Compositions and methods for modulating apolipoprotein C-III expression

Abstract

Provided herein are oligomeric compounds having a conjugate group that target apolipoprotein C-III (ApoCIII). In certain embodiments, the oligomeric compound targeting ApoCIII is conjugated to an N-acetyl galactosamine. Also disclosed herein are conjugated oligomeric compounds targeting ApoCIII for use in reducing ApoCIII to treat, prevent, or ameliorate diseases, disorders or conditions associated with ApoCIII. Certain diseases, disorders, or conditions associated with ApoCIII include inflammatory, cardiovascular, and / or metabolic diseases, disorders, or conditions. The conjugated oligomeric compounds disclosed herein can be used to treat such diseases, disorders, or conditions in an individual in need thereof.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202110394825.6, filing date May 1, 2014, and invention name “Compositions and methods for regulating the expression of apolipoprotein C-III”, which is a divisional application of the Chinese invention patent application with application number 201480035624.5, filing date May 1, 2014, and invention name “Compositions and methods for regulating the expression of apolipoprotein C-III”, which is a national phase application with international application number PCT / US2014 / 036462, which requires: filing date Claims priority to U.S. Provisional Application No. 61 / 818,442, filed May 1, 2013, 61 / 823,826, filed May 15, 2013, 61 / 843,887, filed July 8, 2013, 61 / 871,673, filed August 29, 2013, 61 / 880,790, filed September 20, 2013, 61 / 976,991, filed April 8, 2014, and 61 / 986,867, filed April 30, 2014, the entireties of which are hereby incorporated by reference herein.

[0002] Sequence Listing

[0003] This application is filed in electronic format along with a sequence listing. The sequence listing is provided as a file named 036502-8033CN03-sequncelisting, created on November 21, 2024, and is 347 KB in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Background of the Invention

[0005] The principle behind antisense technology is that antisense compounds hybridize with target nucleic acids and regulate the amount, activity and / or function of target nucleic acids. For example, in some cases, antisense compounds cause changes in the transcription or translation of the target. The regulation of expression can be achieved by, for example, degradation of target mRNA or occupancy-based inhibition. An example of the regulation of RNA target function achieved by degradation is the degradation of target RNA based on RNase H when hybridizing with DNA-like antisense compounds. Another example of the regulation of gene expression achieved by target degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing achieved by utilizing the mechanism of RNA-induced silencing complex (RISC). Another example of the regulation of RNA target function is by occupancy-based mechanisms, such as the mechanisms naturally adopted by microRNAs. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The combination of antisense compounds and microRNAs prevents microRNAs from binding to their messenger RNA targets, thereby interfering with the function of microRNAs. MicroRNA mimics can enhance natural microRNA functions. Certain antisense compounds change the splicing of precursor mRNAs (pre-mRNAs). Regardless of the specific mechanism, sequence specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutic agents to selectively modulate the expression of genes involved in disease pathogenesis.

[0006] Antisense technology is an effective means for modulating the expression of one or more specific gene products and may therefore prove particularly useful in many therapeutic, diagnostic and research applications. Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for the target nucleic acid. In 1998, antisense compounds (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) is the first antisense drug to receive marketing approval from the U.S. Food and Drug Administration (FDA) and is currently a treatment for cytomegalovirus (CMV)-induced retinitis in AIDS patients.

[0007] New chemical modifications improve the effectiveness and efficacy of antisense compounds, revealing the possibility of oral delivery and enhancing subcutaneous administration, reducing the possibility of side effects and producing improvements in patient convenience. Chemical modifications that increase the effectiveness of antisense compounds allow for the use of lower doses, which reduce the possibility of toxicity and reduce the overall cost of treatment. Modifications that increase degradation resistance make clearance from the body slower, thereby allowing less frequent administration. Different types of chemical modifications can be combined in a compound to further optimize the effectiveness of the compound.

[0008] Apolipoprotein C-III (also known as APOC3, APOC-III, ApoCIII, and APO C-III) is a component of HDL and triglyceride-rich lipoproteins (TG). Elevated levels of ApoCIII are associated with elevated TG levels and diseases such as cardiovascular disease, metabolic syndrome, obesity, and diabetes (Chan et al., Int J Clin Pract, 2008, 62:799-809; Onat et al., Atherosclerosis, 2003, 168:81-89; Mendivil et al., Circulation, 2011, 124:2065-2072; Mauger et al., J. Lipid Res, 2006. 47:1212-1218; Chan et al., Clin. Chem, 2002. 278-283; Ooi et al., Clin. Sci, 2008. 114:611-624; Davidsson et al., J. Lipid Res. 2005. 46: 1999-2006; Sacks et al., Circulation, 2000. 102: 1886-1892; Lee et al., Arterioscler Thromb Vasc Biol, 2003. 23: 853-858). ApoCIII inhibits lipoprotein lipase (LPL) and lipolysis by interfering with the binding of lipoproteins to the glycosaminoglycan matrix on the cell surface, thereby slowing the clearance of triglyceride-rich lipoproteins (Shachter, Curr. Opin. Lipidol, 2001, 12, 297-304).

[0009] Antisense technology has emerged as an effective means for reducing the expression of certain gene products and may prove to have unique applicability in many therapeutic, diagnostic, and research applications related to ApoCIII regulation. Antisense compounds targeting ApoCIII and related methods for inhibiting ApoCIII have been previously disclosed (see, e.g., U.S. Patent 7,598,227, U.S. Patent 7,750,141, PCT Publication WO 2004 / 093783, PCT Publication WO 2012 / 149495, and PCT / US14 / 016546, all of which are incorporated herein by reference). Antisense Compound ISIS-APOCIII Targeting ApoCIII Rx It has been tested in Phase I and II clinical trials. However, no antisense compounds targeting ApoCIII have been approved for commercial use, and therefore, there remains a need to provide patients with additional and more effective treatment options. SUMMARY OF THE INVENTION

[0011] In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides conjugated antisense compounds comprising antisense oligonucleotides complementary to nucleic acid transcripts. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in the cell.

[0012] The asialoglycoprotein receptor (ASGP-R) has been previously described. See, for example, Park et al., PNAS Vol. 102, No. 47, pp. 17125-17129 (2005). The receptor is expressed on liver cells, particularly hepatocytes. In addition, it has been shown that compounds comprising clusters of three N-acetylgalactosamine (GalNAc) ligands can bind to ASGP-R, thereby causing the compound to be taken up into cells. See, for example, Khorev et al., Bioorganic and Medicinal Chemistry 16, 9, pp. 5216-5231 (May 2008). Therefore, conjugates comprising the GalNAc clusters have been used to promote the uptake of certain compounds into liver cells (specifically, hepatocytes). For example, it has been shown that certain conjugates containing GalNAc increase the activity of double-stranded siRNA compounds in liver cells in vivo. In such cases, the conjugate containing GalNAc is typically attached to the sense strand of the siRNA duplex. Because the sense strand is discarded before the antisense strand finally hybridizes to the target nucleic acid, there is less 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 8,106,022. Certain conjugate groups described herein are more active and / or easier to synthesize than previously described conjugate groups.

[0013] In certain embodiments of the present invention, the conjugate is linked to a single-stranded antisense compound, including but not limited to RNase H-based antisense compounds and antisense compounds that alter the splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain linked to the antisense compound for a time sufficient to provide a benefit (improved uptake into the cell), but should then be cleaved or otherwise not interfere with subsequent steps necessary for activity, such as hybridization with the target nucleic acid and interaction with RNase H or enzymes associated with splicing or splicing regulation. This balance of properties is even more important in the context of single-stranded antisense compounds than in siRNA compounds where the conjugate can simply be linked to the sense strand. Disclosed herein are conjugated single-stranded antisense compounds that have improved potency in liver cells in vivo compared to the same antisense compound in the absence of the conjugate. The balance of properties required for these compounds, such as improved potency, is surprising given the known properties.

[0014] In certain embodiments, the conjugate groups herein include cleavable moieties. As noted, it is not desirable to be bound by the mechanism, and logically, the conjugate should remain on the compound for a time sufficient to provide enhanced uptake, but thereafter, it is desirable that some parts of the conjugate or ideally all of the conjugate are cracked, thereby releasing the parent compound (e.g., antisense compounds) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. The embodiment utilizes the endogenous nuclease in the cell by making the remainder (cluster) of the conjugate be connected to the antisense oligonucleotide via one or more cleavable bonds (such as those with phosphodiester linkages) through nucleosides. In certain embodiments, clustering is combined with cleavable nucleosides by phosphodiester linkages. In certain embodiments, cleavable nucleosides are connected to antisense oligonucleotides (antisense compounds) by phosphodiester linkages. In certain embodiments, the conjugate groups may include two or three cleavable nucleosides. In the embodiments, the cleavable nucleosides are connected to each other by cleavable bonds (such as those with phosphodiester linkages), are connected to antisense compounds and / or are connected to clustering. Certain conjugates herein do not comprise a cleavable nucleoside but rather a cleavable bond.Sufficient cleavage of the conjugate from the oligonucleotide is shown to be provided by at least one bond susceptible to cleavage in the cell (the cleavable bond).

[0015] In certain embodiments, the conjugated antisense compound is a prodrug. The prodrug is administered to an animal and is ultimately metabolized to a more active form. For example, the conjugated antisense compound is cleaved to remove all or part of the conjugate, thereby producing an active (or more active) form of the antisense compound that lacks all or some of the conjugate.

[0016] In certain embodiments, the conjugate is connected to the 5' end of the oligonucleotide. Some of the 5'-conjugates are more effectively cracked than counterparts with similar conjugate groups connected to the 3' end. In certain embodiments, improved activity can be relevant to improved cracking. In certain embodiments, the oligonucleotide comprising the conjugate at the 5' end has greater usefulness than the oligonucleotide comprising the conjugate at the 3' end (see, for example, embodiments 56, 81, 83 and 84). In addition, 5'-connections allow simpler oligonucleotide synthesis. Usually, oligonucleotides are synthesized in the 3' to 5' direction on a solid support. In order to obtain 3'-conjugated oligonucleotides, usually the 3' nucleosides conjugated in advance are connected to a solid support, then oligonucleotides are constructed routinely. However, the conjugated nucleosides are connected to a solid support to increase the complexity of synthesis. In addition, using the method, the conjugate is then present in the entire synthesis of the oligonucleotide and may degrade during subsequent steps or may limit the type of usable reaction and reagent. Using the structures and techniques described herein for 5'-conjugated oligonucleotides, oligonucleotides can be synthesized using standard automated techniques and the conjugate introduced with the last (5'-most) nucleoside or after cleavage of the oligonucleotide from the solid support.

[0017] In view of the prior art and the present disclosure, those of ordinary skill can simply prepare any conjugate and conjugated oligonucleotide herein.In addition, the synthesis of some of the conjugates disclosed herein and conjugated oligonucleotide is simpler and / or requires fewer steps, and is therefore cheaper than the synthesis of previously disclosed conjugates, thereby providing advantages in manufacturing. For example, the synthesis of some conjugate groups is made up of fewer synthesis steps, thereby causing the conjugate group yield to increase relative to the previously described conjugates. Conjugate groups such as the GalNAc3-10 in Example 46 and the GalNAc3-7 in Example 48 are simpler than previously described conjugates, and the previously described conjugates are such as those described in US8,106,022 or US7,262,177 that require the assembly of more chemical intermediates. Therefore, these and other conjugates described herein are superior to previously described compounds in terms of use with any oligonucleotide, and the oligonucleotides include any chain of single-stranded oligonucleotides and double-stranded oligonucleotides (e.g., siRNA).

[0018] Similarly, disclosed herein are conjugate groups having only one or two GalNAc ligands. As shown, the conjugate groups improve the activity of antisense compounds. The compound is easier to prepare than the conjugate comprising three GalNAc ligands. The conjugate groups comprising one or two GalNAc ligands can be connected to any antisense compound, including any chain of single-stranded oligonucleotides and double-stranded oligonucleotides (e.g., siRNA).

[0019] In certain embodiments, the conjugates herein do not substantially alter certain measures of tolerability. For example, it is shown herein that conjugated antisense compounds are no more immunogenic than the unconjugated parent compound. Because efficacy is improved, embodiments in which tolerability remains the same (or even in which tolerability is only slightly worse compared to the efficacy gain) have improved properties for treatment.

[0020] In certain embodiments, conjugation allows people to change antisense compounds in a way that has less attractive results in the absence of conjugation. For example, in certain embodiments, one or more thiophosphate linkages of a complete thiophosphate antisense compound are replaced by phosphodiester linkages to cause some measured improvements in tolerance. For example, in some cases, the antisense compound with one or more phosphodiester linkages has less immunogenicity than the same compound in which each linkage is a thiophosphate. However, in some cases, as shown in Example 26, replacing one or more thiophosphate linkages with phosphodiester linkages also causes cellular uptake to decrease and / or loss of efficacy. In certain embodiments, the conjugated antisense compound described herein tolerates the linkage changes, wherein when compared with the complete thiophosphate counterparts of the conjugation, uptake and efficacy loss are little or no loss. In fact, in certain embodiments, for example in Examples 44, 57, 59 and 86, the oligonucleotide comprising a conjugate and at least one phosphodiester internucleoside linkage actually shows increased in vivo efficacy, even relative to the complete thiophosphate counterparts also comprising the same conjugate. Furthermore, because conjugation results in a substantial increase in uptake / potency, a slight loss of this substantial gain may be acceptable to achieve improved tolerability. Thus, in certain embodiments, the conjugated antisense compound comprises at least one phosphodiester linkage.

[0021] In certain embodiments, the conjugation of the antisense compounds herein causes an increase in delivery, uptake and activity in hepatocytes. Therefore, more compounds are delivered to liver tissue. However, in certain embodiments, only delivering an increase cannot explain the overall increase in activity. In certain embodiments, more compounds enter hepatocytes. In certain embodiments, even an increase in hepatocyte uptake cannot explain the overall increase in activity. In the embodiments, the productive uptake of the conjugated compound increases. For example, as shown in Example 102, relative to non-parenchymal cells, certain embodiments of the conjugate containing GalNAc increase the enrichment of antisense oligonucleotides in hepatocytes. This enrichment is beneficial to oligonucleotides targeting genes expressed in hepatocytes.

[0022] In certain embodiments, the conjugated antisense compounds herein cause kidney exposure to decrease. For example, as shown in Example 20, the concentration of the antisense oligonucleotide comprising some embodiments of the conjugate containing GalNAc in the kidney is lower than the concentration of the antisense oligonucleotide lacking the conjugate containing GalNAc. This has some beneficial therapeutic significance. For the treatment indication (indication) of the activity not desired in the kidney, the exposure to the kidney has the risk of nephrotoxicity without corresponding benefit. In addition, the high concentration in the kidney usually causes the compound to be lost to urine, thereby resulting in faster clearance. Therefore, for non-kidney targets, kidney accumulation is undesirable.

