presented herein, may be used.

[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 group used in compound 68 is representative and not intended to be limiting, as other protecting groups, including but not limited to those presented herein, may be used.

[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 preparing conjugated oligomer compound 82 containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end using a solid support (Method I) [ka] [ka] GalNAc3-2 has the following structure. [ka]

[0506] GalNAc3 cluster portion of the conjugated group GalNAc3-2 (GalNAc3-2 a By combining ) with any cleavable portion, various conjugated groups can be obtained. GalNAc3-2 a It has the following formula: [ka]

[0507] VIMAD-conjugated oligomer compound 79b was prepared using a standard procedure in automated DNA / RNA synthesis (Dupouy et al., Angew. Chem. Int. E). (See d., 2006, 45, 3623-3627). Phosphoramidite compounds 56 and 60 were prepared according to the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are representative and not intended to be limiting, as oligomeric compounds having a phosphodiester-conjugated group at the 5' end can be prepared using other phosphoramidite construction blocks, including but not limited to those presented herein. By adjusting the order and amount of phosphoramidites added to the solid support, oligomeric compounds described herein with any predetermined sequence and composition can be prepared.

[0508] Example 38: Alternative method for preparing oligomeric compound 82 containing a phosphodiester bonded GalNAc3-2 conjugate at the 5' end (Method II) [ka] The VIMAD-conjugated oligomer compound 79b was prepared using a standard procedure in 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 according to the procedure illustrated in Example 35. This alternative method involves the phosphodiester-conjugated GalNAc3-2 conjugate in the final step of synthesis. This enables a one-step introduction of the phosphodiester conjugate into the oligomeric compound. The phosphoramidites illustrated are representative and not intended to be limiting, as oligomeric compounds having a phosphodiester conjugate at the 5' end can be prepared using other phosphoramidite construction blocks, including but not limited to those presented herein. By adjusting the order and amount of phosphoramidites added to the solid support, oligomeric compounds described herein with any predetermined sequence and composition can be prepared.

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

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

[0511] GalNAc3 cluster portion of the conjugated group GalNAc3-3 (GalNAc3-3 a By combining ) with any cleavable portion, various conjugated groups can be obtained. GalNAc3-3 a It has the following formula: [ka]

[0512] Example 40: General method for preparing oligomeric compound 89 containing a phosphodiester-linked GalNAc3-4 conjugate at the 3' end using a solid support. [ka] [ka] [ka] GalNAc3-4 has the following structure. [ka]

[0513] In the formula, CM is a cleavable portion. In a particular embodiment, the cleavable portion is as follows: [ka]

[0514] GalNAc3 cluster portion of the conjugated group GalNAc3-4 (GalNAc3-4 a By combining ) with any cleavable portion, various conjugated groups can be obtained. GalNAc3-4 a It 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 those 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] The phosphoramidite construction blocks (compounds 60 and 79a) are prepared according to the procedures illustrated in Examples 28 and 36. The phosphoramidites illustrated are representative and not intended to be limiting, as other phosphoramidite construction blocks can be used to prepare oligomeric compounds having a phosphodiester-bonded conjugate at the 3' end with a predetermined sequence and composition. By adjusting the order and amount of phosphoramidites added to the solid support, oligomeric compounds described herein with any predetermined sequence and composition can be prepared.

[0517] Example 41: General method for preparing an ASO containing a phosphodiester-bonded GalNAc3-2 conjugate (see Example 37 where Bx is adenine) at the 5' position using a solid-phase technique (preparation of ISIS 661134) Unless otherwise specified, all reagents and solutions used in the synthesis of oligomeric compounds shall be purchased from commercial sources. Standard phosphoramidite construction blocks and solid supports shall be purchased, for example, T, A, G, and m This compound is used for the incorporation of nucleoside residues, including a C residue. The 5'-terminated phosphodiester-linked GalNAc3-2 conjugate was synthesized using phosphoramidite compounds 56 and 60. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2'-deoxyribonucleoside and 2'-MOE.

[0518] VIMAD solid support packed into the column (110 μmol / g, Guzaev et al.) Using the phosphoramidite coupling method described in al., 2003, ASO synthesis was performed using an ABI 394 synthesizer (1-2 μmol scale) or a GE Healthcar synthesizer. The process was carried out on an e Bioscience AeKTA oligo-pilot synthesizer (40-200 μmol scale). For this coupling step, phosphoramidite was delivered in a quantity exceeding four times the initial packing of the solid support, and phosphoramidite coupling was performed for 10 minutes. 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. During the coupling step, 4,5-dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as the activator. Phosphothioate bonds were introduced by sulfidation with a 0.1 M solution of xanthan hydride in a 1:1 pyridine / CH3CN mixture, with a contact time of 3 minutes. Phosphodiester nucleoside bonds were provided using a solution of 20% tert-butyl hydroperoxide in CH3CN containing 6% water as the oxidizing agent, with a contact time of 12 minutes.

[0519] After the desired sequence was constructed, the cyanoethyl phosphate protecting group was deprotected with 20% diethylamine (v / v) in toluene with a contact time of 45 minutes. The solid support-bound ASO was suspended in aqueous ammonia (28-30% by weight) and heated at 55°C for 6 hours.

[0520] Subsequently, the unbound ASO was filtered and ammonia was removed by boiling. The residue was purified by high-pressure liquid chromatography on a strong anion exchange column (GE Healthcare). Bioscience, Source 30Q, 30 μm, 2.54 × 8 cm, A = 100 mM ammonium acetate in 30% CH3CN aqueous solution, B = 1.5 M NaBr in A, 0–40% B after 60 mins, flow rate 14 mL / min-1, λ = 260 nm). The residue was desalted by HPLC on a reversed-phase column to obtain the desired ASO with an isolation yield of 15–30% based on the initial packing onto the solid support. The ASO was characterized by ion-pair HPLC coupling 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 nucleoside bond (PS), "o" indicates a phosphodiester nucleoside bond (PO), and "o'" indicates -OP(=O)(OH)-. The superscript "m" indicates 5-methylcytosine. GalNAc3-2 a The structure is shown in Example 37.

[0522] Example 42: General method for preparing ASO containing a GalNAc3-3 conjugate at the 5' position using a solid-phase technique (preparation of ISIS 661166) ISIS 661166 was synthesized using a procedure similar to that 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 coupling 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 nucleoside bond (PS), "o" indicates a phosphodiester nucleoside bond (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' term targeting SRB-1 in vivo (see Examples 37 and 41 where Bx is adenine) ISIS 661134 (see Example 41), containing a phosphodiester-linked GalNAc3-2 conjugate at its 5' end, was tested for SRB-1 antisense inhibition in mice in a dose-dependent study. Unconjugated ISIS 440762 and 651900 (GalNAc3-1 conjugate at its 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 either ISIS 440762, 651900, 661134, or a PBS-treated control at the doses shown below. Each treatment group consisted of four animals. Mice were sacrificed 72 hours after the final dose, 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 a standard protocol. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen) before normalization to the PBS-treated control. The following results are presented as the mean percentage of SRB-1 mRNA levels for each treatment group normalized to the PBS-treated control and are denoted as "%PBS". ED was performed using the same method as described above. 50 The following measurements were taken and are presented below.

[0527] As illustrated in Table 22, treatment with antisense oligonucleotides reduced SRB-1 mRNA levels in a dose-dependent manner. In fact, antisense oligonucleotides containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end (ISIS 661134) or a GalNAc3-1 conjugate linked at the 3' end (ISIS 651900) showed a significant improvement in potency compared to unconjugated antisense oligonucleotides (ISIS 440762). Furthermore, ISIS 661134, containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end, was equivalent in potency to ISIS 651900, containing 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 pharmacokinetics (PK) of ASO in the high-dose group (7 mg / kg) were tested and evaluated in the same manner as illustrated in Example 20. Liver samples were cleaved and extracted using a standard protocol. 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. The results showed that ASO (ISIS) containing a phosphodiester-linked GalNAc3-2 conjugate at the 5' end was found. The primary metabolite detected for 661134) was ISIS 440762 (data not shown). No further metabolites were observed at detectable levels. Unlike its counterpart, further metabolites similar to those reported in Table 10a were observed in ASO (ISIS 651900) with a GalNAc3-1 conjugate at its 3' end. These results suggest that the presence of phosphodiester-linked GalNAc3-1 or GalNAc3-2 conjugates can improve the PK profile of ASOs without compromising their strength.

[0530] Example 44: Effect of PO / PS binding on antisense inhibition of ASOs containing a GalNAc3-1 conjugate at the 3' end (see Example 9) that target SRB-1. Each ISIS contains a GalNAc3-1 conjugate at its 3' end, targeting SRB-1. Compounds 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] ASO is a 5-10-5 MOE gapmer, where the gap region contains 10 2'-deoxyribonucleosides, and each wing region contains 5 2'-MOE modified nucleosides. It contains d. ASO was prepared using the same method as illustrated in Example 19 above, and is 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 nucleoside bond (PS), "o" indicates a phosphodiester nucleoside bond (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 either ISIS 353382, 655861, 655862, or a PBS-treated control at the doses shown 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. Mice were sacrificed 72 hours after the final dose, 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 a standard protocol. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen) before normalization to the PBS-treated control. The following results are presented as the mean percentage of SRB-1 mRNA levels for each treatment group normalized to the PBS-treated control and are denoted as "%PBS". ED was performed using the same method as described above. 50 Measure the following and report the results 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. Specifically, antisense oligonucleotides containing a GalNAc3-1 conjugate at the 3' end (ISIS 655861 and 655862) showed a significant improvement in strength compared to unconjugated antisense oligonucleotides (ISIS 353382). Furthermore, ISIS 655862 with mixed PS / PO binding showed an improvement in strength compared to pure PS (ISIS 655861). [Table 24]

[0535] Serum liver transaminase levels, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-infused mice using a standard protocol. Organ weights were also assessed. The results showed no increase in transaminase levels (Table 25) or organ weights (data not shown) in mice treated with ASO compared to PBS controls. Furthermore, ASO with mixed PS / PO binding (ISIS 655862) showed similar transaminase levels compared to pure PS (ISIS 655861). [Table 25]

[0536] Example 45: Preparation of PFP ester (compound 110a) [ka] [ka] Compound 4 (9.5 g, 28.8 mmol) was treated separately with compound 103a or 103b (38 mmol), then treated with TMSOTf (0.5 equivalents) in dichloromethane (200 mL) and a molecular sieve, and stirred at room temperature for 16 hours. At this point, the organic layer was filtered through Celite and 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% → 10% methanol / dichloromethane) to obtain compounds 104a and 104b in yields of over 80%. LC-MS and proton NMR confirmed their structures.

[0537] Compounds 104a and 104b were treated under the same conditions as compounds 100a-d (Example 47) to obtain compounds 105a and 105b in yields of over 90%. LC-MS and proton NMR confirmed their structures.

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

[0539] Compounds 106a and 106b were treated under the same conditions as compounds 96a-d (Example 47) to obtain 107a (60%) and 107b (20%). LC-MS and proton NMR confirmed their structures.

[0540] Compounds 107a and 107b were treated under the same conditions as compounds 97a-d (Example 47) to obtain compounds 108a and 108b in yields of 40-60%. LC-MS and profilometers were used to obtain compounds 108a and 108b. The NMR spectrum matched its structure.

[0541] Compounds 108a (60%) and 108b (40%) were treated under the same conditions as compounds 100a-d (Example 47) to obtain compounds 109a and 109b in yields of over 80%. LC-MS and proton NMR confirmed their structures.

[0542] Compound 109a was treated under the same conditions as compounds 101a-d (Example 47) to obtain compound 110a in a yield of 30-60%. LC-MS and proton NMR confirmed its 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 a standard solid-phase oligonucleotide procedure. The 5'-hexylamino modified oligonucleotides were dissolved in 0.1 M sodium tetraborate (pH 8.5, 200 μL), and 3 equivalents of selected PFP-esterified GalNAc3 clusters dissolved in DMSO (50 μL) were added. If the PFP ester precipitated upon addition to the ASO solution, DMSO was added until all of the PFP ester was present in the solution. After mixing at room temperature for approximately 16 hours, the reaction was completed. The resulting solution was diluted with water to 12 mL and then precipitated on a spin filter at 3000 rpm 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 ammonia water, mixed at room temperature for 2.5 hours, and then concentrated under vacuum to remove most of the ammonia. The conjugated oligonucleotide was purified, desalted by RP-HPLC, and freeze-dried to obtain the GalNAc3 conjugated oligonucleotide. [ka]

[0544] Oligonucleotide 111 is conjugated with GalNAc3-10. The GalNAc3 cluster portion of the conjugated group GalNAc3-10 (GalNAc3-10 a By combining the ) with any cleavable moiety, various conjugated groups can be obtained. In a particular embodiment, the cleavable moiety is -P(=O)(OH)-A, as shown in the following oligonucleotide synthesized with GalNAc3-10 (ISIS 666881). d -P(=O)(OH)-. GalNAc3-10(GalNAc3-10 a The structure of -CM-) is shown below. [ka]

[0545] ISIS 666881 was prepared according to this general procedure. 5'-hexylamino-modified oligonucleotide (ISIS 660254) was synthesized and purified using a standard solid-phase oligonucleotide procedure. 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) were added. If the PFP ester precipitated upon addition to the ASO solution, an additional DMSO (600 μL) was required to completely dissolve the PFP ester. After mixing at room temperature for approximately 16 hours, the reaction was complete. This solution was diluted with water to a total volume of 12 mL and precipitated on a spin filter at 3000 rpm with a mass cutoff of 3000 Da. This process was repeated twice to remove small molecule impurities. This solution was freeze-dried to dryness, redissolved in concentrated ammonia water, mixed at room temperature for 2.5 hours, and then concentrated under vacuum to remove most of the ammonia. The conjugated oligonucleotide was purified, desalted by RP-HPLC, and freeze-dried to obtain ISIS 666881 (42 mg, 4.7 μmol) in 90% by weight yield. [Table 25-2]

[0546] The capital letters indicate the nucleic acid bases of each nucleoside. m C represents 5-methylcytosine. The subscript "e" indicates a 2'-MOE modified nucleoside, "d" indicates a β-D-2'-deoxyribonucleoside, "s" indicates a phosphorothioate nucleoside bond (PS), "o" indicates a phosphodiester nucleoside bond (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 mixture was stirred at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added together with N,N-diisopropylethylamine (12.35 mL, 72 mmol), and the reaction mixture was stirred at room temperature for 16 hours. At this point, the DMF was reduced by more than 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 oil under reduced pressure. The resulting oil was purified by silica gel chromatography (2% → 10% methanol / dichloromethane) to obtain compounds 92a and 92b in approximately 80% yield. LC-MS and proton NMR confirmed their 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 oil under reduced pressure to obtain compounds 93a and 93b in 85–90% yield.

[0549] Compound 7 or 64 (9.6 mmol) was treated with HBTU (3.7 g, 9.6 mmol) and N,N-diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. Either compound 93a or 93b (3 mmol) was added, and the mixture was stirred at room temperature for 16 hours. At this point, the DMF was reduced by more than 75% under reduced pressure, and then mixed... The compound 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 oil under reduced pressure. The resulting oil was purified by silica gel chromatography (5% → 20% methanol / dichloromethane) to obtain compounds 96a-d in 20-40% yield. LC-MS and proton NMR confirmed the structure.

[0550] Compounds 96a-d (0.75 mmol) were individually hydrogenated in ethanol (75 mL) on Raney nickel for 3 hours. At this point, the catalyst was filtered off through Celite, and the ethanol was removed under reduced pressure to obtain compounds 97a-d in 80-90% yield. LC-MS and proton NMR confirmed their structures.

[0551] Compound 23 (0.32 g, 0.53 mmol) was treated with HBTU (0.2 g, 0.53 mmol) and N,N-diisopropylethylamine (0.19 mL, 1.14 mmol) in DMF (30 mL) for 15 minutes. Compounds 97a-d (0.38 mmol) were added individually to this mixture and stirred at room temperature for 16 hours. At this point, the DMF was reduced by more than 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 oil under reduced pressure. The resulting oil was purified by silica gel chromatography (2% → 20% methanol / dichloromethane) to obtain compounds 98a-d in 30-40% yield. LCMS and proton NMR confirmed the structure.

[0552] Compound 99 (0.17 g, 0.76 mmol) was treated with HBTU (0.29 g, 0.76 mmol) and N,N-diisopropylethylamine (0.35 mL, 2.0 mmol) in DMF (50 mL) for 15 minutes. Compounds 97a-d (0.51 mmol) were added individually to this mixture and stirred at room temperature for 16 hours. At this point, the DMF was reduced by more than 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 oil under reduced pressure. The resulting oil was purified by silica gel chromatography (5% → 20% methanol / dichloromethane) to obtain compounds 100a-d in 40-60% yield. LC-MS and proton NMR confirmed the structure.

[0553] Compounds 100a–d (0.16 mmol) were individually hydrogenated in methanol / ethyl acetate (1:1, 50 mL) over 10% Pd(OH)2 / C for 3 hours. At this point, the catalyst was filtered off through Celite, and organic matter was removed under reduced pressure to obtain compounds 101a–d in 80–90% yield. LC-MS and proton NMR confirmed their structures.

[0554] Compounds 101a-d (0.15 mmol) were individually dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmol). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmol) was added dropwise under argon, and the reaction mixture was stirred at room temperature for 30 minutes. At this point, the DMF was reduced by more than 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 oil under reduced pressure. The resulting oil was purified by silica gel chromatography (2% → 5% methanol / dichloromethane) to obtain compounds 102a-d in approximately 80% yield. LC-MS and proton NMR confirmed the structure. [ka]

[0555] An oligomeric compound 102 containing a GalNAc3-8 conjugated group was prepared using the general procedure illustrated in Example 46. a By combining ) with any cleavable portion, various conjugated groups can be obtained. In one preferred embodiment, the cleavable portion is -P(=O)(OH)-A d It is -P(=O)(OH)-.

[0556] GalNAc3-8 (GalNAc3-8 a The structure of -CM-) is shown below. [ka]

[0557] Example 48: Preparation of oligonucleotide 119 containing GalNAc3-7 [ka] [ka] Compound 112 was synthesized according to the procedure described in the reference (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 was added over carbon (0.5 g). The reaction mixture was stirred under hydrogen at room temperature for 12 hours. The reaction mixture was filtered through a Celite pad, and the pad was washed with 1:1 methanol / ethyl acetate. The filtrate and washings were combined, concentrated to dryness, and compound 105a (quantitative) was obtained. This structure was confirmed by LC-MS.

[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. A solution of compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure to obtain oil. The residue was dissolved in CH2Cl2 (100 mL) and washed with saturated NaHCO3 aqueous solution (100 mL) and brine (100 mL). The organic phase was separated, dried (Na2SO4), filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 10-20% MeOH in dichloromethane to obtain compound 114 (1.45 g, 30%). This structure was obtained by LCMS and 1 This was confirmed by 1H NMR analysis.

[0560] Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol / ethyl acetate (4 mL / 4 mL). Palladium was added on a carbon atom (wet, 0.14 g). The reaction mixture was washed with hydrogen and stirred under hydrogen at room temperature for 12 hours. The mixture was then turned over through a Celite pad. The mixture was filtered. This Celite pad was washed with methanol / ethyl acetate (1:1). The filtrate and washings were combined and evaporated under reduced pressure to obtain compound 115 (quantitative). This structure was analyzed by LC-MS and 1 This was confirmed by 1H 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. A solution of compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added, and the reaction mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure, and the residue was dissolved in CH2Cl2. The organic layer was washed with saturated aqueous NaHCO3 and brine, dried over anhydrous Na2SO4, and filtered. The organic layer was concentrated to dryness, and the resulting residue was purified by silica gel column chromatography and eluted with 3-15% MeOH in dichloromethane to obtain compound 116 (0.84 g, 61%). This structure was analyzed by LC-MS and 1 This was confirmed by 1H 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 was added on a wet carbon (0.074 g). The reaction mixture was washed with hydrogen and stirred under hydrogen at room temperature for 12 hours. The reaction mixture was filtered through a Celite pad. This Celite pad was washed with methanol / ethyl acetate (1:1). The filtrate and washings were combined and evaporated under reduced pressure to obtain compound 117 (0.73 g, 98%). This structure was analyzed by LC-MS and 1 This was confirmed by 1H NMR analysis.