[0023] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following formula:

[0024]

[0025] in

[0026] A is an antisense oligonucleotide;

[0027] B is the cleavable part

[0028] C is the conjugate linker

[0029] D is a branched group

[0030] Each E is a tether;

[0031] Each F is a ligand; and

[0032] q is an integer between 1 and 5.

[0033] In the above figure and in similar figures herein, the branching group "D" branches as many times as necessary to accommodate the number of (EF) groups indicated by "q". Thus, when q = 1, the formula is:

[0034] ABCDEF

[0035] When q=2, the formula is:

[0036]

[0037] When q=3, the formula is:

[0038]

[0039] When q=4, the formula is:

[0040]

[0041] When q=5, the formula is:

[0042]

[0043] In certain embodiments, conjugated antisense compounds are provided having the following structure:

[0044] In certain embodiments, conjugated antisense compounds are provided having the following structure:

[0045] In certain embodiments, conjugated antisense compounds are provided having the following structure:

[0046]

[0047] In certain embodiments, conjugated antisense compounds are provided having the following structure:

[0048]

[0049] The present disclosure provides the following non-limiting embodiments:

[0050] In embodiments having more than one particular variable (e.g., more than one "m" or "n"), unless otherwise indicated, each of said particular variables is independently selected. Thus, for structures having more than one n, each n is independently selected, so they may or may not be the same as one another.

[0051] Certain embodiments provide a compound comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides. In certain embodiments, the modified oligonucleotide with the conjugate group consists of 20 linked nucleosides.

[0052] Certain embodiments provide compounds comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence that is complementary to an equal length portion of nucleobases 3533 to 3552 of SEQ ID NO:3, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:3.

[0053] Certain embodiments provide compounds comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence that is complementary to an equal length portion of nucleobases 3514 to 3558 of SEQ ID NO:3, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:3.

[0054] Certain embodiments provide compounds comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence of any one of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221. In certain embodiments, the conjugated modified oligonucleotide has a nucleobase sequence comprising at least 8 consecutive nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221. In certain embodiments, the compound consists of any one of SEQ ID NOs: 19-96, 209-221 and a conjugate group.

[0055] Certain embodiments provide a compound comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence of SEQ ID NO: 87. In certain embodiments, the modified oligonucleotide having the conjugate group has a nucleobase sequence of at least 8 consecutive nucleobases comprising the nucleobase sequence of SEQ ID NO: 87. In certain embodiments, the compound consists of SEQ ID NO: 87 and a conjugate group.

[0056] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures. In certain embodiments, the antisense compounds comprise modified oligonucleotides ISIS 304801 having 5'-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compounds consist of modified oligonucleotides ISIS 304801 having 5'-X, wherein X is a conjugate group comprising GalNAc.

[0057]

[0058] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures: In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 678354. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 678354.

[0059]

[0060] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures: In certain embodiments, the antisense compounds comprise the conjugated modified oligonucleotide ISIS 678357. In certain embodiments, the antisense compounds consist of the conjugated modified oligonucleotide ISIS 678357.

[0061]

[0062] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures. In certain embodiments, the antisense compounds comprise modified oligonucleotides having a core base sequence of SEQ ID NO: 87, wherein the 5'-GalNAc has variability in the sugar pattern of the wing. In certain embodiments, the antisense compounds consist of modified oligonucleotides having a core base sequence of SEQ ID NO: 87, wherein the 5'-GalNAc has variability in the sugar pattern of the wing.

[0063]

[0064] where R 1 is –OCH2CH2OCH3(MOE) and R 2 H; or R 1 and R 2 Together form a bridge, where R 1 For –O- and R 2 is –CH2-, -CH(CH3)- or -CH2CH2-, and R 1 and R 2 directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-;

[0065] And for every pair R on the same ring 3 and R 4 , independently for each ring: R 3 is selected from H and -OCH2CH2OCH3 and R 4 H; or R 3 and R 4 Together form a bridge, where R 3 For –O- and R 4 is –CH2-, -CH(CH3)- or -CH2CH2- and R 3 and R 4 directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-;

[0066] And R 5 selected from H and –CH3;

[0067] And Z is selected from S - and O - .

[0068] The present disclosure provides the following non-limiting numbered embodiments:

[0069] Details

[0070] It should be understood that the above general description and the following detailed description are merely exemplary and explanatory and do not limit the present disclosure. In this article, unless otherwise specifically stated, the use of the singular includes the plural. As used herein, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. In addition, unless otherwise specifically stated, terms such as "element" or "component" cover both elements and components comprising one unit and elements and components comprising more than one subunit.

[0071] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0072] A. definition

[0073] Unless specific definitions are provided, the nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein and the procedures and techniques of these chemical fields are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses and chemical analyses. Some of these techniques and procedures can be found, for example, in "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., 21st 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," 2nd edition, Cold Spring Harbor Laboratory Press, 1989, which references are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data mentioned throughout this disclosure are incorporated by reference in their entirety.

[0074] Unless otherwise indicated, the following terms have the following meanings:

[0075] As used herein, "nucleoside" means a compound comprising a core base portion and a sugar portion. Nucleosides include, but are not limited to, naturally occurring nucleosides (such as those found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

[0076] As used herein, "chemical modification" means the chemical difference of a compound when compared to its naturally occurring counterpart. Chemical modification of oligonucleotides includes nucleoside modifications (including sugar moiety modifications and core base modifications) and internucleoside linkage modifications. In the case of oligonucleotides, chemical modification does not include differences only in the core base sequence.

[0077] As used herein, "furanosyl" means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

[0078] As used herein, "naturally occurring sugar moiety" means ribofuranosyl as found in naturally occurring RNA or deoxyribofuranosyl as found in naturally occurring DNA.

[0079] As used herein, "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

[0080] As used herein, "modified sugar moiety" means a substituted sugar moiety or sugar surrogate.

[0081] As used herein, "substituted sugar moiety" refers to a furanosyl group that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to, furanosyl groups comprising substituents at the 2'-position, 3'-position, 5'-position, and / or 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties.

[0082] As used herein, "2'-substituted sugar moiety" means a furanosyl group comprising a substituent other than H or OH at the 2'-position. Unless otherwise indicated, the 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of the 2'-substituted sugar moiety does not form a bridge with another atom of the furanosyl ring).

[0083] As used herein, "MOE" means -OCH2CH2OCH3.

[0084] As used herein, "2'-F nucleoside" refers to a nucleoside comprising a sugar comprising a fluorine at the 2' position. Unless otherwise indicated, the fluorine in a 2'-F nucleoside is in the ribose position (replacing the OH of the natural ribose sugar).

[0085] As used herein, the term "sugar surrogate" means a structure that does not contain a furanose group and is capable of replacing the naturally occurring sugar portion of a nucleoside, such that the resulting nucleoside subunits can be linked together and / or linked to other nucleosides to form an oligomeric compound that can hybridize with a complementary oligomeric compound. The structure includes a ring that contains a different number of atoms from a furanose group (e.g., a 4-membered ring, a 6-membered ring, or a 7-membered ring); a non-oxygen atom (e.g., carbon, sulfur, or nitrogen) that replaces the oxygen of the furanose group; or both the number of atoms and the oxygen substitution vary. The structure may also include substitutions corresponding to those described for the substituted sugar moiety (e.g., a 6-membered carbocyclic bicyclic sugar surrogate optionally containing additional substituents). Sugar surrogate also includes more complex sugar surrogate (e.g., the non-ring system of peptide nucleic acids). Sugar surrogate includes, but is not limited to, morpholino, cyclohexenyl, and cyclohexanehexol.

[0086] As used herein, "bicyclic sugar moiety" means a modified sugar moiety (including but not limited to furanosyl) comprising 4 to 7 rings, wherein the sugar moiety comprises two atoms connecting the 4 to 7 rings to form a second ring, thereby producing the bridging of a bicyclic structure. In certain embodiments, the 4 to 7 rings are sugar rings. In certain embodiments, the 4 to 7 rings are furanosyl. In some of the embodiments, the bridging has connected the 2'-carbon and 4'-carbon of the furanosyl.

[0087] As used herein, "nucleotide" means a nucleoside that also comprises a phosphate linking group. As used herein, "linked nucleosides" may or may not be linked by a phosphate linkage and therefore include, but are not limited to, "linked nucleotides." As used herein, "linked nucleosides" are nucleosides that are linked in a consecutive order (i.e., without additional nucleosides between those linked nucleosides).

[0088] As used herein, "nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid (ssDNA), double-stranded nucleic acid (dsDNA), small interfering ribonucleic acid (siRNA), and microRNA (miRNA). Nucleic acids may also contain any combination of these elements in a single molecule.

[0089] As used herein, "nucleotide" means a nucleoside that also comprises a phosphate linking group. As used herein, "linked nucleosides" may or may not be linked by a phosphate linkage and therefore include, but are not limited to, "linked nucleotides." As used herein, "linked nucleosides" are nucleosides that are linked in a consecutive order (i.e., without additional nucleosides between those linked nucleosides).

[0090] As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to form a nucleoside that can be incorporated into an oligonucleotide, and wherein the group of atoms is capable of bonding to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. A nucleobase can be naturally occurring or modified. As used herein, "nucleobase sequence" means the order of consecutive nucleobases independent of any sugar, linkage, or nucleobase modification.

[0091] As used herein, the term "unmodified nucleobase" or "naturally occurring nucleobase" means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G); and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

[0092] As used herein, "modified nucleobase" means any nucleobase that is not a naturally occurring nucleobase.

[0093] As used herein, "modified nucleosides" means nucleosides that contain at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides contain modified sugar moieties and / or modified nucleobases.

[0094] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

[0095] As used herein, "constrained ethyl nucleoside" or "cEt" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge.

[0096] As used herein, "locked nucleic acid nucleoside" or "LNA" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge.

[0097] As used herein, "2'-substituted nucleoside" means a nucleoside that includes a substituent other than H or OH at the 2'-position. Unless otherwise indicated, a 2'-substituted nucleoside is not a bicyclic nucleoside.

[0098] As used herein, "deoxynucleoside" means a nucleoside comprising a 2'-H furanosyl sugar moiety as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

[0099] As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.

[0100] As used herein, "oligonucleoside" means an oligonucleotide in which no internucleoside linkage contains a phosphorus atom. As used herein, an oligonucleotide comprises an oligonucleoside.

[0101] As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0102] As used herein, "linker" or "linking group" means a group of atoms that links two or more other groups of atoms together.

[0103] As used herein, "internucleoside linkage" means a covalent linkage between adjacent nucleosides in an oligonucleotide.

[0104] As used herein, "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0105] As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0106] As used herein, "terminal internucleoside linkage" means the linkage between the last two nucleosides of an oligonucleotide or a defined region thereof.

[0107] As used herein, "phosphorus linking group" means a linking group comprising a phosphorus atom. Phosphorus linking groups include, but are not limited to, groups having the formula:

[0108]

[0109] in:

[0110] R a and R d Each is independently O, S, CH2, NH or NJ1, wherein J1 is C1-C6 alkyl or substituted C1-C6 alkyl;

[0111] R b O or S;

[0112] R c is OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino, or substituted amino; and

[0113] J1 is R b O or S.

[0114] Phosphorus linking groups include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, thiophosphoramidate, thiocarbonylalkylphosphonate, phosphotriester, thiocarbonylalkylphosphotriester, and boranophosphate.

[0115] As used herein, "internucleoside phosphorus linking group" means a phosphorus linking group that directly links two nucleosides.

[0116] As used herein, a "non-nucleoside phosphorus linking group" refers to a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-nucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-nucleoside phosphorus linking group links two groups, neither of which is a nucleoside.

[0117] As used herein, "neutral linking group" means a linking group that is uncharged. Neutral linking groups include, but are not limited to, phosphotriester, methylphosphonate, MMI (-CH2-N(CH3)-O-), amide-3 (-CH2-C(=O)-N(H)-), amide-4 (-CH2-N(H)-C(=O)-), methylacetal (-O-CH2-O-) and thiomethylacetal (-S-CH2-O-). Additional neutral linking groups include nonionic linkages comprising siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates and amides (see, for example: Carbohydrate Modifications in Antisense Research; YS Sanghvi and PDCook, eds., ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)). Additional neutral linking groups include nonionic linkages comprising mixed N, O, S and CH2 components.

[0118] As used herein, "internucleoside neutral linking group" means a neutral linking group that directly links two nucleosides.

[0119] As used herein, a "non-nucleoside neutral linking group" refers to a neutral linking group that does not directly connect two nucleosides. In certain embodiments, a non-nucleoside neutral linking group connects a nucleoside to a group other than a nucleoside. In certain embodiments, a non-nucleoside neutral linking group connects two groups, neither of which is a nucleoside.

[0120] 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 conjugate groups and / or end groups. In certain embodiments, the oligomeric compound consists of an oligonucleotide. The oligomeric compound also includes naturally occurring nucleic acids. In certain embodiments, the oligomeric compound comprises a skeleton of one or more connected monomeric subunits, wherein each connected monomeric subunit is directly or indirectly connected to a heterocyclic base moiety. In certain embodiments, the oligomeric compound may also include a monomeric subunit that is not connected to a heterocyclic base moiety, thereby providing an abasic site. In certain embodiments, the linkage connecting the monomeric subunits, sugar moieties or sugar substitutes and the heterocyclic base moiety may be independently modified. In certain embodiments, the linkage-sugar unit that may or may not include a heterocyclic base may be replaced by a monomer in a mimetic such as a peptide nucleic acid.

[0121] As used herein, "terminal group" means one or more atoms attached to either or both of the 3' end or the 5' end of an oligonucleotide. In certain embodiments, the terminal group is a conjugate group. In certain embodiments, the terminal group comprises one or more terminal nucleosides.

[0122] As used herein, "conjugate" or "conjugate group" means an atom or group of atoms that is bound to an oligonucleotide or oligomeric compound. Typically, conjugate groups alter one or more properties of the compound to which they are attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties.

[0123] As used herein, "conjugate linker" or "linker" in the context of a conjugate group means a portion of the conjugate group that comprises any atom or group of atoms and which (1) covalently links an oligonucleotide to another portion of the conjugate group or (2) covalently links two or more portions of the conjugate group.

[0124] Conjugate groups are shown as groups in this article, and are provided for forming a covalently attached key to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3'-oxygen atom of the 3'-hydroxyl of the 3' terminal nucleoside of the oligomeric compound. In certain embodiments, the point of attachment on the oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl of the 5' terminal nucleoside of the oligomeric compound. In certain embodiments, the key for forming a connection to the oligomeric compound is a cleavable bond. In some of the embodiments, the cleavable bond constitutes all or part of a cleavable portion.