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

[0564] An oligomeric compound 119 containing a GalNAc3-7 conjugated group was prepared using the general procedure illustrated in Example 46. The GalNAc3 cluster moiety of the conjugated group GalNAc3-7 (GalNAc3-7 a By combining ) with any cleavable part, various conjugated groups can be obtained. In a particular embodiment, the cleavable part is -P(=O)(OH)-A d It is -P(=O)(OH)-.

[0565] GalNAc3-7 (GalNAc3-7) a The structure of -CM-) is shown below. [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 the mixture was 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. Further 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 into toluene (300 mL) and washed with 1 M NaHSO4 (3 × 150 mL), saturated NaHCO3 aqueous solution (3 × 150 mL), and brine (2 × 100 mL). The organic layer was dried over Na2SO4. The drying agent was filtered off, and the organic layer was concentrated by rotary evaporation. The crude mixture was purified by silica gel column chromatography and eluted with 35-50% toluene in hexane to obtain compound 122 (15.50 g, 78.13%). This structure was analyzed by LC-MS and 1 Confirmed by 1H 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 HCl:hexane). The reaction mixture was concentrated under reduced pressure to half its volume. The remaining solution was cooled in an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with HCl (120 mL), and washed with brine (100 mL). An emulsion was formed and removed after standing overnight. The organic layer was separated and dried (Na2SO4). 4) The mixture was filtered and evaporated to obtain compound 123 (8.42 g). Residual salts are the estimated cause of the excess mass. LC-MS was consistent with this structure. This product was used without further purification. MW calculated value: 574.36, MW measured value: 575.3 [M+H]+ . [ka]

[0568] Compound 126 was synthesized according to the procedure described in the reference (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, N,N-diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol), and subsequently Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere. The ice bath was removed, and the solution was warmed to room temperature. The reaction was complete after 1 hour and determined by TLC (DCM:MeOH:AA at 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in HCl (200 mL) and washed with 1 M NaHSO4 (3 × 100 mL), saturated NaHCO3 aqueous solution (3 × 100 mL), and brine (2 × 100 mL). The organic phase was separated, dried (Na2SO4), filtered, and concentrated. The residue was purified by silica gel column chromatography on a 50% hexane / EtOAC:100% HCl gradient to obtain compound 127 (9.4 g) as a white foam. LCMS and 1 The 1H NMR spectrum was consistent with its 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 the mixture was stirred at room temperature for 1 hour. The reaction mixture was co-evaporated with toluene (30 mL) under reduced pressure to dryness. The resulting residue was then evaporated in acetonitrile (30 mL). Compound 128 (1.67 g) was obtained as trifluoroacetate by evaporating both L) and toluene (40 mL) twice, and was used in the next step without further purification. LC-MS and 1 The 1H NMR spectrum was consistent with its structure. Mass (m / z): 478.2 [M+H] + .

[0571] In a round-bottom flask, 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 and dried under reduced pressure on P2O5 for 4 hours. The mixture was then dissolved in anhydrous DMF (1 mL) and stirred for 5 minutes. A solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-diisopropylethylamine (0.2 mL) was added. The reaction mixture was stirred at room temperature under an argon atmosphere. After 30 minutes, the reaction was complete and determined by LC-MS 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 NaHSO4 (3 × 20 mL), saturated NaHCO3 aqueous solution (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 obtain compound 129 (96.6 mg). LC-MS and 1 The 1H NMR spectrum matches its 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. A small amount of 10% Pd / C (0.015 mg) was added, and the reaction vessel was washed 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 obtain compound 130 (0.08 g). LC-MS and 1 1H NMR was consistent with the structure. This product was used without further purification. Mass (m / z): 838.3 [M+2H] + . 【057...

Claims

1. A compound comprising a modified oligonucleotide and a conjugated group, wherein the modified oligonucleotide consists of 8 to 80 linked nucleosides and has a nucleic acid base sequence that is at least 85%, 90%, 95%, or 100% complementary to Sequence ID No. 2 encoding transthyretin (TTR).

2. The compound according to claim 1, wherein the nucleic acid base sequence of the modified oligonucleotide is complementary within nucleic acid bases 507 to 608 of SEQ ID NO: 2, and the modified oligonucleotide is at least 85%, 90%, 95%, or 100% complementary to SEQ ID NO:

2.

3. The compound according to claim 1, wherein the nucleic acid base sequence of the modified oligonucleotide is complementary to nucleic acid bases 507-526, 508-527, 515-534, 516-535, 580-599, 585-604, 587-606, or 589-608 of SEQ ID NO: 2, and the modified oligonucleotide is at least 85%, 90%, 95%, or 100% complementary to SEQ ID NO:

2.

4. The compound according to claim 1, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleic acid base sequence containing at least eight consecutive nucleic acid bases from any of the nucleic acid base sequences of SEQ ID NOs. 12, 13, 14, 15, 16, 17, 18, or 19.

5. The compound according to claim 4, wherein the modified oligonucleotide has a nucleic acid base sequence comprising the sequence listed in SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, or 19.

6. The compound according to claim 4, wherein the modified oligonucleotide has a nucleic acid base sequence consisting of the sequences listed in SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, or 19.

7. The compound according to any one of claims 1 to 6, wherein the modified oligonucleotide consists of 20 linked nucleosides.

8. The compound according to any one of claims 1 to 7, wherein the modified oligonucleotide comprises at least one modified sugar.

9. The compound according to claim 8, wherein the modified sugar is a dicyclic sugar.

10. The aforementioned bicyclic sugar is 4'-(CH ２ )-O-2'(LNA), 4'-(CH ２ ) ２ -O-2'(ENA), and 4'-CH(CH ３ The compound according to claim 9, selected from the group consisting of )-O-2'(cEt).

11. The compound according to claim 8, wherein the modified sugar is 2'-O-methoxyethyl.

12. The compound according to any one of claims 1 to 11, wherein the modified oligonucleotide comprises at least one modified nucleic acid base.

13. The compound according to claim 12, wherein the modified nucleic acid base is 5-methylcytosine.

14. Modified oligonucleotides consisting of 20 linked nucleosides having nucleic acid base sequences listed in SEQ ID NOs. 12, 13, 14, 15, 16, 17, 18, or 19. The modified oligonucleotide contains cydo, A gap segment consisting of 10 linked deoxynucleosides, A 5' wing segment consisting of five linked nucleosides, It includes a 3' wing segment consisting of five linked nucleosides, The compound according to any one of claims 1 to 13, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of the 5' wing segment contains 2'-O-methoxyethyl sugar, each nucleoside of the 3' wing segment contains 2'-O-methoxyethyl sugar, the internucleoside bond is a phosphorothioate bond, and each cytosine is 5-methylcytosine.

15. The compound according to any one of claims 1 to 14, wherein the compound is single-chain.

16. The compound according to any one of claims 1 to 14, wherein the compound is double-chain.

17. The compound according to any one of claims 1 to 16, wherein the modified oligonucleotide comprises at least one modified nucleoside bond.

18. The compound according to claim 17, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

19. The compound according to claim 18, wherein the modified oligonucleotide comprises at least one phosphodiester nucleoside bond.

20. The compound according to claim 18, wherein the modified oligonucleotide comprises at least two phosphodiester nucleoside interbonding bonds.

21. The compound according to claim 18, wherein the modified oligonucleotide comprises at least three phosphodiester nucleoside interbonding groups.

22. The compound according to claim 18, wherein the modified oligonucleotide comprises at least four phosphodiester nucleoside interbonding groups.

23. The compound according to claim 18, wherein the modified oligonucleotide comprises at least five phosphodiester nucleoside interbonding groups.

24. The compound according to claim 18, wherein the modified oligonucleotide comprises at least six phosphodiester nucleoside interbonding bonds.

25. The compound according to claim 18, wherein the modified oligonucleotide comprises at least seven phosphodiester nucleoside interbonding groups.

26. The compound according to any one of claims 19 to 25, wherein each nucleoside bond of the modified oligonucleotide is selected from phosphodiester nucleoside bonds and phosphorothioate nucleoside bonds.

27. The compound according to claim 18, wherein each nucleoside bond of the modified oligonucleotide contains a phosphorothioate nucleoside bond, and is a phosphorothioate nucleoside bond.

28. A compound consisting of ISIS 304299 and a conjugated group.

29. A compound consisting of ISIS 420915 and a conjugated group.

30. A compound consisting of ISIS 420921 and a conjugated group.

31. A compound consisting of ISIS 420922 and a conjugated group.

32. A compound consisting of ISIS 420950 and a conjugated group.

33. A compound consisting of ISIS 420955 and a conjugated group.

34. A compound consisting of ISIS 420957 and a conjugated group.

35. A compound consisting of ISIS 420959 and a conjugated group.

36. The compound according to any one of claims 1 to 35, wherein the conjugated group is linked to the modified oligonucleotide at the 5' end of the modified oligonucleotide.

37. The compound according to any one of claims 1 to 35, wherein the conjugated group is linked to the modified oligonucleotide at the 3' end of the modified oligonucleotide.

38. The compound according to any one of claims 1 to 37, wherein the conjugated group comprises exactly one ligand.

39. The compound according to any one of claims 1 to 37, wherein the conjugated group comprises exactly two ligands.

40. The compound according to any one of claims 1 to 37, wherein the conjugated group comprises three or more ligands.

41. The compound according to any one of claims 1 to 37, wherein the conjugated group comprises exactly three ligands.

42. Each ligand is a polysaccharide, modified polysaccharide, mannose, galactose, mannose derivatives, galactose derivatives, D-mannopyranose, L-mannopyranose, D-arabinose, L-galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannopyranose, β-D-mannopyranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, cy Alkaline acid, α-D-galactosamine, N-acetylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamide-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycoyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranose, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside, 2,5-anhydro-D- A compound according to any one of claims 38 to 41, selected from allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

43. The compound according to claim 42, wherein each ligand is N-acetylgalactosamine.

44. The aforementioned conjugated group is as follows: 【Chemistry 1】 A compound according to any one of claims 1 to 37, comprising:

45. The aforementioned conjugated group is as follows: 【Chemistry 2】 A compound according to any one of claims 1 to 37, comprising:

46. The aforementioned conjugated group is as follows: 【Transformation 3】 A compound according to any one of claims 1 to 37, comprising:

47. The aforementioned conjugated group is as follows: 【Chemistry 4】 A compound according to any one of claims 1 to 37, comprising:

48. The aforementioned conjugated group is as follows: 【Transformation 5】 A compound according to any one of claims 1 to 37, comprising:

49. The compound according to any one of claims 1 to 48, wherein the conjugated group comprises at least one phosphorus-binding group or a neutral-binding group.

50. The aforementioned conjugated group is as follows: 【Transformation 6】 【Transformation 7】 【Transformation 8】 Includes a structure selected from among, In the formula, n is between 1 and 12. The compound according to any one of claims 1 to 49, wherein m is 1 to 12.

51. The aforementioned conjugated group is as follows: 【Chemistry 9】 It has a tether having a structure selected from among the following, In the formula, L is either a phosphorus-bonding group or a neutral-bonding group. Z １ However, C(=O)O-R ２ And, Z ２ is H, C １ ~C ６ alkyl, or substituted C １ ~C ６ alkyl, and R ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is each m １ However, independently, they are between 0 and 20, and at least one m １ The compound according to any one of claims 1 to 49, wherein the value is greater than 0 for each tether.

52. The conjugated groups are as follows: 【Chemistry 10】 It has a tether having a structure selected from among the following, During the ceremony, Z ２ However, H or CH ３ And, each m １ However, independently, they are between 0 and 20, and at least one m １ The compound according to any one of claims 1 to 51, wherein the value is greater than 0 for each tether.

53. The aforementioned conjugated group is as follows: 【Chemistry 11】 It has a tether having a structure selected from among the following, In the formula, n is between 1 and 12. The compound according to any one of claims 1 to 51, wherein m is 1 to 12.

54. The compound according to any one of claims 1 to 53, wherein the conjugated group is covalently bonded to the modified oligonucleotide.

55. The aforementioned compound is defined by the following formula 【Chemistry 12】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, C is the aforementioned conjugate linker, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

56. The aforementioned compound is defined by the following formula 【Chemistry 13】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, C is the aforementioned conjugate linker, D is the branching base, Each E is a tether, Each F is an aqueous solution, Each n is independently either 0 or 1. The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

57. The aforementioned compound is defined by the following formula 【Chemistry 14】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, C is the aforementioned conjugate linker, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

58. The aforementioned compound is defined by the following formula 【Chemistry 15】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, C is the aforementioned conjugate linker, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

59. The aforementioned compound is defined by the following formula 【Chemistry 16】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, C is the aforementioned conjugate linker, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

60. The aforementioned compound is defined by the following formula 【Chemistry 17】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

61. The aforementioned compound is defined by the following formula [Chemistry 18] It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

62. The aforementioned compound is defined by the following formula 【Chemistry 19】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 1 to 54, wherein q is an integer from 1 to 5.

63. The aforementioned conjugate linker is as follows: 【Chemistry 20】 It has a structure in which it is selected from among In the formula, each L is independently either a phosphorus bond group or a neutral bond group. The compound according to any one of claims 1 to 62, wherein each n is independently 1 to 20.

64. The aforementioned conjugate linker is as follows: 【Chemistry 21】 A compound according to any one of claims 1 to 62, having a structure selected from among them.

65. The aforementioned conjugate linker has the following structure 【Chemistry 22】 A compound according to any one of claims 1 to 62, having the following characteristics.

66. The aforementioned conjugate linker is as follows: 【Chemistry 23】 A compound according to any one of claims 1 to 62, having a structure selected from among them.

67. The aforementioned conjugate linker is as follows: 【Chemistry 24】 A compound according to any one of claims 1 to 62, having a structure selected from among them.

68. The aforementioned conjugate linker is as follows: 【Chemistry 25】 A compound according to any one of claims 1 to 63, having a structure selected from among them.

69. The compound according to any one of claims 1 to 63, wherein the conjugated linker comprises pyrrolidine.

70. The compound according to any one of claims 1 to 64, wherein the conjugated linker does not contain pyrrolidine.

71. The compound according to any one of claims 1 to 63 or 69 to 70, wherein the conjugated linker contains PEG.

72. The compound according to any one of claims 1 to 63 or 69 to 71, wherein the conjugated linker comprises an amide.

73. The compound according to any one of claims 1 to 63 or 69 to 72, wherein the conjugated linker comprises at least two amides.

74. The compound according to any one of claims 1 to 63 or 71, wherein the conjugated linker does not contain an amide.

75. The compound according to any one of claims 1 to 63 or 69 to 73, wherein the conjugated linker comprises a polyamide.

76. The conjugated linker comprises an amine, as described in any one of claims 1 to 63 or 69 to 75. A compound of [unclear].

77. The compound according to any one of claims 1 to 63 or 69 to 76, wherein the conjugated linker contains one or more disulfide bonds.

78. The compound according to any one of claims 1 to 63 or 69 to 77, wherein the conjugated linker includes a protein-binding portion.

79. The compound according to claim 78, wherein the protein-binding portion contains a lipid.

80. The protein-binding portion is cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. The compound according to claim 78, selected from vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosome lysants, steroids (e.g., ubaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-derived lithocholic acid), or cationic lipids.

81. The compound according to claim 78, wherein the protein-binding portion is selected from C16-C22 long-chain saturated or unsaturated fatty acids, cholesterol, cholic acid, vitamin E, adamantane, or 1-pentafluoropropyl.

82. The aforementioned conjugate linker is as follows: 【Chemistry 26】 It has a structure in which it is selected from among The compound according to any one of claims 1 to 63, wherein each n is independently 1 to 20 and p is 1 to 6.

83. The aforementioned conjugate linker is as follows: 【Chemistry 27】 It has a structure in which it is selected from among The compound according to any one of claims 1 to 63, wherein each n is independently 1 to 20 in the formula.

84. The aforementioned conjugate linker is as follows: 【Chemistry 28】 A compound according to any one of claims 1 to 63, having a structure selected from among them.

85. The aforementioned conjugate linker is as follows: 【Chemistry 29】 It has a structure in which it is selected from among The compound according to any one of claims 1 to 63, wherein n is 1 to 20 in the formula.

86. The aforementioned conjugate linker is as follows: 【Transformation 30】 A compound according to any one of claims 1 to 63, having a structure selected from among them.

87. The aforementioned conjugate linker is as follows: 【Chemistry 31】 It has a structure in which it is selected from among The compound according to any one of claims 1 to 63, wherein each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

88. The aforementioned conjugate linker has the following structure 【Chemistry 32】 A compound according to any one of claims 1 to 63, having the following characteristics.

89. The branching base has the following structure 【Transformation 33】 Having one of the following, In the formula, each A １ However, independently, O, S, C=O, or NH, The compound according to any one of claims 1 to 88, wherein each n is independently 1 to 20.

90. The branching base has the following structure 【Transformation 34】 Having one of the following, In the formula, each A １ However, independently, O, S, C=O, or NH, The compound according to any one of claims 1 to 88, wherein each n is independently 1 to 20.

91. The branching base has the following structure 【Chemistry 35】 A compound according to any one of claims 1 to 88, having the following characteristics.

92. The branching base has the following structure 【Transformation 36】 A compound according to any one of claims 1 to 88, having the following characteristics.

93. The branching base has the following structure 【Chemistry 37】 A compound according to any one of claims 1 to 88, having the following characteristics.

94. The branching base has the following structure 【Transformation 38】 A compound according to any one of claims 1 to 88, having the following characteristics.

95. The compound according to any one of claims 1 to 88, wherein the branched group contains an ether.

96. The branching base has the following structure 【Chemistry 39】 It has, Each n is independently between 1 and 20. The compound according to any one of claims 1 to 88, wherein m is 2 to 6.

97. The branching base has the following structure 【Chemistry 40】 A compound according to any one of claims 1 to 88, having the following characteristics.

98. The branching base has the following structure 【Chemistry 41】 A compound according to any one of claims 1 to 88, having the following characteristics.

99. The aforementioned branching base, 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 or 【Chemistry 46】 Includes, In the formula, each j is an integer between 1 and 3. The compound according to any one of claims 1 to 88, wherein each n is an integer from 1 to 20.

100. The aforementioned branching base, 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 [Transformation 50] or 【Chemistry 51】 A compound according to any one of claims 1 to 88, comprising:

101. Each tether is as follows: 【Chemistry 52】 Selected from among, In the formula, L is selected from phosphorus-binding groups and neutral-binding groups. Z １ However, C(=O)O-R ２ And, Z ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is R ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is each m １ However, independently, they are between 0 and 20, and at least one m １ The compound according to any one of claims 1 to 100, wherein the compound is greater than 0 for each tether.

102. Each tether is as follows: 【Chemistry 53】 Selected from among, During the ceremony, Z ２ However, H or CH ３ And, each m １ However, independently, they are between 0 and 20, and at least one m ２ The compound according to any one of claims 1 to 100, wherein the compound is greater than 0 for each tether.