[0125] In certain embodiments, the conjugate group comprises a cleavable portion (e.g., a cleavable bond or a cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. The carbohydrate cluster portion comprises: a targeting portion and optionally a conjugate linker. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligands. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups and is designated "GalNAc3". In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated "GalNAc4". Specific carbohydrate cluster portions (having specific chain links, branching groups, and conjugate linker groups) are described herein and are designated by Roman numerals followed by the subscript "a". Thus, "GalNAc3-1" a " refers to a specific carbohydrate cluster portion of a conjugate group having three GalNac groups and clearly identified tethering, branching, and linking groups. The carbohydrate cluster fragment is linked to the oligomeric compound via a cleavable moiety (e.g., a cleavable bond or a cleavable nucleoside).

[0126] As used herein, "cleavable moiety" means a bond or group that can be split under physiological conditions. In certain embodiments, the cleavable moiety is cleaved inside a cell or subcellular compartment (such as a lysosome). In certain embodiments, the cleavable moiety is cleaved by an endogenous enzyme such as a nuclease. In certain embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.

[0127] As used herein, "cleavable bond" means any chemical bond that can be split. In certain embodiments, the cleavable bond is selected from the group consisting of an amide, a polyamide, an ester, an ether, one or two esters of a phosphodiester, a phosphate, a carbamate, a disulfide, or a peptide.

[0128] As used herein, "carbohydrate cluster" means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (For examples of carbohydrate conjugate clusters, see, e.g., Maier et al., "Synthesis of Antisense Oligon ucleotides Conjugated to a Multivalent Carbohydrate Cluster for Celullar Targeting," Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, 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).

[0129] As used herein, "modified carbohydrate" means any carbohydrate that has one or more chemical modifications relative to a naturally occurring carbohydrate.

[0130] As used herein, "carbohydrate derivative" means any compound that can be synthesized using a carbohydrate as a starting material or intermediate.

[0131] As used herein, "carbohydrate" means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

[0132] As used herein, "protecting group" means any compound or protecting group known to those skilled in the art. Non-limiting examples of protecting groups can be found in "Protective Groups in Organic Chemistry", TW Greene, PGM Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

[0133] As used herein, "single-stranded" refers to an oligomeric compound that does not hybridize to its complement and lacks sufficient self-complementarity to form a stable self-duplex.

[0134] As used herein, "double-stranded" means a pair of oligomeric compounds that hybridize to each other or a single self-complementary oligomeric compound that forms a hairpin structure. In certain embodiments, the double-stranded oligomeric compound comprises a first and a second oligomeric compound.

[0135] As used herein, "antisense compound" means a compound comprising or consisting of an oligonucleotide, at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, thereby resulting in at least one antisense activity.

[0136] As used herein, "antisense activity" means any detectable and / or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity comprises modulation of the amount or activity of a target nucleic acid transcript (e.g., mRNA). In certain embodiments, antisense activity comprises modulation of the splicing of a pre-mRNA.

[0137] As used herein, "RNase H-based antisense compound" means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.

[0138] As used herein, "RISC-based antisense compound" refers to an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to the RNA-induced silencing complex (RISC).

[0139] As used herein, "detecting" or "measuring" means a test or assay for detecting or measuring. The detection and / or measurement may result in a value of zero. Thus, if a test for detecting or measuring results in a finding of no activity (an activity of zero), a step of detecting or measuring activity has nonetheless been performed.

[0140] As used herein, "detectable and / or measurable activity" means a statistically significant activity that is not zero.

[0141] As used herein, "substantially unchanged" means that a particular parameter has little or no change, particularly relative to another parameter that has changed more. In certain embodiments, a parameter is substantially unchanged when it changes by less than 5%. In certain embodiments, a parameter is substantially unchanged if it changes by less than two-fold while another parameter changes by at least ten-fold. For example, in certain embodiments, antisense activity is a change in the amount of target nucleic acid. In certain such embodiments, if the amount of non-target nucleic acid changes much less than the amount of target nucleic acid, then the amount of non-target nucleic acid is substantially unchanged, but the change is not necessarily zero.

[0142] As used herein, "expression" refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (eg, splicing, polyadenylation, addition of a 5'-cap), and translation.

[0143] As used herein, "target nucleic acid" refers to a nucleic acid molecule to which an antisense compound is intended to hybridize to produce the desired antisense activity. An antisense oligonucleotide has sufficient complementarity to its target nucleic acid to allow hybridization under physiological conditions.

[0144] As used herein, "nucleobase complementarity" or "complementarity" when referring to a nucleobase means a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, a complementary nucleobase means a nucleobase of an antisense compound that can base pair with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of the antisense compound can hydrogen bond with a nucleobase at a certain position of the target nucleic acid, the position of the hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary on the nucleobase pair. Nucleobases comprising certain modifications can maintain the ability to pair with corresponding nucleobases, and therefore still be able to have nucleobase complementarity.

[0145] As used herein, "non-complementarity" with respect to nucleobases means nucleobase pairs that do not form hydrogen bonds with each other.

[0146] As used herein, " complementarity " about oligomeric compounds (for example, connecting nucleosides, oligonucleotides or nucleic acids) means the ability of described oligomeric compounds or its region and another oligomeric compounds or its region to hybridize by core base complementarity.Complementary oligomeric compounds do not necessarily all have core base complementarity on each nucleoside.On the contrary, some mispairings are tolerated.In certain embodiments, complementary oligomeric compounds or regions are complementary on 70% of the core bases (70% complementary).In certain embodiments, complementary oligomeric compounds or regions are 80% complementary.In certain embodiments, complementary oligomeric compounds or regions are 90% complementary.In certain embodiments, complementary oligomeric compounds or regions are 95% complementary.In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.

[0147] As used herein, "mismatch" means a nucleobase of a first oligomeric compound that is unable to pair with a nucleobase at a corresponding position of a second oligomeric compound when the first and second oligomeric compounds are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.

[0148] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). Although not limited to a specific mechanism, the most common mechanism of pairing involves hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementary nucleobases.

[0149] As used herein, "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.

[0150] As used herein, "fully complementary" with respect to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or a contiguous portion thereof. Thus, a fully complementary region does not contain mismatched or unhybridized nucleobases in either strand.

[0151] As used herein, "complementarity percentage" means the percentage of nucleobases of an oligomeric compound that are complementary to an equal length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

[0152] As used herein, "percent identity" means the number of nucleobases in a first nucleic acid that are of the same type (independent of chemical modification) as the nucleobase at the corresponding position in a second nucleic acid divided by the total number of nucleobases in the first nucleic acid.

[0153] As used herein, "modulation" means a change in the amount or quality of a molecule, function or activity when compared to the amount or quality of the molecule, function or activity before modulation. For example, modulation includes a change in gene expression, either an increase (stimulation or induction) or a decrease (inhibition or reduction). As another example, modulation of expression can include a change in splice site selection for pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice variant compared to the amount in the absence of modulation.

[0154] As used herein, "chemical motif" refers to a pattern of chemical modifications in an oligonucleotide or a region thereof. A motif can be defined by modifications on certain nucleosides and / or certain linker groups of an oligonucleotide.

[0155] As used herein, "nucleoside motif" means a pattern of nucleoside modifications in an oligonucleotide or region thereof. The linkages of such oligonucleotides may be modified or unmodified. Unless otherwise indicated, motifs describing only nucleosides herein are intended to be nucleoside motifs. Therefore, in such cases, the linkages are not limited.

[0156] As used herein, "sugar motif" refers to the pattern of sugar modifications in an oligonucleotide or region thereof.

[0157] As used herein, "linkage motif" refers to the pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, herein, only motifs describing linkages are intended to be linkage motifs. Therefore, in such cases, nucleosides are not limited.

[0158] As used herein, "nucleobase modification motif" means a pattern of modifications along the nucleobases of an oligonucleotide. Unless otherwise indicated, the nucleobase modification motif is independent of the nucleobase sequence.

[0159] As used herein, "sequence motif" means the pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modification and thus may have any combination of chemical modifications, including no chemical modification.

[0160] As used herein, "modification type" with respect to a nucleoside or a "type" of nucleosides means the chemical modification of the nucleoside and includes both modified and unmodified nucleosides. Thus, unless otherwise indicated, a "nucleoside having a first type of modification" may be an unmodified nucleoside.

[0161] As used herein, "differently modified" means chemical modifications or chemical substituents that are different from one another, including the absence of modifications. Thus, for example, an MOE nucleoside and an unmodified DNA nucleoside are "differently modified," even though the DNA nucleoside is unmodified. Similarly, DNA and RNA are "differently modified," even though both are naturally occurring, unmodified nucleosides. Nucleosides that are identical but contain different nucleobases are not differently modified. For example, a nucleoside containing a 2'-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside containing a 2'-OMe modified sugar and an unmodified thymidine nucleobase are not differently modified.

[0162] As used herein, "the same type of modification" refers to modifications that are identical to one another, including the absence of modifications. Thus, for example, two unmodified DNA nucleosides have the "same type of modification," even if the DNA nucleosides are unmodified. The nucleosides having the same type of modification may comprise different core bases.

[0163] As used herein, "individual regions" means portions of an oligonucleotide wherein the chemical modification or motif of chemical modifications of any adjacent portions comprises at least one difference allowing the individual regions to be distinguished from one another.

[0164] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, the sterile saline is pharmaceutical grade saline.

[0165] As used herein, the term "metabolic disorder" means a disease or condition primarily characterized by dysregulation of metabolism - the complex series of chemical reactions involved in the breakdown of food to produce energy.

[0166] As used herein, the term "cardiovascular disorder" means a disease or condition primarily characterized by impaired heart or blood vessel function.

[0167] As used herein, the term "monocyclic or polycyclic ring system" is intended to include all ring systems selected from single or polycyclic radical ring systems, wherein the rings are fused or linked and is intended to include monocyclic and mixed ring systems individually selected from the following: aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. The monocyclic and polycyclic structures may contain rings, each of which has the same level of saturation or each independently has a different degree 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 as well as rings containing only C ring atoms, and the rings may be present in mixed motifs, such as, for example, benzimidazole, in which one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The monocyclic or polycyclic ring system may be further substituted with substituents, such as, for example, phthalimide having two =O groups attached to one of the rings. The monocyclic or polycyclic ring system can be linked to the parent molecule using various strategies, such as directly through a ring atom, through fusion of multiple ring atoms, through a substituent, or through a bifunctional linking moiety.

[0168] As used herein, "prodrug" means an inactive or less active form of a compound that, when administered to a subject, is metabolized to form the active or more active compound (eg, drug).

[0169] As used herein, "substituent" and "substituent group" refer to an atom or group that replaces an atom or group of a specified parent compound. For example, the substituent of a modified nucleoside is any atom or group that is different from the atom or group found in a naturally occurring nucleoside (for example, the 2'-substituent of the modification is any atom or group other than H or OH on the 2'-position of a nucleoside). The substituent may be protected or unprotected. In certain embodiments, the compounds of the present disclosure have a substituent at one position of the parent compound or at more than one position. The substituent may also be further substituted with other substituents and may be directly connected or connected to the parent compound via a linking group such as an alkyl or alkyl group.

[0170] Similarly, as used herein, a "substituent" with respect to a chemical functional group means an atom or group of atoms different from the atoms or group 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 is an atom or group other than hydrogen that replaces one hydrogen atom of an unsubstituted methyl). Unless otherwise indicated, groups suitable as substituents include, but are not limited to, halogen, hydroxy, alkyl, alkenyl, alkynyl, acyl (-C(O)R aa ), carboxyl (-C(O)O-R aa ), aliphatic group, cycloaliphatic group, alkoxy, substituted oxy (-O-R aa ), aryl, aralkyl, heterocyclic group, heteroaryl, heteroarylalkyl, amino (-N(R bb )(R cc )), imino (=NR bb ), amido (-C(O)N(R bb )(R cc ) or -N(R bb )C(O)R aa ), azido (-N3), nitro (-NO2), cyano (-CN), carbamido (-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 )), guanidino (-N(R bb )C(=NR bb )N(R bb )(R cc )), amidino (-C(=NR bb )N(R bb )(R cc ) or -N(R bb )C(=NR bb )(R aa )), mercapto (-SR bb ), sulfinyl (-S(O)R bb ), sulfonyl (-S(O)2R bb ), and sulfamoyl (-S(O)2N(R bb )(R cc )- or -N(R bb )S(O)2Rbb ). Each R aa 、R bb and R cc is independently H, an optionally attached chemical functional group, or another substituent having a preferred list including, but not limited to, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic, and heteroarylalkyl. Selected substituents within the compounds described herein are present to a certain degree of recursion.

[0171] As used herein, "alkyl" as used herein means a saturated straight or branched chain hydrocarbon group containing up to twenty-four carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. Alkyl groups typically contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms (C1-C1 12 Alkyl groups), of which 1 to about 6 carbon atoms are more preferred.

[0172] As used herein, "alkenyl" means a straight or branched chain hydrocarbon 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-butene-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups typically include 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more additional substituents.

[0173] As used herein, "alkynyl" means a straight or branched hydrocarbon group containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include 2 to about 24 carbon atoms, more typically 2 to about 12 carbon atoms, with 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more additional substituents.

[0174] As used herein, "acyl" refers to a group formed by removing a hydroxyl group from an organic acid and has the general formula -C(O)-X, where X is typically aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, and the like. Acyl groups as used herein may optionally include additional substituents.

[0175] As used herein, "alicyclic" means a ring system in which the ring is aliphatic. The ring system may comprise one or more rings, at least one of which is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms. As used herein, alicyclics may optionally include additional substituents.

[0176] As used herein, "aliphatic" means a straight or branched chain hydrocarbon radical containing up to twenty-four carbon atoms, wherein the degree of saturation between any two carbon atoms is a single bond, a double bond, or a triple bond. Aliphatic groups preferably contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms, with 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Aliphatic groups interrupted by heteroatoms include, but are not limited to, polyalkoxy groups, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include additional substituents.

[0177] As used herein, "alkoxy" refers to a group formed between an alkyl group and an oxygen atom, wherein the oxygen atom is used to connect the alkoxy group to the parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. As used herein, the alkoxy group may optionally include additional substituents.

[0178] As used herein, "aminoalkyl" refers to a C1-C 12 Alkyl. The alkyl portion of the group forms a covalent bond with the parent molecule. The amino group may be located at any position and the aminoalkyl group may be substituted with additional substituents on the alkyl and / or amino portion.

[0179] As used herein, "aralkyl" and "arylalkyl" refer to a group covalently attached to a C1-C 12 The alkyl group of the resulting aralkyl (or arylalkyl) forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, and the like. Aralkyl groups as used herein may optionally include additional substituents attached to the alkyl group, the aryl group, or the two groups forming the group.