103. Each tether is as follows: 【Chemistry 54】 Selected from among, In the formula, n is between 1 and 12. The compound according to any one of claims 1 to 100, wherein m is 1 to 12.

104. The compound according to any one of claims 1 to 100, wherein at least one tether contains ethylene glycol.

105. The compound according to any one of claims 1 to 100 or 102, wherein at least one tether comprises an amide.

106. The compound according to any one of claims 1 to 100 or 102, wherein at least one tether comprises a polyamide.

107. The compound according to any one of claims 1 to 100 or 102, wherein at least one tether contains an amine.

108. The compound according to any one of claims 1 to 100 or 102 to 107, wherein at least two tethers are different from each other.

109. The compound according to any one of claims 1 to 100 or 102 to 107, wherein all of the tethers are identical to each other.

110. Each tether is as follows: 【Transformation 55】 Selected from among, In the formula, each n is independently between 1 and 20. The compound according to any one of claims 1 to 100, wherein each p is 1 to about 6.

111. Each tether is as follows: 【Transformation 56】 A compound according to any one of claims 1 to 100, selected from among them.

112. Each tether has the following structure 【Chemistry 57】 It has, The compound according to any one of claims 1 to 100, wherein each n is independently 1 to 20 in the formula.

113. Each tether has the following structure 【Transformation 58】 A compound according to any one of claims 1 to 100, having the following characteristics.

114. The aforementioned tether is as follows: 【Chemistry 59】 or 【Transformation 60】 It has a structure in which it is selected from among The compound according to any one of claims 1 to 100, wherein each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

115. The aforementioned tether is as follows: 【Chemistry 61】 A compound according to any one of claims 1 to 100, having a structure selected from among the following.

116. The compound according to any one of claims 1 to 115, wherein the ligand is galactose.

117. The compound according to any one of claims 1 to 115, wherein the ligand is mannose-6-phosphate.

118. Each ligand is as follows: 【Transformation 62】 Selected from among, In the formula, each R １ The compound according to any one of claims 1 to 115, wherein the compound is selected from OH and NHCOOH.

119. Each ligand is as follows: 【Transformation 63】 A compound according to any one of claims 1 to 115, selected from among them.

120. Each ligand has the following structure 【Chemistry 64】 A compound according to any one of claims 1 to 115, having the following characteristics.

121. Each ligand has the following structure 【Transformation 65】 A compound according to any one of claims 1 to 115, having the following characteristics.

122. The compound according to any one of claims 1 to 121, wherein the conjugated group includes a cell targeting moiety.

123. The aforementioned conjugated group has the following structure 【Chemical Formula 66】 It includes a cell targeting region having, The compound according to claim 122, wherein each n is independently 1 to 20 in the formula.

124. The cell target portion has the following structure 【Transformation 67】 A compound according to any one of claims 122, having the following characteristics.

125. The cell target portion has the following structure 【Transformation 68】 It has, The compound according to claim 122, wherein each n is independently 1 to 20 in the formula.

126. The cell target portion has the following structure 【Transformation 69】 The compound according to claim 122, having the following characteristics.

127. The cell target portion is as follows: 【Transformation 70】 The compound according to claim 122, comprising:

128. The cell target portion is as follows: 【Chemistry 71】 The compound according to claim 122, comprising:

129. The cell target portion has the following structure 【Chemistry 72】 The compound according to claim 122, having the following characteristics.

130. The cell target portion has the following structure 【Transformation 73】 The compound according to claim 122, having the following characteristics.

131. The cell target portion is as follows: 【Chemistry 74】 The compound according to claim 122, comprising:

132. The cell target portion has the following structure 【Chemistry 75】 The compound according to claim 122, having the following characteristics.

133. The cell target portion is as follows: 【Transformation 76】 The compound according to claim 122, comprising:

134. The cell target portion is as follows: 【Chemical 77】 The compound according to claim 122, comprising:

135. The cell target portion is as follows: 【Transformation 78】 The compound according to claim 122, comprising:

136. The cell target portion has the following structure 【Transformation 79】 The compound according to claim 122, having the following characteristics. The cell target portion has the following structure 【Chemistry 80】 The compound according to claim 122, having the following characteristics.

137. The cell target portion has the following structure 【Chemistry 81】 The compound according to claim 122, having the following characteristics.

138. The cell target portion has the following structure 【Chemistry 82】 The compound according to claim 122, having the following characteristics.

139. The cell target portion has the following structure 【Chemistry 83】 The compound according to claim 122, having the following characteristics.

140. The cell target portion is as follows: 【Chemical 84】 The compound according to claim 122, comprising:

141. The cell target portion is as follows: 【Chemical 85】 The compound according to claim 122, comprising:

142. The cell target portion is as follows: 【Chemical 86】 The compound according to claim 122, comprising:

143. The cell target portion is as follows: 【Transformation 87】 The compound according to claim 122, comprising:

144. The cell target portion has the following structure 【Chemical 88】 The compound according to claim 122, having the following characteristics.

145. The cell target portion is as follows: 【Chemistry 89】 The compound according to claim 122, comprising:

146. The cell target portion has the following structure 【Chemistry 90】 The compound according to claim 122, having the following characteristics.

147. The cell target portion is as follows: 【Chemistry 91】 Includes, In the formula, each Y is O, S, substituted or unsubstituted C. １ ~C １０ The compound according to claim 122, selected from alkyl, amino, substituted amino, azide, alkenyl, or alkynyl.

148. The aforementioned conjugated group is as follows: 【Chemistry 92】 Includes, In the formula, each Y is O, S, substituted or unsubstituted C. １ ~C １０ A compound according to any one of claims 1 to 121, selected from alkyl, amino, substituted amino, azide, alkenyl, or alkynyl.

149. The cell target portion has the following structure 【Chemistry 93】 It has, In the formula, each Y is O, S, substituted or unsubstituted C. １ ~C １０ The compound according to claim 122, selected from alkyl, amino, substituted amino, azide, alkenyl, or alkynyl.

150. The aforementioned conjugated group is as follows: 【Chemical 94】 A compound according to any one of claims 1 to 149, comprising:

151. The aforementioned conjugated group is as follows: 【Chemical 95】 A compound according to any one of claims 1 to 149, comprising:

152. The aforementioned conjugated group is as follows: 【Chemistry 96】 A compound according to any one of claims 1 to 149, comprising:

153. The aforementioned conjugated group is as follows: 【Chemistry 97】 A compound according to any one of claims 1 to 149, comprising:

154. The compound according to any one of claims 1 to 153, wherein the conjugated group comprises a cleavable portion selected from phosphodiesters, amides, deoxynucleosides, or esters.

155. The compound according to any one of claims 1 to 154, wherein the conjugated group includes a portion that can be cleaved by a phosphodiester.

156. The compound according to any one of claims 1 to 152, wherein the conjugated group does not contain a cleavable portion, and the conjugated group includes a phosphorothioate bond between the conjugated group and the oligonucleotide.

157. The compound according to any one of claims 1 to 156, wherein the conjugated group includes a portion that can be amide-cleaved.

158. The compound according to any one of claims 1 to 156, wherein the conjugated group includes a portion that can be esterified.

159. The above compound has the following structure 【Chem.98】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

160. The above compound has the following structure 【Chem.99】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

161. The above compound has the following structure 【Chemistry 100】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, Z is H or a bonded solid support. The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

162. The above compound has the following structure 【Chemistry 101】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, Z is H or a bonded solid support. The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

163. The above compound has the following structure 【Chemical Engineering 102】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

164. The above compound has the following structure 【Chemistry 103】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

165. The above compound has the following structure 【Chemical 104】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

166. The above compound has the following structure 【Chemistry 105】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

167. The above compound has the following structure 【Chemistry 106】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

168. The above compound has the following structure 【Chemistry 107】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

169. The above compound has the following structure 【Chemistry 108】 It has, where Q １３ is H or O(CH ２ ) ２ -OCH ３ and A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

170. The above compound has the following structure 【Chemistry 109】 It has, where Q １３ is H or O(CH ２ ), ２ -OCH ３ and A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

171. The above compound has the following structure 【Chemical 110】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

172. The above compound has the following structure 【Chemistry 111】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

173. The above compound has the following structure 【Chemistry 112】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

174. The aforementioned conjugated group is as follows: 【Chemistry 113】 Includes, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

175. The aforementioned conjugated group is as follows: 【Chemistry 114】 Includes, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

176. The aforementioned conjugated group is as follows: 【Chemical 115】 Includes, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 1 to 158, wherein Bx is a heterocyclic base moiety.

177. B ｘ The compound according to any one of claims 159 to 176, wherein the compound is selected from adenine, guanine, thymine, uracil, cytosine, or 5-methylcytosine.

178. B ｘ The compound according to any one of claims 159 to 177, wherein is adenine.

179. B ｘ The compound according to any one of claims 159 to 177, wherein is thymine.

180. Q １３ O(CH ２ ) ２ - OCH ３ The compound according to any one of claims 159 to 176.

181. Q １３ The compound according to any one of claims 159 to 176, wherein is H.

182. A composition comprising the compound or a salt thereof according to any one of claims 1 to 181, and at least one of pharmaceutically acceptable carriers or diluents.

183. A prodrug comprising the compound according to any one of claims 1 to 181.

184. A method comprising administering to an animal the compound according to any one of claims 1 to 181, the composition according to claim 182, or the prodrug according to claim 183.

185. The method according to claim 184, wherein the animal is a human.

186. The method according to claim 184 or 185, wherein administration of the compound prevents, treats, improves or delays the progression of transthyretin amyloidosis.

187. The method according to any one of claims 184 to 186, comprising co-administering the compound or composition and a second agent.

188. The method according to claim 187, wherein the compound or composition and the second agent are administered simultaneously.

189. The method according to any one of claims 184 to 188, wherein the administration is directed to the choroid plexus.

190. A method for reducing transthyretin mRNA or protein expression in an animal, comprising administering to the animal the compound according to any one of claims 1 to 181, the composition according to claim 182, or the prodrug according to claim 183, thereby reducing transthyretin mRNA or protein expression in the animal.

191. The method according to claim 190, wherein the animal is a human.

192. The method according to claim 190 or 191, wherein a reduction in transthyretin mRNA or protein expression prevents, treats, improves or delays the progression of transthyretin amyloidosis.

193. The method according to any one of claims 190 to 192, comprising co-administering the compound or composition and a second drug.

194. The method according to claim 193, wherein the compound or composition and the second agent are administered simultaneously.

195. The method according to any one of claims 190 to 194, wherein the compound or composition is administered to the choroid plexus.

196. A method for treating transthyretin amyloidosis in a subject, comprising administering to the subject a therapeutically effective amount of the compound according to any one of claims 1 to 181, the composition according to claim 182, or the prodrug according to claim 183.

197. Administration of the aforementioned compound or composition may result in emotional instability, dyscoordination, nystagmus, spastic paraplegia, lack of muscle coordination, visual impairment, insomnia, paresthesia, myoclonus, blindness, speech disorders, carpal tunnel syndrome, epilepsy, subarachnoid hemorrhage, stroke and cerebral hemorrhage, hydrocephalus, ataxia, as well as spastic paralysis, coma, sensory neuropathy, paresthesia, hypoesthesia, motor neuropathy, autonomic neuropathy, orthostatic hypotension, cyclic constipation, cyclic diarrhea, nausea, vomiting, decreased sweating, impotence, delayed gastric emptying, urinary retention, urinary incontinence, progressive heart disease, fatigue, shortness of breath, weight loss, loss of appetite, and anesthesia. The method according to claim 196, which alleviates at least one symptom associated with transthyretin amyloidosis, selected from the group consisting of stabbing pain, weakness, tongue hypertrophy, nephrotic syndrome, congestive heart failure, exertional dyspnea, peripheral edema, arrhythmia, palpitations, altered consciousness, syncope, postural hypotension, peripheral nerve disease, sensorimotor disorder, lower limb neuropathy, upper limb neuropathy, hyperalgesia, changes in temperature sensation, weakness of the lower limbs, cachexia, peripheral edema, hepatomegaly, purpura, diastolic dysfunction, ventricular premature contractions, cranial nerve disorders, decreased deep tendon reflexes, intravitreal amyloid deposition, vitreous opacity, dry eye, glaucoma, scallop-like appearance of the pupil, and swelling of the lower limbs due to fluid retention.

198. The method according to claim 196 or 197, comprising co-administering the compound or composition and a second agent.

199. The method according to claim 198, wherein the compound or composition and the second agent are administered simultaneously.

200. The method according to any one of claims 196 to 199, wherein the compound or composition is administered to the choroid plexus.

201. The method according to any one of claims 196 to 200, wherein the subject is a human.

202. A compound comprising a modified oligonucleotide and a conjugated group, wherein the modified oligonucleotide consists of 8 to 80 linked nucleosides and has a nucleic acid base sequence that is at least 85%, 90%, 95%, or 100% complementary to Sequence ID No. 1, which encodes hepatitis B virus (HBV).

203. The compound according to claim 202, wherein the nucleic acid base sequence of the modified oligonucleotide is complementary to the nucleic acid bases 1583-1602, 1780-1799, 411-427, 1266-1285, 1577-1596, 1585-1604, 1583-1598, 1264-1279, or 1780-1797 of SEQ ID NO: 1, and the modified oligonucleotide is at least 85%, 90%, 95%, or 100% complementary to SEQ ID NO:

1.

204. The compound according to claim 202, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleic acid base sequence containing at least eight consecutive nucleic acid bases from any of the nucleic acid base sequences of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, or 11.

205. The compound according to claim 204, wherein the modified oligonucleotide has a nucleic acid base sequence comprising the sequence listed in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, or 11.

206. The compound according to claim 204, wherein the modified oligonucleotide has a nucleic acid base sequence consisting of the sequences listed in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, or 11.

207. The modified oligonucleotide is, A gap segment consisting of linked deoxynucleosides, A 5' wing segment consisting of linked nucleosides, It includes a 3' wing segment consisting of linked nucleosides, The compound according to any one of claims 202 to 206, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of the 5' wing segment contains 2'-O-methoxyethyl sugar, each nucleoside of the 3' wing segment contains 2'-O-methoxyethyl sugar, the internucleoside bond is a phosphorothioate bond, and each cytosine is 5-methylcytosine thing.

208. The modified oligonucleotide is, A gap segment consisting of linked deoxynucleosides, A 5' wing segment consisting of linked nucleosides, It includes a 3'-wing segment consisting of linked nucleosides, The compound according to any one of claims 202 to 207, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of the 5' wing segment contains 2'-O-methoxyethyl sugar or a restricted ethyl sugar, each nucleoside of the 3' wing segment contains 2'-O-methoxyethyl sugar or a restricted ethyl sugar, the internucleoside bond is a phosphorothioate bond, and each cytosine is 5-methylcytosine.

209. The compound according to any one of claims 202 to 208, wherein the compound is single-chain.

210. The compound according to any one of claims 202 to 208, wherein the compound is double-chain.

211. The compound according to any one of claims 202 to 210, wherein the modified oligonucleotide comprises at least one modified nucleoside bond.

212. The compound according to claim 211, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

213. The compound according to claim 212, wherein the modified oligonucleotide comprises at least one phosphodiester nucleoside bond.

214. The compound according to claim 212, wherein the modified oligonucleotide comprises at least two phosphodiester nucleoside interbonding groups.

215. The compound according to claim 212, wherein the modified oligonucleotide comprises at least three phosphodiester nucleoside interbonding groups.

216. The compound according to claim 212, wherein the modified oligonucleotide comprises at least four phosphodiester nucleoside interbonding groups.

217. The compound according to claim 212, wherein the modified oligonucleotide comprises at least five phosphodiester nucleoside interbonding groups.

218. The compound according to claim 212, wherein the modified oligonucleotide comprises at least six phosphodiester nucleoside interbonding groups.

219. The compound according to claim 212, wherein the modified oligonucleotide comprises at least seven phosphodiester nucleoside interbonding bonds.

220. The compound according to any one of claims 213 to 219, wherein each nucleoside bond of the modified oligonucleotide is selected from phosphodiester nucleoside bonds and phosphorothioate nucleoside bonds.

221. Claim 212, wherein each nucleoside bond of the modified oligonucleotide includes a phosphorothioate nucleoside bond, and is a phosphorothioate nucleoside bond. A compound of [unclear].

222. A compound consisting of ISIS 505358 and a conjugated group.

223. A compound consisting of ISIS 509934 and a conjugated group.

224. A compound consisting of ISIS 510100 and a conjugated group.

225. A compound consisting of ISIS 552023 and a conjugated group.

226. A compound consisting of ISIS 552024 and a conjugated group.

227. A compound consisting of ISIS 552032 and a conjugated group.

228. A compound consisting of ISIS 552859 and a conjugated group.

229. A compound consisting of ISIS 552925 and a conjugated group.

230. A compound consisting of ISIS 577119 and a conjugated group.

231. The compound according to any one of claims 202 to 230, wherein the conjugated group is linked to the modified oligonucleotide at the 5' end of the modified oligonucleotide.

232. The compound according to any one of claims 202 to 230, wherein the conjugated group is linked to the modified oligonucleotide at the 3' end of the modified oligonucleotide.

233. The compound according to any one of claims 202 to 232, wherein the conjugated group comprises exactly one ligand.

234. The compound according to any one of claims 202 to 232, wherein the conjugated group comprises exactly two ligands.

235. The compound according to any one of claims 202 to 232, wherein the conjugated group comprises three or more ligands.

236. The compound according to any one of claims 202 to 232, wherein the conjugated group comprises exactly three ligands.

237. Each ligand is a polysaccharide, modified polysaccharide, mannose, galactose, mannose derivatives, galactose derivatives, D-mannopyranose, L-mannopyranose, D-arabinose, L-galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-g Glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, α-D-galactosamine, N-acetylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamide-2,3-di-O-methyl-D-mannol A compound according to any one of claims 202 to 236, selected from pyranose, 2-deoxy-2-sulfamino-D-glucopyranose, N-glycoyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranose, 4-thio-β-D-galactopyranoside, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside, 2,5-anhydro-D-alononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

238. The compound according to claim 237, wherein each ligand is N-acetylgalactosamine.

239. The aforementioned conjugated group is as follows: 【Chemistry 116】 A compound according to any one of claims 202 to 238, comprising:

240. The aforementioned conjugated group is as follows: 【Chemistry 117】 A compound according to any one of claims 202 to 238, comprising:

241. The aforementioned conjugated group is as follows: 【Chemistry 118】 A compound according to any one of claims 202 to 238, comprising:

242. The aforementioned conjugated group is as follows: 【Chemical 119】 A compound according to any one of claims 202 to 238, comprising:

243. The aforementioned conjugated group is as follows: 【Chemical 120】 A compound according to any one of claims 202 to 238, comprising:

244. The compound according to any one of claims 202 to 243, wherein the conjugated group comprises at least one phosphorus-binding group or a neutral-binding group.

245. The aforementioned conjugated group is as follows: 【Chemistry 121】 【Chemistry 122】 【Chemical 123】 Includes a structure selected from among, In the formula, n is between 1 and 12. The compound according to any one of claims 202 to 244, wherein m is 1 to 12.

246. The aforementioned conjugated group is as follows: 【Chemistry 124】 It has a tether having a structure selected from among the following, In the formula, L is either a phosphorus-bonding group or a neutral-bonding group. Z １ However, C(=O)O-R ２ And, Z ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is R ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is each m １ However, independently, they are between 0 and 20, and at least one m １ The compound according to any one of claims 202 to 245, wherein the value is greater than 0 for each tether.

247. The conjugated groups are as follows: 【Chemistry 125】 It has a tether having a structure selected from among the following, During the ceremony, Z ２ However, H or CH ３ And, each m １ However, independently, they are between 0 and 20, and at least one m １ The compound according to any one of claims 202 to 246, wherein the value is greater than 0 for each tether.