[0180] As used herein, "aryl" and "aromatic" refer to a monocyclic or polycyclic carbocyclic ring system radical having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Preferred aromatic ring systems have from about 5 to about 20 carbon atoms in one or more rings. As used herein, aryl groups may optionally include additional substituents.

[0181] As used herein, "halo" and "halogen" mean an atom selected from fluorine, chlorine, bromine, and iodine.

[0182] As used herein, "heteroaryl" and "heteroaromatic" refer to groups comprising monocyclic or polycyclic aromatic rings, ring systems, or fused ring systems, wherein at least one ring is aromatic and includes one or more heteroatoms. Heteroaryl is also intended to include fused ring systems, including systems wherein one or more fused rings do not contain heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thienyl, furyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. A heteroaryl group may be attached to the parent molecule directly or via a linking moiety such as an aliphatic group or a heteroatom. As used herein, a heteroaryl group may optionally include additional substituents.

[0183] As used herein, "conjugate compound" means any atom, group of atoms, or linked group of atoms suitable for use as a conjugate group. In certain embodiments, the conjugate compound may possess or impart one or more properties, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties.

[0184] As used herein, unless otherwise indicated or modified, the term "double-stranded" refers to two separate oligomeric compounds that hybridize to each other. The double-stranded compound may have one or more non-hybridized nucleosides (overhangs) and / or one or more internal non-hybridized nucleosides (mismatches) at one or both ends of one or both strands, provided that there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.

[0185] As used herein, "5' target site" refers to the nucleotide of the target nucleic acid that is complementary to the 5'-most nucleotide of a particular antisense compound.

[0186] As used herein, "about" means within ±10% of a value. For example, if it is stated that a marker can be increased by about "50%," it means that the marker can be increased by 45%-55%.

[0187] As used herein, "concomitant administration" refers to the simultaneous administration of two pharmaceutical agents by any means at the same time, wherein the pharmacological effects of both agents manifest in the patient. Concomitant administration does not require that the two agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same administration route. The effects of the two agents do not need to manifest at the same time. The effects need only overlap for a period of time and need not be coextensive.

[0188] As used herein, "administering" or "administration" means providing a pharmaceutical agent to an individual and includes, but is not limited to, administration by a medical professional or self-administration. Administration of a pharmaceutical agent to an individual can be continuous, long-term, short-term, or intermittent. Administration can be parenteral or non-parenteral.

[0189] As used herein, "agent" means an active substance that provides a therapeutic benefit when administered to an animal. "First agent" means a therapeutic compound provided herein. For example, a first agent can be an antisense oligonucleotide targeting apoCIII. "Second agent" means a second therapeutic compound of the invention (e.g., a second antisense oligonucleotide targeting apoCIII) and / or a non-apoCIII therapeutic compound.

[0190] As used herein, "amelioration" or "ameliorate" or "ameliorating" refers to a reduction in at least one indicator, sign or symptom of a disease, disorder or condition of interest. The severity of an indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0191] As used herein, "animal" refers to human and non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and gorillas.

[0192] As used herein, "ApoCIII," "apolipoprotein C-III," or "ApoC3" refers to any nucleic acid or protein sequence encoding ApoCIII. For example, in certain embodiments, ApoCIII includes a DNA sequence encoding ApoCIII, an RNA sequence transcribed from DNA encoding ApoCIII (including genomic DNA containing introns and exons), an mRNA sequence encoding ApoCIII, or a peptide sequence encoding ApoCIII.

[0193] As used herein, "ApoCIII nucleic acid" means any nucleic acid encoding ApoCIII. For example, in certain embodiments, ApoCIII nucleic acids include DNA sequences encoding ApoCIII, RNA sequences transcribed from DNA encoding ApoCIII (including genomic DNA containing introns and exons), and mRNA sequences encoding ApoCIII.

[0194] As used herein, "ApoCIII specific inhibitor" refers to any agent that can specifically inhibit the expression of ApoCIII mRNA and / or the expression or activity of ApoCIII protein at the molecular level. For example, ApoCIII specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents that can inhibit the expression of ApoCIII mRNA and / or ApoCIII protein. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is an oligonucleotide targeting ApoCIII. In certain embodiments, the oligonucleotide targeting ApoCIII is a modified oligonucleotide targeting ApoCIII. In certain embodiments, the oligonucleotide targeting ApoCIII is a modified oligonucleotide targeting ApoCIII having a conjugate group. In certain embodiments, oligonucleotides targeting ApoCIII have sequences as shown in SEQ ID NOs: 19-96, 209-221, or other sequences (e.g., as disclosed in PCT Publication No. WO 2004 / 093783 or PCT Publication No. WO 2012 / 149495, all of which are incorporated herein by reference). In certain embodiments, ApoCIII-specific inhibitors can affect components of the lipogenic or glucogenic pathways by specifically modulating ApoCIII mRNA levels and / or ApoCIII protein expression. Similarly, in certain embodiments, ApoCIII-specific inhibitors can affect other molecular processes in animals.

[0195] As used herein, "ApoCIII mRNA" means mRNA encoding ApoCIII protein.

[0196] As used herein, "ApoCIII protein" means any protein sequence encoding ApoCIII.

[0197] As used herein, "atherosclerosis" means hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called "atheromas" or "plaques," which are composed primarily of cholesterol and other fats, calcium, and scar tissue, and damage the inner lining of the arteries.

[0198] As used herein, "coronary heart disease (CHD)" refers to the narrowing of the small blood vessels that supply the heart with blood and oxygen, usually as a result of atherosclerosis.

[0199] As used herein, "diabetes mellitus" or "diabetes" is a syndrome characterized by a metabolic disorder or abnormally high blood sugar (hyperglycemia) caused by insufficient insulin levels or decreased insulin sensitivity. Characteristic symptoms are excessive urine production (polyuria) caused by high blood sugar levels, excessive thirst and increased fluid intake (polydipsia) in an attempt to compensate for the increased urination, blurred vision caused by the effects of high blood sugar on the optical system of the eye, unexplained weight loss, and lethargy.

[0200] As used herein, "diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia" means a condition characterized by type 2 diabetes, low HDL-C, elevated triglycerides (TG), and elevated small, dense LDL particles.

[0201] As used herein, "diluent" refers to an ingredient in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, a diluent in an injectable composition can be a liquid, such as a saline solution.

[0202] As used herein, "dyslipidemia" refers to a disorder of lipid and / or lipoprotein metabolism, including overproduction or deficiency of lipids and / or lipoproteins. Dyslipidemia can be manifested as an increase in lipids such as chylomicrons, cholesterol, and triglycerides, and lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[0203] As used herein, "dosage unit" means the form of the medicament provided, such as a pill, tablet, or other dosage unit known in the art. In certain embodiments, the dosage unit is a vial containing a lyophilized antisense oligonucleotide. In certain embodiments, the dosage unit is a vial containing a reconstituted antisense oligonucleotide.

[0204] As used herein, "dose" means a specified amount of a medicament provided in a single administration or within a specified time. In certain embodiments, a dose can be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments, when subcutaneous administration is desired, the desired dose requires a volume that is not easily accommodated by a single injection, and therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, a medicament is administered by infusion over an extended period of time or continuously. A dose can be stated as the amount of the medicament per hour, per day, per week, or per month. A dose can be expressed as mg / kg or g / kg.

[0205] As used herein, "effective amount" or "therapeutically effective amount" means an amount of an agent sufficient to achieve a desired physiological result in an individual in need of the active agent. The effective amount may vary between individuals and depends on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0206] As used herein, "Fredrickson Type I" is also referred to as "lipoprotein lipase deficiency," "LPLD," "familial chylomicronemia syndrome," or "FCS," and there are several forms: Type 1a (also known as Buerger-Gruestz syndrome) is a lipoprotein lipase deficiency usually caused by LPL deficiency or alterations in ApoC-II; Type Ib (also known as familial apoprotein CII deficiency) is a condition caused by a deficiency of apoprotein C-II, an activator of lipoprotein lipase; and Type Ic is a chylomicronemia caused by circulating inhibitors of lipoprotein lipase. Type I is a rare condition that usually presents in childhood. It is characterized by severe chylomicron elevation and extremely elevated triglyceride levels (consistently reaching well above 1000 mg / dL and often rising to 10,000 mg / dL or more), accompanied by episodes of abdominal pain, recurrent acute pancreatitis, eruptive cutaneous xanthomatosis, and hepatosplenomegaly. Patients rarely develop atherosclerosis, probably because their plasma lipoprotein particles are too large to enter the arterial intima (Nordestgaard et al., J Lipid Res, 1988, 29:1491-1500; Nordestgaard et al., Arteriosclerosis, 1988, 8:421-428). Type I is usually caused by mutations in the LPL gene or its cofactor ApoC-II, resulting in the inability of affected individuals to produce enough functionally active LPL. Patients are homozygous or compound heterozygous for the mutations. Type I Fredrickson may also be due to mutations in GPIHBP1, APOA5, or LMF1, or other genes, resulting in poorly functioning LPL. Brunzell, in: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, eds. Gene Reviews TM[Internet]. Seattle (WA): University of Washington, Seattle; 1993-2013. October 12, 1999 [Updated on December 15, 2011]. In addition, in some cases, type I Fredrickson's disease can be attributed to the presence of LPL inhibitors (e.g., anti-LPL antibodies) in individuals that cause dysfunctional LPL. The incidence of type I Fredrickson's disease is approximately 1 in 1,000,000 in the general population and is higher in South Africa and Eastern Quebec due to a founder effect. Patients have little or no response to TG-lowering drugs (Tremblay et al., J Clin Lipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010, 20:742-747) and therefore use dietary fat restriction to 20 grams / day or less to control the symptoms of this rare disease.

[0207] As used herein, "fully complementary" or "100% complementary" means that every nucleobase of a nucleobase sequence of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain embodiments, the first nucleic acid is an antisense compound and the second nucleic acid is a target nucleic acid.

[0208] As used herein, "glucose" refers to a monosaccharide that is used by cells as an energy source and as an inflammatory mediator. "Plasma glucose" refers to the glucose present in plasma.

[0209] As used herein, "high-density lipoprotein-C" or "HDL-C" refers to cholesterol associated with high-density lipoprotein particles. The concentration of HDL-C in serum (or plasma) is typically measured in mg / dL or nmol / L. "Serum HDL-C" and "plasma HDL-C" refer to HDL-C in serum and plasma, respectively.

[0210] As used herein, "HMG-CoA reductase inhibitors" means agents that act by inhibiting the enzyme HMG-CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

[0211] As used herein, "hypercholesterolemia" means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol, and VLDL-cholesterol according to the guidelines of the National Cholesterol Educational Program (NCEP) Expert Panel Report on the Detection, Evaluation, or Treatment of High Cholesterol in Adults (see Arch. Int. Med. (1988) 148, 36-39).

[0212] As used herein, "hyperlipidemia" or "hyperlipemia" is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests as abnormally high concentrations of fat. The lipid fractions in circulating blood are divided into cholesterol, low-density lipoproteins, very low-density lipoproteins, chylomicrons, and triglycerides. The Fredrickson classification of hyperlipidemia is based on the pattern of TG and cholesterol-rich lipoprotein particles as measured by electrophoresis or ultracentrifugation, and is generally used to characterize the main causes of hyperlipidemia, such as hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965, 31: 321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1): 34–42).

[0213] As used herein, "hypertriglyceridemia" refers to a condition characterized by elevated triglyceride levels. Hypertriglyceridemia is a consequence of increased production and / or decreased or delayed catabolism of triglyceride (TG)-rich lipoproteins VLDL and, to a lesser extent, chylomicrons (CM). Its causes include primary (i.e., genetic) and secondary (other potential causes such as diabetes, metabolic syndrome / insulin resistance, obesity, physical inactivity, smoking, excessive alcohol, and a very high carbohydrate diet) factors, or most commonly, a combination of both (Yuan et al. CMAJ, 2007, 176: 1113-1120). Hypertriglyceridemia is a common clinical trait associated with an increased risk of cardiometabolic diseases (Hegele et al. 2009, Hum Mol Genet, 18:4189-4194; Hegele and Pollex 2009, Mol Cell Biochem, 326:35-43) and the development of acute pancreatitis in its most severe form (Toskes 1990, Gastroenterol Clin North Am, 19:783-791; Gaudet et al. 2010, Atherosclerosis Supplements, 11:55-60; Catapano et al. 2011, Atherosclerosis, 217S:S1-S44; Tremblay et al. 2011, J Clin Lipidol, 5:37-44). Examples of cardiometabolic diseases include, but are not limited to, diabetes, metabolic syndrome / insulin resistance, and hereditary conditions such as familial chylomicronemia syndrome (FCS), familial mixed hyperlipidemia, and familial hypertriglyceridemia. Critical high TG levels (150-199 mg / dL) are typically found in the general population and are a common component of metabolic syndrome / insulin resistance. The same applies to high TG levels (200-499 mg / dL), except that underlying genetic factors play an increasingly important role in the etiology as plasma TG levels increase. Very high TG levels (≥500 mg / dL) are also typically associated with elevated CM levels and are accompanied by an increased risk of acute pancreatitis. If TG levels exceed 880 mg / dL (>10 mmol), the risk of pancreatitis is considered clinically significant, and the European Atherosclerosis Society / European Society of Cardiology (EAS / ESC) 2011 guidelines state that actions to prevent acute pancreatitis are mandatory (Catapano et al. 2011, Atherosclerosis, 217S:S1-S44).According to the EAS / ESC 2011 guidelines, hypertriglyceridemia is the cause of approximately 10% of all pancreatitis cases, and the development of pancreatitis can occur at TG levels between 440-880 mg / dL. Based on evidence from clinical studies showing that elevated TG levels are an independent risk factor for atherosclerotic CVD, guidelines from the National Cholesterol Education Program Adult Treatment Panel III (NCEP 2002, Circulation, 106: 3143-421) and the American Diabetes Association (ADA 2008, Diabetes Care, 31: S12-S54.) recommend a target TG level of less than 150 mg / dL to reduce cardiovascular risk.

[0214] As used herein, "identifying" or "selecting an animal with a metabolic or cardiovascular disease" means identifying or selecting a subject that is susceptible to or has been diagnosed with a metabolic disease, cardiovascular disease, or metabolic syndrome; or, identifying or selecting a subject that has any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome, including but not limited to hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, above normal body weight, and / or above normal body fat content, or any combination thereof. The identification can be achieved by any method, including but not limited to standard clinical trials or evaluations, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) blood glucose, measuring serum or circulating (plasma) triglycerides, measuring blood pressure, measuring body fat content, measuring body weight, etc.

[0215] As used herein, "improved cardiovascular outcomes" means a reduction in the incidence of, or risk of, adverse cardiovascular events. Examples of adverse cardiovascular events include, but are not limited to, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial arrhythmia.

[0216] As used herein, "immediately adjacent" means that there are no intervening elements between immediately adjacent elements, eg, regions, fragments, nucleotides and / or nucleosides.

[0217] As used herein, "increasing HDL" or "elevating HDL" means increasing the level of HDL in an animal following administration of at least one compound of the invention, as compared to the level of HDL in the absence of any compound.

[0218] As used herein, "individual" or "subject" or "animal" means a human or non-human animal selected for treatment or therapy.

[0219] As used herein, "subject in need thereof" refers to a human or non-human animal selected for treatment or therapy in need of such treatment or therapy.

[0220] As used herein, "induce," "inhibit," "enhance," "elevate," "increase," "decrease," "lower," and the like refer to a quantitative difference between two states. For example, "an amount effective to inhibit the activity or expression of apoCIII" means that the level of apoCIII activity or expression in a treated sample will be different from the level of apoCIII activity or expression in an untreated sample. The terms apply, for example, to expression levels and activity levels.

[0221] As used herein, "inflammatory condition" refers to a disease, disease state, syndrome, or other condition that results in inflammation. For example, rheumatoid arthritis and liver fibrosis are inflammatory conditions. Other examples of inflammatory conditions include sepsis, myocardial ischemia / reperfusion injury, adult respiratory distress syndrome, nephritis, transplant rejection, inflammatory bowel disease, multiple sclerosis, arteriosclerosis, atherosclerosis, and vasculitis.

[0222] As used herein, "inhibiting expression or activity" refers to a reduction or blocking of the expression or activity of an RNA or protein and does not necessarily mean a complete elimination of expression or activity.

[0223] As used herein, "insulin resistance" is defined as a condition in which normal amounts of insulin are insufficient to produce a normal insulin response from fat, muscle, and liver tissue. Insulin resistance in adipocytes leads to hydrolysis of stored triglycerides, which increases free fatty acids in plasma. Insulin resistance in muscle reduces glucose uptake, while insulin resistance in the liver reduces glucose storage, both of which increase blood glucose. High plasma levels of insulin and glucose caused by insulin resistance often lead to metabolic syndrome and type 2 diabetes.

[0224] As used herein, "insulin sensitivity" is a measure of how efficiently an individual processes glucose. Individuals with high insulin sensitivity process glucose efficiently, while individuals with low insulin sensitivity do not process glucose efficiently.

[0225] As used herein, "lowering lipids" means a decrease in one or more lipids (e.g., LDL, VLDL) in a subject. "Increasing lipids" means an increase in lipids (e.g., HDL) in a subject. Lowering lipids or increasing lipids can occur over time at one or more doses.

[0226] As used herein, "lipid-lowering therapy" or "lipid-lowering agent" means a treatment regimen provided to a subject to reduce one or more lipids in the subject. In certain embodiments, lipid-lowering therapy is provided to reduce one or more of apo(a), apoCIII, CETP, apoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small, dense LDL particles, and Lp(a) in the subject. Examples of lipid-lowering therapy include, but are not limited to, apoB inhibitors, statins, fibrates, and MTP inhibitors.

[0227] As used herein, "lipoproteins" such as VLDL, LDL, and HDL refer to a group of proteins found in serum, plasma, and lymph and are important for lipid transport. The chemical composition of each lipoprotein is somewhat different, for example, with HDL having a higher protein to lipid ratio and VLDL having a lower protein to lipid ratio.

[0228] As used herein, "lipoprotein lipase" or "LPL" refers to an enzyme that hydrolyzes triglycerides (TGs) found in lipoproteins such as CM or VLDL into free fatty acids and monoacylglycerols. LPL requires apo C-II as a cofactor to function in hydrolyzing TGs. LPL is primarily produced in skeletal muscle, adipose tissue, and cardiac muscle. The hydrolysis and removal of TGs from CM and VLDL generally prevents excessive postprandial increases in CM mass and TGs.

[0229] As used herein, "lipoprotein lipase deficient," "lipoprotein lipase deficiency," "LPL deficiency," or "LPLD" is also referred to as "Fredrickson type I dyslipidemia," "chylomicronemia," "familial chylomicronemia syndrome," or "FCS." Although subjects with LPLD typically lack LPL or LPL activity required for efficient breakdown of fatty acids such as TG, these subjects may still have very low LPL activity or express very low levels of LPL. In some instances, subjects with LPLD may express LPL or have LPL activity that is at most about or no more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% active. In other instances, subjects with LPLD have no measurable LPL or LPL activity. One embodiment of LPLD encompasses subjects with " HyperlipoproteinemiaThe term "type Ia" refers to a subject who is unable to produce sufficient functional lipoprotein lipase to effectively break down fatty acids such as TG. The inability to break down TG results in hypertriglyceridemia in the subject and often hyperTG and chylomicronemia persist and can be seen as lipemia more than 12 hours after a meal. Type Ia is usually caused by one or more mutations in the LPL gene. As disclosed herein, LPLD also encompasses subjects with a dysfunctional lipoprotein lipase, such as those with " Hyperlipoproteinemia Type Ib" (also known as Fredrickson type Ib) and " Hyperlipoproteinemia Type Ib is caused by a deficiency of apoprotein C-II, an activator of lipoprotein lipase. Type Ic is due to circulating inhibitors of lipoprotein lipase. Like type Ia, type Ib / 1c subjects are also unable to break down triglycerides, resulting in hypertriglyceridemia, and hypertriglyceridemia and chylomicronemia often persist for more than 12 hours after a meal and can be seen as lipemia. In certain embodiments, LPLD is associated with at least one mutation in the LPL gene, such as P207L, G188L, or D9N, or other mutations affecting LPL (Brunzell, In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, eds. GeneReviews. TM [Internet].Seattle (WA): University of Washington, Seattle; 1993-2013. October 12, 1999 [updated on October 12, 2011].

[0230] As used herein, "low-density lipoprotein-cholesterol (LDL-C)" refers to the cholesterol carried in low-density lipoprotein particles. The concentration of LDL-C in serum (or plasma) is usually measured in mg / dL or nmol / L. "Serum LDL-C" and "plasma LDL-C" refer to LDL-C in serum and plasma, respectively.

[0231] As used herein, "major risk factors" refer to factors that contribute to a high risk of a particular disease or condition. In certain embodiments, the major risk factors for coronary heart disease include, but are not limited to, smoking, high blood pressure, high LDL, low HDL-C, family history of coronary heart disease, age, and other factors disclosed herein.

[0232] As used herein, "metabolic disorder" or "metabolic disease" refers to a condition characterized by changes or disturbances in metabolic function. "Metabolic" and "metabolism" are terms well known in the art and generally include the entire range of biochemical processes occurring in a living organism. Metabolic disorders include, but are not limited to, dyslipidemia caused by hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome, and type 2 diabetes.

[0233] As used herein, "metabolic syndrome" means a condition characterized by the clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference greater than 102 cm in men or greater than 88 cm in women; serum triglycerides of at least 150 mg / dL; HDL-C less than 40 mg / dL in men or less than 50 mg / dL in women; blood pressure of at least 130 / 85 mmHg; and fasting glucose of at least 110 mg / dL. These determinants can be easily measured in clinical practice (JAMA, 2001, 285: 2486-2497).

[0234] "Parenteral administration" means administration by injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intracerebroventricular administration. Administration can be continuous, long-term, short-term, or intermittent.

[0235] As used herein, "peptide" means a molecule formed by linking at least two amino acids by an amide bond. Peptides refer to polypeptides and proteins.

[0236] As used herein, "pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to apoCIII is a pharmaceutical agent.

[0237] As used herein, "pharmaceutical composition" or "composition" means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can include one or more active agents and a pharmaceutical carrier, such as a sterile aqueous solution.

[0238] As used herein, "pharmaceutically acceptable derivatives" include derivatives of the compounds described herein, such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labeled variations, pharmaceutically acceptable salts, and other derivatives known in the art.

[0239] As used herein, "pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects. The term "pharmaceutically acceptable salt" or "salt" includes salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic or organic acids and bases). "Pharmaceutically acceptable salts" of the compounds described herein can be prepared by methods known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and widely accepted for therapeutic administration to humans. Therefore, in one embodiment, the compounds described herein are in the form of sodium salts.

[0240] As described herein, "portion" means a defined number of consecutive (i.e., connected) core bases of a nucleic acid. In certain embodiments, a portion is a defined number of consecutive core bases of a target nucleic acid. In certain embodiments, a portion is a defined number of consecutive core bases of an antisense compound.

[0241] As used herein, "prevent" or "preventing" means delaying or arresting the onset or development of a disease, disorder, or condition for a period of time ranging from minutes to indefinitely. Prevention also means reducing the risk of developing a disease, disorder, or condition.

[0242] As used herein, "increase" means an increase in amount. For example, increasing plasma HDL levels means increasing the amount of HDL in plasma.

[0243] As used herein, "lower" means to decrease to a smaller degree, size, amount or number. For example, lowering plasma triglyceride levels means reducing the amount of triglycerides in the plasma.

[0244] As used herein, a "region" or "target region" is defined as a portion of a target nucleic acid having at least one identifiable structure, function, or characteristic. For example, a target region may comprise a 3'UTR, a 5'UTR, an exon, an intron, an exon / intron junction, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid region. Structurally defined regions of apoCIII can be obtained from sequence databases such as NCBI by accession number and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from the 5' target site of one target segment within the target region to the 3' target site of another target segment within the target region.

[0245] As used herein, "second agent" or "second therapeutic agent" refers to an agent that can be used in combination with a "first agent." Second therapeutic agents may include, but are not limited to, antisense oligonucleotides targeting apoCIII. Second agents may also include anti-apoCIII antibodies, apoCIII peptide inhibitors, cholesterol-lowering agents, lipid-lowering agents, blood sugar-lowering agents, and anti-inflammatory agents.

[0246] As used herein, a "segment" is defined as a smaller subdivision of a region within a nucleic acid. For example, Refers to the sequence of nucleotides of a target nucleic acid to which one or more antisense compounds are targeted. The "5' target site" refers to the 5'-most nucleotide of a target segment. The "3' target site" refers to the 3'-most nucleotide of a target segment. Alternatively, the "start site" may refer to the 5'-most nucleotide of a target segment and the "stop site" may refer to the 3'-most nucleotide of a target segment. A target segment may also begin at the "start site" of one sequence and end at the "stop site" of another sequence.

[0247] As used herein, "statin" means an agent that inhibits the activity of HMG-CoA reductase.

[0248] As used herein, "subcutaneous administration" means administration just beneath the skin.

[0249] As used herein, "subject" means a human or non-human animal selected for treatment or therapy.

[0250] As used herein, "symptoms of a cardiovascular disease or disorder" means phenomena that are caused by and accompany a cardiovascular disease or disorder and serve as an indicator of the cardiovascular disease or disorder. For example, angina; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower limbs; cyanosis; fatigue; fainting; tingling in the face; tingling in the limbs; limpness or muscle cramps; abdominal distension; or fever are symptoms of a cardiovascular disease or disorder.

[0251] As used herein, "targeting" or "targeted" refers to the process of designing and selecting antisense compounds that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0252] As used herein, "therapeutically effective amount" means an amount of an agent that provides a therapeutic benefit to an individual.

[0253] As used herein, "therapeutic lifestyle changes" means dietary and lifestyle changes aimed at reducing fat / adipose tissue mass and / or cholesterol. Such changes will reduce the risk of developing heart disease and may include recommendations for daily dietary intake of total calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrates, protein, cholesterol, insoluble fiber, and recommendations for physical activity.

[0254] As used herein, "treat" or "treating" refers to the administration of a compound described herein to effect an alteration or improvement in a disease, disorder, or condition.

[0255] As used herein, "triglyceride" or "TG" means a lipid or neutral fat composed of glycerol bound to three fatty acid molecules.

[0256] As used herein, "type 2 diabetes" (also referred to as "type 2 diabetes mellitus," "diabetes mellitus, type 2," "non-insulin-dependent diabetes mellitus," "NIDDM," "obesity-related diabetes," or "adult-onset diabetes") is a metabolic disorder primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.

[0257] Certain embodiments

[0258] Certain embodiments provide compounds and methods for reducing ApoCIII mRNA and protein expression. In certain embodiments, the compound is an ApoCIII-specific inhibitor for treating, preventing, or ameliorating ApoCIII-related diseases. In certain embodiments, the compound is an antisense oligonucleotide targeting ApoCIII. In certain embodiments, the compound comprises a modified oligonucleotide targeting ApoCIII and a conjugate group.

[0259] In certain embodiments, the compound comprises an siRNA or antisense oligonucleotide targeting apolipoprotein C-III (ApoC-III) known in the art and a conjugate group described herein. Examples of antisense oligonucleotides targeting ApoC-III suitable for conjugation include, but are not limited to, those disclosed in U.S. Patent Application Publication No. US2013 / 0317085, which is incorporated herein by reference in its entirety. In certain embodiments, the compound comprises an siRNA or antisense oligonucleotide having SEQ ID NOs 19-96 and SEQ ID NOs 20-29 disclosed in US2013 / 0317085. The antisense oligonucleotides having the nucleobase sequence of any one of the above and the conjugate groups described herein. The nucleobase sequences of all SEQ ID NOs mentioned above are incorporated herein by reference.

[0260] In certain embodiments, the modified oligonucleotide having a conjugate group has a nucleobase sequence comprising at least 8 consecutive nucleobases of a sequence selected from any of the sequences in U.S. Patent 7,598,227, U.S. Patent 7,750,141, PCT Publication WO 2004 / 093783, or PCT Publication WO 2012 / 149495, each of which is incorporated herein by reference. In certain embodiments, the modified oligonucleotide has a sequence selected from any of the sequences disclosed in U.S. Patent 7,598,227, U.S. Patent 7,750,141, PCT Publication WO 2004 / 093783, or PCT Publication WO 2012 / 149495, each of which is incorporated herein by reference.

[0261] Certain embodiments provide compounds comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides. In certain embodiments, the modified oligonucleotide with a conjugate group consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25 linked nucleosides. In certain embodiments, the modified oligonucleotide with a conjugate group comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 linked nucleosides. In certain embodiments, the modified oligonucleotide with a conjugate group consists of 20 linked nucleosides.