248. The aforementioned conjugated group is as follows: 【Chemistry 126】 It has a tether having a structure selected from among the following, In the formula, n is between 1 and 12. The compound according to any one of claims 202 to 247, wherein m is 1 to 12.

249. The compound according to any one of claims 202 to 248, wherein the conjugated group is covalently bonded to the modified oligonucleotide.

250. The aforementioned compound is defined by the following formula 【Chemistry 127】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, C is the aforementioned conjugate linker, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

251. The aforementioned compound is defined by the following formula 【Chemistry 128】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, C is the aforementioned conjugate linker, D is the branching base, Each E is a tether, Each F is an aqueous solution, Each n is independently either 0 or 1. The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

252. The aforementioned compound is defined by the following formula 【Chemistry 129】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, C is the aforementioned conjugate linker, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

253. The aforementioned compound is defined by the following formula 【Chemistry 130】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, C is the aforementioned conjugate linker, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

254. The aforementioned compound is defined by the following formula 【Chemistry 131】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, C is the aforementioned conjugate linker, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

255. The aforementioned compound is defined by the following formula 【Chemistry 132】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

256. The aforementioned compound is defined by the following formula 【Chemistry 133】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, B is the portion that can be cut, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

257. The aforementioned compound is defined by the following formula 【Chemistry 134】 It has a structure represented by, During the ceremony, A is the modified oligonucleotide, D is the branching base, Each E is a tether, Each F is an aqueous solution, The compound according to any one of claims 202 to 249, wherein q is an integer from 1 to 5.

258. The aforementioned conjugate linker is as follows: 【Chemistry 135】 It has a structure in which it is selected from among In the formula, each L is independently either a phosphorus bond group or a neutral bond group. The compound according to any one of claims 202 to 257, wherein each n is independently 1 to 20.

259. The aforementioned conjugate linker is as follows: 【Transformation 136】 A compound according to any one of claims 202 to 257, having a structure selected from among them.

260. The aforementioned conjugate linker has the following structure 【Chemistry 137】 A compound according to any one of claims 202 to 257, having the following characteristics.

261. The aforementioned conjugate linker is as follows: 【Chemistry 138】 A compound according to any one of claims 202 to 257, having a structure selected from among them.

262. The aforementioned conjugate linker is as follows: 【Chemistry 139】 A compound according to any one of claims 202 to 257, having a structure selected from among them.

263. The aforementioned conjugate linker is as follows: [Chemical 140] A compound according to any one of claims 202 to 257, having a structure selected from among them.

264. The compound according to any one of claims 202 to 263, wherein the conjugated linker comprises pyrrolidine.

265. The compound according to any one of claims 202 to 263, wherein the conjugated linker does not contain pyrrolidine.

266. The compound according to any one of claims 202 to 265, wherein the conjugated linker contains PEG.

267. The compound according to any one of claims 202 to 266, wherein the conjugated linker comprises an amide.

268. The compound according to any one of claims 202 to 266, wherein the conjugated linker comprises at least two amides.

269. The compound according to any one of claims 202 to 266, wherein the conjugated linker does not contain an amide.

270. The compound according to any one of claims 202 to 269, wherein the conjugated linker comprises a polyamide.

271. The compound according to any one of claims 202 to 270, wherein the conjugated linker contains an amine.

272. The compound according to any one of claims 202 to 271, wherein the conjugated linker contains one or more disulfide bonds.

273. The compound according to any one of claims 202 to 272, wherein the conjugated linker includes a protein-binding portion.

274. The compound according to claim 273, wherein the protein-binding portion contains a lipid.

275. The protein-binding portion is cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. The compound according to claim 273, selected from vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosome lysants, steroids (e.g., ubaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-derived lithocholic acid), or cationic lipids.

276. The compound according to claim 273, wherein the protein-binding portion is selected from C16-C22 long-chain saturated or unsaturated fatty acids, cholesterol, cholic acid, vitamin E, adamantane, or 1-pentafluoropropyl.

277. The aforementioned conjugate linker is as follows: 【Chemistry 141】 It has a structure in which it is selected from among The compound according to any one of claims 202 to 276, wherein each n is independently 1 to 20 and p is 1 to 6.

278. The aforementioned conjugate linker is as follows: 【Chemistry 142】 It has a structure in which it is selected from among The compound according to any one of claims 202 to 277, wherein each n is independently 1 to 20 in the formula.

279. The aforementioned conjugate linker is as follows: 【Chemistry 143】 A compound according to any one of claims 202 to 277, having a structure selected from among them.

280. The aforementioned conjugate linker is as follows: 【Chemistry 144】 It has a structure in which it is selected from among The compound according to any one of claims 202 to 277, wherein n is 1 to 20 in the formula.

281. The aforementioned conjugate linker is as follows: 【Chemistry 145】 A compound according to any one of claims 202 to 277, having a structure selected from among them.

282. The aforementioned conjugate linker is as follows: 【Chemistry 146】 It has a structure in which it is selected from among The compound according to any one of claims 202 to 277, wherein each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

283. The aforementioned conjugate linker has the following structure 【Chemistry 147】 A compound according to any one of claims 202 to 277, having the following characteristics.

284. The branching base has the following structure 【Chemistry 148】 Having one of the following, In the formula, each A １ However, independently, O, S, C=O, or NH, The compound according to any one of claims 202 to 283, wherein each n is independently 1 to 20.

285. The branching base has the following structure 【Chemistry 149】 Having one of the following, In the formula, each A １ However, independently, O, S, C=O, or NH, The compound according to any one of claims 202 to 283, wherein each n is independently 1 to 20.

286. The branching base has the following structure [Chemical 150] A compound according to any one of claims 202 to 283, having the following characteristics.

287. The branching base has the following structure 【Chemistry 151】 A compound according to any one of claims 202 to 283, having the following characteristics.

288. The branching base has the following structure 【Chemistry 152】 A compound according to any one of claims 202 to 283, having the following characteristics.

289. The branching base has the following structure 【Chemistry 153】 A compound according to any one of claims 202 to 283, having the following characteristics.

290. The compound according to any one of claims 202 to 283, wherein the branched group contains an ether.

291. The branching base has the following structure 【Chemistry 154】 It has, Each n is independently between 1 and 20. The compound according to any one of claims 202 to 283, wherein m is 2 to 6.

292. The branching base has the following structure 【Chemistry 155】 A compound according to any one of claims 202 to 283, having the following characteristics.

293. The branching base has the following structure 【Chemistry 156】 A compound according to any one of claims 202 to 283, having the following characteristics.

294. The aforementioned branching base is as follows: 【Chemistry 157】 【Chemistry 158】 【Chemistry 159】 [Chemical 160] or 【Chemistry 161】 Includes, In the formula, each j is an integer between 1 and 3. The compound according to any one of claims 202 to 283, wherein each n is an integer from 1 to 20.

295. The aforementioned branching base is as follows: 【Chemistry 162】 【Chemical 163】 【Chemistry 164】 【Chemistry 165】 or 【Chemistry 166】 A compound according to any one of claims 202 to 283, comprising:

296. Each tether is as follows: 【Chemistry 167】 Selected from among, In the formula, L is selected from phosphorus-binding groups and neutral-binding groups. Z １ However, C(=O)O-R ２ And, Z ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is R ２ However, H, C １ ~C ６ Alkyl or substituted C １ ~C ６ Alki is each m １ However, independently, they are between 0 and 20, and at least one m １ The compound according to any one of claims 202 to 295, wherein the value is greater than 0 for each tether.

297. Each tether is as follows: 【Chemical 168】 Selected from among, During the ceremony, Z ２ However, H or CH ３ And, each m ２ However, independently, they are between 0 and 20, and at least one m ２ The compound according to any one of claims 202 to 295, wherein the value is greater than 0 for each tether.

298. Each tether is as follows: 【Chemistry 169】 Selected from among, In the formula, n is between 1 and 12. The compound according to any one of claims 202 to 295, wherein m is 1 to 12.

299. The compound according to any one of claims 202 to 295, wherein at least one tether contains ethylene glycol.

300. The compound according to any one of claims 202 to 295 or 297, wherein at least one tether comprises an amide.

301. The compound according to any one of claims 202 to 295 or 297, wherein at least one tether comprises a polyamide.

302. The compound according to any one of claims 202 to 295 or 297, wherein at least one tether contains an amine.

303. The compound according to any one of claims 202 to 295 or 297, wherein at least two tethers are different from each other.

304. The compound according to any one of claims 202 to 295 or 297, wherein all of the tethers are identical to each other.

305. Each tether is as follows: 【Chemistry 170】 Selected from among, In the formula, each n is independently between 1 and 20. The compound according to any one of claims 202 to 304, wherein each p is 1 to about 6.

306. Each tether is as follows: 【Chemistry 171】 A compound according to any one of claims 202 to 304, selected from among them.

307. Each tether has the following structure 【Chemistry 172】 It has, The compound according to any one of claims 202 to 304, wherein each n is independently 1 to 20 in the formula.

308. Each tether has the following structure 【Chemistry 173】 A compound according to any one of claims 202 to 304, having the following characteristics.

309. The aforementioned tether is as follows: 【Chemistry 174】 or 【Chemistry 175】 It has a structure in which it is selected from among The compound according to any one of claims 202 to 304, wherein each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

310. The aforementioned tether is as follows: 【Chemistry 176】 A compound according to any one of claims 202 to 304, having a structure selected from among them.

311. The compound according to any one of claims 202 to 310, wherein the ligand is galactose.

312. The compound according to any one of claims 202 to 310, wherein the ligand is mannose-6-phosphate.

313. Each ligand is as follows: 【Chemistry 177】 Selected from among, In the formula, each R １ The compound according to any one of claims 202 to 310, wherein the compound is selected from OH and NHCOOH.

314. Each ligand is as follows: 【Chemistry 178】 A compound according to any one of claims 202 to 310, selected from among them.