[0262] Certain embodiments provide compounds comprising a modified oligonucleotide having a conjugate group that targets ApoCIII and has a sequence complementary to any one of the sequences listed in GENBANK Accession No. NM_000040.1 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_033899.8 truncated from nucleotides 20262640 to 20266603 (incorporated herein as SEQ ID NO: 2), and / or GenBank Accession No. NT_035088.1 truncated from nucleotides 6238608 to 6242565 (incorporated herein as SEQ ID NO: 3). In certain embodiments, the modified oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% complementary to any one of SEQ ID NOs: 1-3. In certain embodiments, the compound comprises a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-3. In certain embodiments, the compound comprises a modified oligonucleotide targeted to an ApoCIII segment and a conjugate group, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleobases that are complementary to an equal length portion of any one of the target segments shown in Tables 121 and 124. In the tables, " refers to the 5' most nucleotide of the target segment and "terminator site" refers to the 3' most nucleotide of the target segment. The target segment can range from the start site to the end site of each sequence listed in the table. Alternatively, the target segment can range from the start site of one sequence and end at the end site of another sequence. For example, as shown in the table, the target segment can range from 3533 to 3552, from the start site to the end site of SEQ ID NO:87. In another example, as shown in the table, the target segment can range from 3514 to 3558, from the start site of SEQ ID NO:83 to the end site of SEQ ID NO:88. In certain embodiments, the antisense compound comprises at least 8 nucleobases of the sequence of SEQ ID NO:87. In certain embodiments, the antisense compound comprises the sequence of SEQ ID NO:87. In certain embodiments, the antisense compound consists of the sequence of SEQ ID NO:87. In certain embodiments, the antisense compound is ISIS 304801.

[0263] Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to any one of SEQ ID NOs: 1-3. Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to any one of the target segments disclosed herein.

[0264] Certain embodiments provide compounds comprising a modified oligonucleotide that targets ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases that are complementary to an equal length portion of nucleobases 3533 to 3552 of SEQ ID NO:3, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:3.

[0265] Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 consecutive nucleobases that are complementary to an equal length portion of nucleobases 3514 to 3558 of SEQ ID NO:3, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:3.

[0266] Certain embodiments provide compounds comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221. In certain embodiments, the conjugated modified oligonucleotide has a nucleobase sequence comprising at least 8 consecutive nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221. In certain embodiments, the compound consists of any one of SEQ ID NOs: 19-96, 209-221 and a conjugate group.

[0267] Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 87, or 20 consecutive nucleobases comprising the nucleobase sequence of SEQ ID NO: 87. In certain embodiments, the modified oligonucleotide having a conjugate group has a nucleobase sequence of at least 8 consecutive nucleobases comprising the nucleobase sequence of SEQ ID NO: 87. In certain embodiments, the compound consists of SEQ ID NO: 87 and a conjugate group.

[0268] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures. In certain embodiments, the antisense compounds comprise modified oligonucleotides ISIS 304801 having 5'-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compounds consist of modified oligonucleotides ISIS 304801 having 5'-X, wherein X is a conjugate group comprising GalNAc.

[0269]

[0270] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures: In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 678354. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 678354.

[0271]

[0272] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures: In certain embodiments, the antisense compounds comprise the conjugated modified oligonucleotide ISIS 678357. In certain embodiments, the antisense compounds consist of the conjugated modified oligonucleotide ISIS 678357.

[0273]

[0274] In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structures. In certain embodiments, the antisense compounds comprise modified oligonucleotides having a core base sequence of SEQ ID NO: 87, wherein the 5'-GalNAc has variability in the sugar pattern of the wing. In certain embodiments, the antisense compounds consist of modified oligonucleotides having a core base sequence of SEQ ID NO: 87, wherein the 5'-GalNAc has variability in the sugar pattern of the wing.

[0275]

[0276] where R 1 is –OCH2CH2OCH3(MOE) and R 2 H; or R 1 and R 2 Together form a bridge, where R 1 For –O- and R 2 is –CH2-, -CH(CH3)- or -CH2CH2-, and R 1 and R 2 directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-;

[0277] And for every pair R on the same ring 3 and R 4 , independently for each ring: R 3 is selected from H and -OCH2CH2OCH3 and R 4 H; or R 3 and R 4 Together form a bridge, where R 3 For –O- and R 4 is –CH2-, -CH(CH3)- or -CH2CH2- and R 3 and R 4 directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and -O-CH2CH2-;

[0278] And R 5 selected from H and –CH3;

[0279] And Z is selected from S - and O - .

[0280] Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide is single-stranded.

[0281] Certain embodiments provide a compound comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein at least one internucleoside linkage is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 internucleoside linkages of the modified oligonucleotide are phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 phosphodiester internucleoside linkages. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is selected from phosphodiester internucleoside linkages and phosphorothioate internucleoside linkages. Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein at least one nucleoside comprises a modified nucleobase. In certain embodiments, the modified nucleobase is 5-methylcytosine.

[0282] Certain embodiments provide compounds comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide comprises at least one modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified sugar comprises a 2'-O-methoxyethyl, a constrained ethyl, a 3'-fluoro-HNA, or a 4'-(CH2) n -O-2' bridge, where n is 1 or 2.

[0283] Certain embodiments provide a compound comprising a modified oligonucleotide targeting ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises: (a) a gap segment consisting of linked deoxynucleosides; (b) a 5' wing segment consisting of linked nucleosides; (c) a 3' wing segment consisting of linked nucleosides, and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.

[0284] Certain embodiments provide a compound comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides, and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is 5-methylcytosine.

[0285] Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of any one of SEQ ID NOs: 19-96, 209-221, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides, and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is 5-methylcytosine.

[0286] Certain embodiments provide compounds comprising a modified oligonucleotide targeted to ApoCIII and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NO: 87, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides, and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is 5-methylcytosine.

[0287] Certain embodiments provide modified oligonucleotides and conjugate groups that target ApoCIII, wherein the modified oligonucleotide consists of 20 linked nucleosides and has the nucleobase sequence of SEQ ID NO: 87, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides, and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is 5-methylcytosine.

[0288] In certain embodiments, the conjugate group is attached to the modified oligonucleotide at the 5' end of the modified oligonucleotide.In certain embodiments, the conjugate group is attached to the modified oligonucleotide at the 3' end of the modified oligonucleotide.

[0289] In certain embodiments, the conjugate group comprises exactly one ligand. In certain embodiments, the conjugate group comprises one or more ligands. In certain embodiments, the conjugate group comprises exactly two ligands. In certain embodiments, the conjugate group comprises two or more ligands. In certain embodiments, the conjugate group comprises three or more ligands. In certain embodiments, the conjugate group comprises exactly three ligands. In certain embodiments, each ligand is selected from the group consisting of: a polysaccharide, a modified polysaccharide, mannose, galactose, a mannose derivative, a galactose derivative, 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-pyranose Mannose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-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-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3 ,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose. In certain embodiments, each ligand is N-acetylgalactosamine.

[0290] In certain embodiments, the conjugate group comprises:

[0291]

[0292] In certain embodiments, the conjugate group comprises:

[0293]

[0294] In certain embodiments, the conjugate group comprises:

[0295]

[0296] In certain embodiments, the conjugate group comprises:

[0297]

[0298] In certain embodiments, the conjugate group comprises:

[0299]

[0300] In certain embodiments, the conjugate group comprises at least one phosphorus linking group or a neutral linking group.

[0301] In certain embodiments, the conjugate group comprises a structure selected from the group consisting of:

[0302]

[0303] where n is 1 to 12, and

[0304] Wherein m is 1 to 12.

[0305] In certain embodiments, the conjugate group comprises a tether having a structure selected from:

[0306]

[0307] wherein L is a phosphorus linking group or a neutral linking group;

[0308] Z1 is C(=O)O-R2;

[0309] Z2 is H, C1-C6 alkyl or substituted C1-C6 alkyl;

[0310] R2 is H, C1-C6 alkyl or substituted C1-C6 alkyl; and

[0311] Each ml is independently from 0 to 20, wherein for each tether, at least one ml is greater than 0.

[0312] In certain embodiments, the conjugate group comprises a tether having a structure selected from:

[0313]

[0314] wherein Z2 is H or CH3; and

[0315] Each ml is independently from 0 to 20, wherein for each tether, at least one ml is greater than 0.

[0316] In certain embodiments, the conjugate group comprises a tether having a structure selected from:

[0317]

[0318] where n is 1 to 12, and

[0319] Wherein m is 1 to 12.

[0320] In certain embodiments, the conjugate group is covalently linked to the modified oligonucleotide.

[0321] In certain embodiments, the compound has a structure represented by the following formula:

[0322]

[0323] in

[0324] A is the modified oligonucleotide;

[0325] B is the cleavable part

[0326] C is the conjugate linker

[0327] D is a branched group

[0328] Each E is a tether;

[0329] Each F is a ligand; and

[0330] q is an integer between 1 and 5.

[0331] In certain embodiments, the compound has a structure represented by the following formula:

[0332]

[0333] in:

[0334] A is the modified oligonucleotide;

[0335] B is the cleavable part

[0336] C is the conjugate linker

[0337] D is a branched group

[0338] Each E is a tether;

[0339] Each F is a ligand;

[0340] Each n is independently 0 or 1; and

[0341] q is an integer between 1 and 5.

[0342] In certain embodiments, the compound has a structure represented by the following formula:

[0343]

[0344] in

[0345] A is the modified oligonucleotide;

[0346] B is the cleavable part;

[0347] C is a conjugate linker;

[0348] Each E is a tether;

[0349] Each F is a ligand; and

[0350] q is an integer between 1 and 5.

[0351] In certain embodiments, the compound has a structure represented by the following formula:

[0352]

[0353] in

[0354] A is the modified oligonucleotide;

[0355] C is a conjugate linker;

[0356] D is a branched group;

[0357] Each E is a tether;

[0358] Each F is a ligand; and

[0359] q is an integer between 1 and 5.

[0360] In certain embodiments, the compound has a structure represented by the following formula:

[0361]

[0362] in

[0363] A is the modified oligonucleotide;

[0364] C is a conjugate linker;

[0365] Each E is a tether;

[0366] Each F is a ligand; and

[0367] q is an integer between 1 and 5.

[0368] In certain embodiments, the compound has a structure represented by the following formula:

[0369]

[0370] in

[0371] A is the modified oligonucleotide;

[0372] B is the cleavable part;

[0373] D is a branched group;

[0374] Each E is a tether;

[0375] Each F is a ligand; and

[0376] q is an integer between 1 and 5.

[0377] In certain embodiments, the compound has a structure represented by the following formula:

[0378]

[0379] in

[0380] A is the modified oligonucleotide;

[0381] B is the cleavable part;

[0382] Each E is a tether;

[0383] Each F is a ligand; and

[0384] q is an integer between 1 and 5.

[0385] In certain embodiments, the compound has a structure represented by the following formula:

[0386]

[0387] in

[0388] A is the modified oligonucleotide;

[0389] D is a branched group;

[0390] Each E is a tether;

[0391] Each F is a ligand; and

[0392] q is an integer between 1 and 5.

[0393] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0394]

[0395] wherein each L is independently a phosphorus linking group or a neutral linking group; and

[0396] Each n is independently 1 to 20.

[0397] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0398]

[0399] In certain embodiments, the conjugate linker has the following structure:

[0400]

[0401] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0402]

[0403] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0404]

[0405] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0406]

[0407] In certain embodiments, the conjugate linker comprises pyrrolidine.In certain embodiments, the conjugate linker does not comprise pyrrolidine.

[0408] In certain embodiments, the conjugate linker comprises PEG.

[0409] In certain embodiments, the conjugate linker comprises an amide. In certain embodiments, the conjugate linker comprises at least two amides. In certain embodiments, the conjugate linker does not comprise an amide. In certain embodiments, the conjugate linker comprises a polyamide.

[0410] In certain embodiments, the conjugate linker comprises an amine.

[0411] In certain embodiments, the conjugate linker comprises one or more disulfide bonds.

[0412] In certain embodiments, the conjugate linker comprises a protein binding moiety. In certain embodiments, the protein binding moiety comprises a lipid. In certain embodiments, the protein-binding moiety is selected from the group consisting of: cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-di-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folic acid, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, a polysaccharide), an endosomal component, a steroid (e.g., ursolic acid, agave sapogenin, diosgenin), a terpene (e.g., a triterpene, such as sarsasapogenin, agastrointestinal, epigastrointestinal derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is selected from: C16 to C22 long chain saturated or unsaturated fatty acids, cholesterol, bile acid, vitamin E, adamantane, or 1-pentafluoropropyl.

[0413] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0414]

[0415] wherein each n is independently 1 to 20; and p is 1 to 6.

[0416] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0417]

[0418] wherein each n is independently 1 to 20.

[0419] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0420]

[0421] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0422]

[0423] Wherein n is 1 to 20.

[0424] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0425]

[0426] In certain embodiments, the conjugate linker has a structure selected from the group consisting of:

[0427] wherein each n is independently 0, 1, 2, 3, 4, 5, 6 or 7.

[0428] In certain embodiments, the conjugate linker has the following structure:

[0429]

[0430] In certain embodiments, the branched group has one of the following structures:

[0431]

[0432] wherein each A1 is independently O, S, C=O or NH; and

[0433] Each n is independently 1 to 20.

[0434] In certain embodiments, the branched group has one of the following structures:

[0435]

[0436] wherein each A1 is independently O, S, C=O or NH; and

[0437] Each n is independently 1 to 20.

[0438] In certain embodiments, the branched group has the following structure:

[0439]

[0440] In certain embodiments, the branched group has the following structure:

[0441]

[0442] In certain embodiments, the branched group has the following structure:

[0443]

[0444] In certain embodiments, the branched group has the following structure:

[0445]

[0446] In certain embodiments, the branching group comprises an ether.

[0447] In certain embodiments, the branched group has the following structure:

[0448] each n is independently 1 to 20; and

[0449] m is 2 to 6.

[0450] In certain embodiments, the branched group has the following structure:

[0451]

[0452] In certain embodiments, the branched group has the following structure:

[0453]

[0454] In certain embodiments, the branched group comprises:

[0455]

[0456] wherein each j is an integer from 1 to 3; and

[0457] wherein each n is an integer from 1 to 20.

[0458] In certain embodiments, the branched group comprises:

[0459]

[0460] In certain embodiments, each tether is selected from the group consisting of:

[0461]

[0462] wherein L is selected from a phosphorus linking group and a neutral linking group;

[0463] Z1 is C(=O)O-R2;

[0464] Z2 is H, C1-C6 alkyl or substituted C1-C6 alkyl;

[0465] R2 is H, C1-C6 alkyl or substituted C1-C6 alkyl; and

[0466] Each ml is independently from 0 to 20, wherein for each tether, at least one ml is greater than 0.

[0467] In certain embodiments, each tether is selected from the group consisting of:

[0468]

[0469] wherein Z2 is H or CH3; and

[0470] Each m2 is independently from 0 to 20, wherein for each tether, at least one m2 is greater than 0.

[0471] In certain embodiments, each tether is selected from the group consisting of:

[0472]

[0473] where n is 1 to 12, and

[0474] Wherein m is 1 to 12.