315. Each ligand has the following structure 【Chemistry 179】 A compound according to any one of claims 202 to 310, having the following characteristics.

316. Each ligand has the following structure 【Chemistry 180】 A conjugated antisense compound according to any one of claims 202 to 310, having the following characteristics.

317. The compound according to any one of claims 202 to 317, wherein the conjugated group comprises a cell targeting moiety.

318. The aforementioned conjugated group has the following structure 【Chemistry 181】 It includes a cell targeting region having, The compound according to claim 317, wherein each n is independently 1 to 20 in the formula.

319. The cell target portion has the following structure 【Chemistry 182】 The compound according to claim 317, having the following characteristics.

320. The cell target portion has the following structure 【Chemistry 183】 It has, The compound according to claim 317, wherein each n is independently 1 to 20 in the formula.

321. The cell target portion has the following structure 【Chemistry 184】 The compound according to claim 317, having the following characteristics.

322. The cell target portion is as follows: 【Chemistry 185】 The compound according to claim 317, comprising:

323. The cell target portion is as follows: 【Chemical 186】 The compound according to claim 317, comprising:

324. The cell target portion has the following structure 【Chemistry 187】 The compound according to claim 317, having the following characteristics.

325. The cell target portion has the following structure 【Chemical 188】 The compound according to claim 317, having the following characteristics.

326. The cell target portion is as follows: 【Chemical 189】 The compound according to claim 37, comprising:

327. The cell target portion has the following structure 【Chemistry 190】 The compound according to claim 317, having the following characteristics.

328. The cell target portion is as follows: 【Chemistry 191】 The compound according to claim 317, comprising:

329. The cell target portion is as follows: 【Chemistry 192】 The compound according to claim 317, comprising:

330. The cell target portion is as follows: 【Chemistry 193】 The compound according to claim 317, comprising:

331. The cell target portion has the following structure 【Chemistry 194】 The compound according to claim 317, having the following characteristics. The cell target portion has the following structure 【Chemistry 195】 The compound according to claim 317, having the following characteristics.

332. The cell target portion has the following structure 【Chemistry 196】 The compound according to claim 317, having the following characteristics.

333. The cell target portion has the following structure 【Chemistry 197】 The compound according to claim 317, having the following characteristics.

334. The cell target portion has the following structure 【Chemistry 198】 The compound according to claim 317, having the following characteristics.

335. The cell target portion is as follows: 【Chemistry 199】 The compound according to claim 317, comprising:

336. The cell target portion is as follows: 【Chemistry 200】 The compound according to claim 317, comprising:

337. The cell target portion is as follows: 【Chemical Engineering 201】 The compound according to claim 317, comprising:

338. The cell target portion is as follows: 【Chemical Engineering 202】 The compound according to claim 317, comprising:

339. The cell target portion has the following structure 【Chemical 203】 The compound according to claim 317, having the following characteristics.

340. The cell target portion is as follows: 【Chemical 204】 The compound according to claim 317, comprising:

341. The cell target portion has the following structure 【Chemical 205】 The compound according to claim 317, having the following characteristics.

342. The cell target portion is as follows: 【Chemical 206】 Includes, In the formula, each Y is O, S, substituted or unsubstituted C. １ ~C １０ The compound according to claim 317, selected from alkyl, amino, substituted amino, azide, alkenyl, or alkynyl.

343. The aforementioned conjugated group is as follows: 【Chemical 207】 Includes, In the formula, each Y is O, S, substituted or unsubstituted C. １ ~C １０ A compound according to any one of claims 202 to 317, selected from alkyl, amino, substituted amino, azide, alkenyl, or alkynyl.

344. The cell target portion has the following structure 【Chemical 208】 It has, In the formula, each Y is O, S, substituted or unsubstituted C. １ ~C １０ The compound according to claim 317, selected from alkyl, amino, substituted amino, azide, alkenyl, or alkynyl.

345. The aforementioned conjugated group is as follows: 【Chemical Engineering 209】 A compound according to any one of claims 202 to 317, comprising:

346. The aforementioned conjugated group is as follows: 【Chemical 210】 A compound according to any one of claims 202 to 317, comprising:

347. The aforementioned conjugated group is as follows: 【Chemistry 211】 A compound according to any one of claims 202 to 317, comprising:

348. The aforementioned conjugated group is as follows: 【Chemical Engineering 212】 A compound according to any one of claims 202 to 317, comprising:

349. The compound according to any one of claims 202 to 348, wherein the conjugated group comprises a cleavable portion selected from phosphodiesters, amides, deoxynucleosides, or esters.

350. The compound according to any one of claims 202 to 348, wherein the conjugated group includes a phosphodiester cleavable portion.

351. The compound according to any one of claims 202 to 348, wherein the conjugated group does not contain a cleavable portion, and the conjugated group includes a phosphorothioate bond between the conjugated group and the oligonucleotide.

352. The compound according to any one of claims 202 to 351, wherein the conjugated group includes a portion that can be amide-cleaved.

353. The compound according to any one of claims 202 to 351, wherein the conjugated group includes a portion that can be esterified.

354. The above compound has the following structure 【Chemistry 213】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

355. The above compound has the following structure 【Chemical 214】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

356. The above compound has the following structure 【Chemical 215】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, Z is H or a bonded solid support. The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

357. The above compound has the following structure 【Chemical 216】 It has, In the formula, each n is independently between 1 and 20. Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, Z is H or a bonded solid support. The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

358. The above compound has the following structure 【Chemical 217】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

359. The above compound has the following structure 【Chemistry 218】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

360. The above compound has the following structure 【Chemical 219】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

361. The above compound has the following structure 【Chemical 220】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

362. The above compound has the following structure 【Chemistry 221】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

363. The above compound has the following structure 【Chemistry 222】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

364. The above compound has the following structure 【Chemistry 223】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

365. The above compound has the following structure 【Chemistry 224】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

366. The above compound has the following structure 【Chemical 225】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

367. The above compound has the following structure 【Chemistry 226】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

368. The above compound has the following structure 【Chemistry 227】 It has, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

369. The aforementioned conjugated group is as follows: 【Chemistry 228】 Includes, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

370. The aforementioned conjugated group is as follows: 【Chemistry 229】 Includes, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

371. The aforementioned conjugated group is as follows: 【Chemistry 230】 Includes, In the formula, Q １３ However, H or O (CH ２ ) ２ - OCH ３ And, A is the modified oligonucleotide, The compound according to any one of claims 202 to 353, wherein Bx is a heterocyclic base moiety.

372. B ｘ The compound according to any one of claims 354 to 371, wherein the compound is selected from adenine, guanine, thymine, uracil, cytosine, or 5-methylcytosine.

373. B ｘ The compound according to any one of claims 354 to 372, wherein is adenine.

374. B ｘ The compound according to any one of claims 354 to 372, wherein is thymine.

375. Q １３ O(CH ２ ) ２ - OCH ３ The compound according to any one of claims 354 to 371.

376. Q １３ The compound according to any one of claims 354 to 371, wherein is H.

377. A composition comprising the compound or a salt thereof according to any one of claims 202 to 376, and at least one of pharmaceutically acceptable carriers or diluents.

378. A prodrug comprising the compound described in any one of claims 202 to 376.

379. A method for treating an HBV-related disease, disorder, or condition in a subject, comprising administering to the subject the compound according to any one of claims 202 to 376, the composition according to claim 377, or the prodrug according to claim 378, wherein the disease, disorder, or condition is jaundice, hepatitis, hepatic fibrosis, inflammation, cirrhosis, hepatic failure, liver cancer, diffuse hepatoinflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or liver disease-related transplantation.

380. A method for reducing the HBV antigen level in a subject infected with HBV, comprising administering to the subject the compound according to any one of claims 202 to 376, the composition according to claim 377, or the prodrug according to claim 378, thereby reducing the HBV antigen level in the subject.

381. The method according to claim 380, wherein the HBV antigen is HBsAG.

382. The method according to claim 380, wherein the HBV antigen is HBeAG.

383. The following structure 【Chemistry 231】 A compound containing, A compound in which X is a conjugated group containing GalNAc.

384. The following structure 【Chemistry 232】 Compounds containing these compounds.

385. The following structure 【Chemical 233】 Compounds containing these compounds.

386. The following structure 【Chemistry 234】 A compound containing, In the formula, R １ However, -OCH ２ CH ２ OCH ３ (MOE) and R ２ However, is it H or R １ and R ２ Either they come together to form a bridge, and therefore, R １ However, it is -O-, and R ２ However, -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 - Select from R １ and R ２ They are directly connected, R of each ring on the same ring ３ and R ４ For each pair, independently for each ring, R ３ However, H and -OCH ２ CH ２ OCH ３ Selected from, and R ４ However, is it H or R ３ and R ４ Either they come together to form a bridge, and therefore, R ３ However, it is -O-, and R ４ However, -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 - Select from R ３ and R ４ They are directly connected, R ５ However, H and -CH ３ Selected from, Z is S － and O － A compound selected from among them.

387. The following structure 【Chemical 235】 A compound containing, A compound in which X is a conjugated group containing GalNAc.

388. The following structure 【Chemistry 236】 Compounds containing these compounds.

389. The following structure 【Chemistry 237】 Compounds containing these compounds.

390. The following structure 【Chemical 238】 A compound containing, In the formula, R １ However, -OCH ２ CH ２ OCH ３ (MOE) and R ２ However, is it H or R １ and R ２ Either they come together to form a bridge, and therefore, R １ However, it is -O-, and R ２ However, -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 - Select from R １ and R ２ They are directly connected, R of each ring on the same ring ３ and R ４ For each pair, independently for each ring, R ３ However, H and -OCH ２ CH ２ OCH ３ Selected from, and R ４ However, is it H or R ３ and R ４ Either they come together to form a bridge, and therefore, R ３ However, it is -O-, and R ４ However, -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 - Select from R ３ and R ４ They are directly connected, R ５ However, H and -CH ３ Selected from, Z is S － and O － A compound selected from among them.

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