[0475] In certain embodiments, at least one tether comprises ethylene glycol.

[0476] In certain embodiments, at least one tether comprises an amide. In certain embodiments, at least one tether comprises a polyamide.

[0477] In certain embodiments, at least one tether comprises an amine.

[0478] In certain embodiments, at least two tethers are different from each other. In certain embodiments, all tethers are identical to each other.

[0479] In certain embodiments, each tether is selected from the group consisting of:

[0480]

[0481] wherein each n is independently 1 to 20; and

[0482] Each p is from 1 to about 6.

[0483] In certain embodiments, each tether is selected from the group consisting of:

[0484]

[0485] In certain embodiments, each tether has the following structure:

[0486]

[0487] wherein each n is independently 1 to 20.

[0488] In certain embodiments, each tether has the following structure:

[0489]

[0490] In certain embodiments, the tether has a structure selected from the group consisting of:

[0491] wherein each n is independently 0, 1, 2, 3, 4, 5, 6 or 7.

[0492] In certain embodiments, the tether has a structure selected from the group consisting of:

[0493]

[0494] In certain embodiments, the ligand is galactose.

[0495] In certain embodiments, the ligand is mannose-6-phosphate.

[0496] In certain embodiments, each ligand is selected from:

[0497]

[0498] wherein each R1 is selected from OH and NHCOOH.

[0499] In certain embodiments, each ligand is selected from:

[0500]

[0501] In certain embodiments, each ligand has the following structure:

[0502]

[0503] In certain embodiments, each ligand has the following structure:

[0504]

[0505] In certain embodiments, the conjugate group comprises a cell targeting moiety.

[0506] In certain embodiments, the conjugate group comprises a cell targeting moiety having the following structure:

[0507]

[0508] wherein each n is independently 1 to 20.

[0509] In certain embodiments, the cell targeting moiety has the following structure:

[0510]

[0511] In certain embodiments, the cell targeting moiety has the following structure:

[0512]

[0513] wherein each n is independently 1 to 20.

[0514] In certain embodiments, the cell targeting moiety has the following structure:

[0515]

[0516] In certain embodiments, the cell targeting moiety comprises:

[0517]

[0518] In certain embodiments, the cell targeting moiety comprises:

[0519]

[0520] In certain embodiments, the cell targeting moiety has the following structure:

[0521]

[0522] In certain embodiments, the cell targeting moiety has the following structure:

[0523]

[0524] In certain embodiments, the cell targeting moiety comprises:

[0525]

[0526] In certain embodiments, the cell targeting moiety has the following structure:

[0527]

[0528] In certain embodiments, the cell targeting moiety comprises:

[0529]

[0530] In certain embodiments, the cell targeting moiety comprises:

[0531]

[0532] In certain embodiments, the cell targeting moiety comprises:

[0533]

[0534] In certain embodiments, the cell targeting moiety has the following structure:

[0535]

[0536] In certain embodiments, the cell targeting moiety has the following structure:

[0537]

[0538] In certain embodiments, the cell targeting moiety has the following structure:

[0539]

[0540] In certain embodiments, the cell targeting moiety has the following structure:

[0541]

[0542] In certain embodiments, the cell targeting moiety has the following structure:

[0543]

[0544] In certain embodiments, the cell targeting moiety comprises:

[0545]

[0546] In certain embodiments, the cell targeting moiety comprises:

[0547]

[0548] In certain embodiments, the cell targeting moiety comprises:

[0549]

[0550] In certain embodiments, the cell targeting moiety comprises:

[0551]

[0552] In certain embodiments, the cell targeting moiety has the following structure:

[0553]

[0554] In certain embodiments, the cell targeting moiety comprises:

[0555]

[0556] In certain embodiments, the cell targeting moiety has the following structure:

[0557]

[0558] In certain embodiments, the cell targeting moiety comprises:

[0559]

[0560] wherein each Y is selected from O, S, substituted or unsubstituted C1-C 10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

[0561] In certain embodiments, the conjugate group comprises:

[0562]

[0563] wherein each Y is selected from O, S, substituted or unsubstituted C1-C 10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

[0564] In certain embodiments, the cell targeting moiety has the following structure:

[0565]

[0566] wherein each Y is selected from O, S, substituted or unsubstituted C1-C 10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

[0567] In certain embodiments, the conjugate group comprises:

[0568]

[0569] In certain embodiments, the conjugate group comprises:

[0570]

[0571] In certain embodiments, the conjugate group comprises:

[0572]

[0573] In certain embodiments, the conjugate group comprises:

[0574]

[0575] In certain embodiments, the conjugate group comprises a cleavable moiety selected from the group consisting of a phosphodiester, an amide, or an ester.

[0576] In certain embodiments, the conjugate group comprises a phosphodiester cleavable moiety.

[0577] In certain embodiments, the conjugate group does not comprise a cleavable moiety, and wherein the conjugate group comprises a phosphorothioate linkage between the conjugate group and the oligonucleotide.

[0578] In certain embodiments, the conjugate group comprises an amide cleavable moiety.

[0579] In certain embodiments, the conjugate group comprises an ester cleavable moiety.

[0580] In certain embodiments, the compound has the structure:

[0581]

[0582] wherein each n is independently 1 to 20;

[0583] Q 13 is H or O(CH2)2-OCH3;

[0584] A is the modified oligonucleotide; and

[0585] Bx is a heterocyclic base moiety.

[0586] In certain embodiments, the compound has the structure:

[0587]

[0588] wherein each n is independently 1 to 20;

[0589] Q 13 is H or O(CH2)2-OCH3;

[0590] A is the modified oligonucleotide; and

[0591] Bx is a heterocyclic base moiety.

[0592] In certain embodiments, the compound has the structure:

[0593] wherein each n is independently 1 to 20;

[0594] Q 13 is H or O(CH2)2-OCH3;

[0595] A is the modified oligonucleotide;

[0596] Z is H or an attached solid support; and

[0597] Bx is a heterocyclic base moiety.

[0598] In certain embodiments, the compound has the structure:

[0599]

[0600] wherein each n is independently 1 to 20;

[0601] Q 13 is H or O(CH2)2-OCH3;

[0602] A is the modified oligonucleotide;

[0603] Z is H or an attached solid support; and

[0604] Bx is a heterocyclic base moiety.

[0605] In certain embodiments, the compound has the structure:

[0606]

[0607] where Q 13 is H or O(CH2)2-OCH3;

[0608] A is the modified oligonucleotide; and

[0609] Bx is a heterocyclic base moiety.

[0610] In certain embodiments, the compound has the structure:

[0611] where Q 13 is H or O(CH2)2-OCH3;

[0612] A is the modified oligonucleotide; and

[0613] Bx is a heterocyclic base moiety.

[0614] In certain embodiments, the compound has the structure:

[0615]

[0616] where Q 13 is H or O(CH2)2-OCH3;

[0617] A is the modified oligonucleotide; and

[0618] Bx is a heterocyclic base moiety.

[0619] In certain embodiments, the compound has the structure:

[0620] where Q 13 is H or O(CH2)2-OCH3;

[0621] A is the modified oligonucleotide; and

[0622] Bx is a heterocyclic base moiety.

[0623] In certain embodiments, the compound has the structure:

[0624]

[0625] where Q 13 is H or O(CH2)2-OCH3;

[0626] A is the modified oligonucleotide; and

[0627] Bx is a heterocyclic base moiety.

[0628] In certain embodiments, the compound has the structure:

[0629] where Q 13 is H or O(CH2)2-OCH3;

[0630] A is the modified oligonucleotide; and

[0631] Bx is a heterocyclic base moiety.

[0632] In certain embodiments, the compound has the structure:

[0633]

[0634] where Q 13 is H or O(CH2)2-OCH3;

[0635] A is the modified oligonucleotide; and

[0636] Bx is a heterocyclic base moiety.

[0637] In certain embodiments, the compound has the structure:

[0638]

[0639] where Q 13 is H or O(CH2)2-OCH3;

[0640] A is the modified oligonucleotide; and

[0641] Bx is a heterocyclic base moiety.

[0642] In certain embodiments, the compound has the structure:

[0643]

[0644] where Q 13 is H or O(CH2)2-OCH3;

[0645] A is the modified oligonucleotide; and

[0646] Bx is a heterocyclic base moiety.

[0647] In certain embodiments, the compound has the structure:

[0648]

[0649] where Q 13 is H or O(CH2)2-OCH3;

[0650] A is the modified oligonucleotide; and

[0651] Bx is a heterocyclic base moiety.

[0652] In certain embodiments, the compound has the structure:

[0653]

[0654] where Q 13 is H or O(CH2)2-OCH3;

[0655] A is the modified oligonucleotide; and

[0656] Bx is a heterocyclic base moiety.

[0657] In certain embodiments, the conjugate group comprises:

[0658]

[0659] where Q 13 is H or O(CH2)2-OCH3;

[0660] A is the modified oligonucleotide; and

[0661] Bx is a heterocyclic base moiety.

[0662] In certain embodiments, the conjugate group comprises:

[0663]

[0664] where Q 13 is H or O(CH2)2-OCH3;

[0665] A is the modified oligonucleotide; and

[0666] Bx is a heterocyclic base moiety.

[0667] In certain embodiments, the conjugate group comprises:

[0668]

[0669] where Q 13 is H or O(CH2)2-OCH3;

[0670] A is the modified oligonucleotide; and

[0671] Bx is a heterocyclic base moiety.

[0672] In certain embodiments, B xis selected from adenine, guanine, thymine, uracil, cytosine or 5-methylcytosine. In certain embodiments, B x In certain embodiments, B x In certain embodiments, Q 13 In certain embodiments, Q 13 For H.

[0673] Certain embodiments of the present invention provide prodrugs comprising the compositions or compounds disclosed herein.

[0674] In certain embodiments, the compound is in salt form. In certain embodiments, the compound further comprises a pharmaceutically acceptable carrier or diluent. In certain embodiments, the compound comprises a modified oligonucleotide targeting ApoCIII and a conjugate group or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

[0675] Certain embodiments provide compositions and methods comprising administering to an animal a conjugated antisense compound or composition disclosed herein. In certain embodiments, administering the conjugated antisense compound prevents, treats, ameliorates, or slows the progression of cardiovascular, metabolic, and / or inflammatory diseases.

[0676] Certain embodiments provide compositions and methods for treating ApoCIII-related diseases, disorders or conditions. In certain embodiments, ApoCIII levels are elevated in animals. In certain embodiments, the composition is a compound comprising an ApoCIII specific inhibitor. In certain embodiments, the ApoCIII specific inhibitor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is a modified oligonucleotide targeting ApoCIII. In certain embodiments, the antisense compound is a modified oligonucleotide targeting ApoCIII and a conjugate group. In certain embodiments, the modified oligonucleotide targeting ApoCIII with a conjugate group is used to treat, prevent, improve inflammatory, cardiovascular and / or metabolic diseases, disorders or conditions, or slow down their progression. In certain embodiments, the compositions and methods for therapy include administering an ApoCIII specific inhibitor to an individual in need thereof.

[0677] Certain embodiments provide conjugated antisense compounds and compositions and methods for reducing ApoCIII levels.In certain embodiments, ApoCIII levels are reduced in the liver, adipose tissue, heart, skeletal muscle, or small intestine.

[0678] In certain embodiments, reducing ApoCIII levels in a tissue, organ, or subject increases HDL levels. In certain embodiments, HDL levels increase by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, or at least 5% from baseline HDL levels.

[0679] In certain embodiments, reducing the ApoCIII levels in a tissue, organ, or subject reduces TG levels. In certain embodiments, the subject has a triglyceride level of ≥100 mg / dL, ≥200 mg / dL, ≥300 mg / dL, ≥400 mg / dL, ≥440 mg / dL, ≥500 mg / dL, ≥600 mg / dL, ≥700 mg / dL, ≥800 mg / dL, ≥880 mg / dL, ≥900 mg / dL, ≥1000 mg / dL, ≥1100 mg / dL, ≥1200 mg / dL, ≥1300 mg / dL, ≥1400 mg / dL, ≥1500 mg / dL, ≥1600 mg / dL, ≥1700 mg / dL, ≥1800 mg / dL, ≥1900 mg / dL, ≥2000 mg / dL.

[0680] In certain embodiments, TG levels (postprandial or fasting) are reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, or at least 1% from baseline TG levels. In certain embodiments, TG levels (postprandial or fasting) are reduced to ≤1900 mg / dL, ≤1800 mg / dL, ≤1700 mg / dL, ≤1600 mg / dL, ≤1500 mg / dL, ≤1400 mg / dL, ≤1300 mg / dL, ≤1200 mg / dL, ≤1100 mg / dL, ≤1000 mg / dL, ≤900 mg / dL, ≤80 0mg / dL, ≤750mg / dL, ≤700mg / dL, ≤650mg / dL, ≤600mg / dL, ≤550mg / dL, ≤500mg / dL, ≤450mg / dL, ≤400mg / dL, ≤350mg / dL, ≤300mg / dL, ≤250mg / dL, ≤200mg / dL, ≤150mg / dL or ≤100mg / dL.

[0681] In certain embodiments, reducing the level of ApoCIII in a tissue, organ, or subject improves the LDL to HDL ratio or the TG to HDL ratio.

[0682] In certain embodiments, reducing ApoCIII levels in a tissue, organ, or subject improves insulin sensitivity.

[0683] In certain embodiments, reducing ApoCIII levels in a tissue, organ, or subject increases chylomicron clearance.

[0684] Certain embodiments provide compositions and methods for reducing ApoCIII mRNA or protein expression in an animal, comprising administering to the animal a conjugated antisense compound or composition disclosed herein to reduce ApoCIII mRNA or protein expression in the animal.

[0685] Certain embodiments provide conjugated antisense compounds and compositions and methods for preventing, treating, delaying, slowing down the progression and / or improving ApoCIII-related diseases, disorders and conditions in subjects in need thereof. In certain embodiments, the diseases, disorders and conditions include inflammatory, cardiovascular and / or metabolic diseases, disorders and conditions. Some of the cardiovascular diseases, disorders or conditions include but are not limited to chylomicronemia, hypertriglyceridemia, aortic valve stenosis, aneurysms (e.g., abdominal aortic aneurysms), angina pectoris, arrhythmias, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, myocardial infarction, peripheral vascular disease (e.g., peripheral arterial disease, peripheral arterial occlusive disease), Fredrickson type I dyslipidemia, FCS, LPL deficiency, retinal vascular occlusion or stroke. Some of the metabolic diseases, disorders or conditions include but are not limited to hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia. Some of the inflammatory diseases, disorders or conditions include, but are not limited to, pancreatitis, aortic stenosis, coronary artery disease (CAD), Alzheimer's disease and thromboembolic diseases, disorders or conditions. Some thromboembolic diseases, disorders or conditions include, but are not limited to, stroke, thrombosis (e.g., venous thromboembolism), myocardial infarction and peripheral vascular disease. Some embodiments provide conjugated antisense compounds and compositions and methods for preventing, treating, delaying, slowing progression and / or improving hypertriglyceridemia. Some embodiments provide conjugated antisense compounds and compositions and methods for preventing, treating, delaying, slowing progression and / or improving chylomicronemia. Some embodiments provide conjugated antisense compounds and compositions and methods for preventing, treating, delaying, slowing progression and / or improving pancreatitis.

[0686] Certain embodiments provide methods for reducing at least one symptom of a cardiovascular disease, disorder, or condition. In certain embodiments, symptoms include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling of the lower extremities, cyanosis, fatigue, fainting, tingling in the face, tingling in the extremities, lameness or muscle cramps, abdominal distension, and fever. In certain embodiments, symptoms of a metabolic disease, disorder, or condition include, but are not limited to, frequent urination, abnormal thirst, extreme hunger, abnormal weight loss, extreme fatigue, irritability, frequent infections, blurred vision, slow-healing wounds / bruises, tingling / numbness in the hands / feet, and recurrent skin, gum, or bladder infections. Certain embodiments provide methods for reducing at least one symptom of hypertriglyceridemia. Certain embodiments provide methods for reducing at least one symptom of chylomicronemia. Certain embodiments provide methods for reducing at least one symptom of pancreatitis.

[0687] In certain embodiments, modulation of ApoCIII expression occurs in a cell, tissue, or organ. In certain embodiments, modulation occurs in a cell, tissue, or organ of an animal. In certain embodiments, modulation is a reduction in ApoCIII mRNA levels. In certain embodiments, modulation is a reduction in ApoCIII protein levels. In certain embodiments, both ApoCIII mRNA and protein are reduced. The reduction can occur in a time-dependent or dose-dependent manner.

[0688] In certain embodiments, the subject or animal is a human.

[0689] In certain embodiments, the compound is administered parenterally. In other embodiments, the parenteral administration is subcutaneous.

[0690] In certain embodiments, the conjugated antisense compound is co-administered with a second agent or therapy.In certain embodiments, the conjugated antisense compound or composition and the second agent are administered concomitantly.

[0691] In certain embodiments, the second agent is a hypoglycemic agent. In certain embodiments, the second agent is an LDL, TG, or cholesterol-lowering agent. In certain embodiments, the second agent is an anti-inflammatory agent. In certain embodiments, the second agent is an Alzheimer's disease drug. In certain embodiments, the second agent can be, but is not limited to, a nonsteroidal anti-inflammatory drug (NSAID such as aspirin), niacin (e.g., Niaspan), nicotinic acid, an apoB inhibitor (e.g., Mipomersen), a CETP inhibitor (e.g., Anacetrapib), an apo(a) inhibitor, a thyroid hormone analog (e.g., Eprotirome), an HMG-CoA reductase inhibitor (e.g., statins), fibrates (e.g., Gemfibrozil), and a microsomal triglyceride transfer protein inhibitor (e.g., Lomitapide). The agents or therapies can be administered together or simultaneously. The agents or therapies can be administered sequentially or subsequently.

[0692] Certain embodiments provide compositions and conjugated antisense compounds targeting ApoCIII as described herein for reducing ApoCIII levels in animals. Certain embodiments provide the use of compounds targeting ApoCIII for reducing ApoCIII levels in animals. Certain embodiments provide the use of compounds targeting ApoCIII for increasing HDL levels in animals. Certain embodiments provide the use of compounds targeting ApoCIII for increasing HDL chylomicron clearance in animals. Certain embodiments provide the use of compounds targeting ApoCIII for treating, preventing, or ameliorating diseases, disorders, or conditions associated with ApoCIII. Certain embodiments provide the use of compounds targeting ApoCIII for treating, preventing, or ameliorating hypertriglyceridemia. Certain embodiments provide the use of compounds targeting ApoCIII for treating, preventing, or ameliorating chylomicronemia (e.g., FCS and / or LPLD). Certain embodiments provide the use of compounds targeting ApoCIII for treating, preventing, or ameliorating pancreatitis.

[0693] Certain embodiments provide the use of the compositions and conjugated antisense compounds targeting ApoCIII described herein for the preparation of a medicament for reducing ApoCIII levels in an animal. Certain embodiments provide the use of the compositions and compounds for the preparation of a medicament for treating, preventing, or ameliorating a disease, disorder, or condition associated with ApoCIII.

[0694] Certain embodiments provide for the use of the compositions and conjugated antisense compounds described herein for the preparation of a medicament for treating, ameliorating, delaying, or preventing one or more of diseases associated with ApoCIII.

[0695] Certain embodiments provide kits for treating, preventing, or ameliorating a disease, disorder, or condition as described herein, wherein the kit comprises: (i) an ApoCIII-specific inhibitor as described herein; and optionally (ii) a second agent or therapy as described herein.

[0696] The kits of the invention may further include instructions for using the kit to treat, prevent, or ameliorate a disease, disorder, or condition as described herein via a combination therapy as described herein.

[0697] B. Certain compounds

[0698] In certain embodiments, the present invention provides conjugated antisense compounds comprising an antisense oligonucleotide and a conjugate.

[0699] a. Certain antisense oligonucleotides

[0700] In certain embodiments, the invention provides antisense oligonucleotides.Described antisense oligonucleotides comprise connection nucleosides, and each nucleoside comprises sugar moiety and core base.Can consider the structure of described antisense oligonucleotide according to chemical signature (for example, modification and modified pattern) and core base sequence (for example, the sequence of antisense oligonucleotide, identity and sequence of target nucleic acid).

[0701] i. Certain chemical characteristics

[0702] In certain embodiments, the antisense oligonucleotide comprises one or more modifications. In certain embodiments, the antisense oligonucleotide comprises one or more modified nucleosides and / or modified internucleoside linkages. In certain embodiments, the modified nucleosides comprise modified sugar moieties and / or modified core bases.

[0703] 1. Certain sugar moieties

[0704] In certain embodiments, the compounds of the present disclosure include one or more modified nucleosides, and the modified nucleosides include modified sugar moieties. Relative to oligonucleotides that only include nucleosides containing naturally occurring sugar moieties, the compounds comprising one or more sugar-modified nucleosides can have desirable properties, such as enhanced nuclease stability or increased binding affinity to the target nucleic acid. In certain embodiments, the modified sugar moieties are substituted sugar moieties. In certain embodiments, the modified sugar moieties are sugar surrogates. The sugar surrogates can include one or more substitutions corresponding to those substituted sugar moieties.

[0705] In certain embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2' position and / or the 5' position. Examples of sugar substituents suitable for 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 is selected from allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, O-C1-C 10 Substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn) and O-CH2-C(=O)-N(Rm)(Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C1-C 10 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 more than one non-bridging sugar substituent, for example, a 2'-F-5'-methyl sugar moiety (for additional 5',2'-disubstituted sugar moieties and nucleosides, see, for example, PCT International Application WO 2008 / 101157).

[0706] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In certain embodiments, the 2'-substituted nucleosides comprise a 2'-substituent selected from the group consisting of halo, allyl, amino, azido, 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-alkylene-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ) or O-CH2-C(=O)-N(R m )(R n ), where each R m and R n are independently H, an amino protecting group or a substituted or unsubstituted C1-C 10 These 2'-substituents may be further substituted by one or more substituents independently selected from the group consisting of hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), mercapto, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0707] In certain embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from the group consisting of: F, NH2, N3, OCF 3、 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 ), where each R m and R n are independently H, an amino protecting group or a substituted or unsubstituted C1-C 10 alkyl.

[0708] In certain embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from the group consisting of: F, OCF 3、 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.

[0709] In certain embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from the group consisting of: F, O-CH3, and OCH2CH2OCH3.

[0710] The sugar moiety of some modifications comprises a bridging sugar substituent that forms a second ring, thereby producing a bicyclic sugar moiety. In some of the embodiments, the bicyclic sugar moiety comprises a bridge between the 4' furanose ring atom and the 2' furanose ring atom. Examples of the 4' to 2' sugar substituents include, but are not limited to: -[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 analogs thereof (see, e.g., U.S. Patent No. 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., WO2009 / 006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., WO 2008 / 006478, published December 11, 2008); 2008 / 150729); 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 Alkyl; 4'-CH2-N(R)-O-2', where R is H, C1-C 12 Alkyl or protecting group (see, U.S. Patent 7,427,672 issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and the like (see, published PCT International Application WO 2008 / 154401 published on December 8, 2008).

[0711] In certain embodiments, the 4' to 2' bridge independently comprises 1 to 4 linked groups independently selected from the group consisting of: -[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 )-;

[0712] in:

[0713] x is 0, 1, or 2;

[0714] n is 1, 2, 3, or 4;

[0715] Each R a and R b are independently H, a protecting group, a hydroxyl group, a 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 20 aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfinyl (S(=O)-J1); and

[0716] Each J1 and J2 is independently H, 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 20 Aryl, acyl (C (= O) -H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl or a protecting group.

[0717] Nucleosides containing a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4'-CH2-O-2') BNA, (B) β-D-methyleneoxy (4'-CH2-O-2') BNA (also known as locked nucleic acid or LNA), (C) ethyleneoxy (4'-(CH2)2-O-2') BNA, (D) aminooxy (4'-CH2-ON(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-O-2') BNA, and (F) oxyamino (4'-CH2-N(R)-O-2') BNA. ')BNA, (F) methyl(methyleneoxy) (4'-CH(CH3)-O-2')BNA (also known as constrained ethyl or cEt), (G) methylene-thio (4'-CH2-S-2')BNA, (H) methylene-amino (4'-CH2-N(R)-2')BNA, (I) methylcarbocycle (4'-CH2-CH(CH3)-2')BNA, and (J) propylenecarbocycle (4'-(CH2)3-2')BNA.

[0718]

[0719] wherein Bx is a nucleobase moiety and R is independently H, a protecting group or a C1-C 12 alkyl.

[0720] Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et 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., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26)8362-8379 (July 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol. l., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461 and 7,399,845; WO 2004 / 106356, WO 1994 / 14226, WO 2005 / 021570 and WO 2007 / 134181; U.S. Patent Publication Nos. US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618; U.S. Patent Serial Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and PCT International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922.

[0721] In certain embodiments, bicyclic sugar moiety and the nucleoside that mixes described bicyclic sugar moiety are further defined by isomeric configuration.For example, the nucleoside that comprises 4 '-2 ' methylene-oxy bridged can be α-L configuration or be β-D configuration.Previously, α-L-methyleneoxy (4 '-CH2-O-2 ') bicyclic nucleoside is mixed in the antisense oligonucleotide of demonstration antisense activity (Frieden etc., Nucleic Acids Research, 2003,21,6365-6372).

[0722] In certain embodiments, the substituted sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., a 5'-substituted sugar and a 4'-2' bridged sugar). (See, PCT International Application WO 2007 / 134181 published on November 22, 2007, wherein LNA is substituted with, for example, a 5'-methyl or 5'-vinyl group).

[0723] In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain embodiments, the oxygen atom of a naturally occurring sugar is substituted by, for example, a sulfur, carbon, or nitrogen atom. In certain embodiments, the modified sugar moiety further comprises a bridging substituent and / or a non-bridging substituent as described above. For example, some sugar surrogate comprises a 4'-sulfur atom and a 2'-position (see, for example, U.S. patent application US2005 / 0130923 published on June 16, 2005) and / or a substitution on the 5' position. By way of example, carbocyclic bicyclic nucleosides with 4'-2' bridging have been described (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).

[0724] In certain embodiments, the sugar surrogate comprises a ring having other than 5 atoms. For example, in certain embodiments, the sugar surrogate comprises a morpholino. Morpholino compounds and their use in oligomeric compounds have been reported in many patents and published articles (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 surrogate having the following structure:

[0725]

[0726] In certain embodiments, morpholinos can be modified, for example, by adding or changing various substituents from the above morpholino structures. Such sugar surrogates are referred to herein as "modified morpholinos."

[0727] For another example, in certain embodiments, the sugar substitute comprises a six-membered tetrahydropyran. The tetrahydropyran may be further modified or substituted. Nucleosides comprising the modified tetrahydropyran include, but are not limited to, hexitol nucleic acids (HNA), anitol nucleic acids (ANA), mannitol nucleic acids (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002) 10: 841-854), fluoro-HNA (F-HNA), and compounds having Formula VI:

[0728]

[0729] wherein independently for each of the at least one tetrahydropyranoside analog of Formula VI:

[0730] Bx is the nucleobase moiety;

[0731] T3 and T4 are each independently an internucleoside linking group that links the tetrahydropyranoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group that links the tetrahydropyranoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;

[0732] 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; and

[0733] Each of R1 and R2 is independently selected from the following: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein X is O, S or NJ1, and each J1, J2 and J3 is independently H or C1-C6 alkyl.

[0734] In certain embodiments, a modified THP nucleoside of Formula VI is provided, wherein Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are each H. In certain embodiments, at least one of Q1, Q2, Q3, Q4, Q5, Q6, and Q7 is 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, wherein 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.

[0735] Many other bicyclic and tricyclic sugar surrogate ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0736] Also provided are combinations of modifications, including but not limited to, 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008 / 101157 published on 8 / 21 / 08 for other disclosed 5',2'-disubstituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923 published on June 16, 2005) or alternatively 5'-substitution of bicyclic nucleic acids (see PCT International Application WO 2007 / 134181 published on 11 / 22 / 07, wherein the 4'-CH2-O-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, for example, Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0737] In certain embodiments, the disclosure provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified core bases and / or modified linkages. Select specific modification so that the resulting oligonucleotide has the characteristics of hope. In certain embodiments, the oligonucleotide comprises one or more RNA sample nucleosides. In certain embodiments, the oligonucleotide comprises one or more DNA sample nucleotides.

[0738] 2. Certain nucleobase modifications

[0739] In certain embodiments, the nucleosides of the present disclosure comprise one or more unmodified nucleobases.In certain embodiments, the nucleosides of the present disclosure comprise one or more modified nucleobases.

[0740] In certain embodiments, the modified nucleobase is selected from the group consisting of universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and thymine. Adenine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, specifically 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, mixed bases, size-expanded bases, and fluorinated bases as defined herein. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytosine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytosine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one); G-clamps such as substituted phenoxazine cytosines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytosine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytosine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., eds., CRC Press, 1993, 273-288.

[0741] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include but are not limited to U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525, 5,830,653 and 6,005,096, certain of which are common with the present application and each of which is incorporated herein by reference in its entirety.

[0742] 3. Certain internucleoside linkages

[0743] In certain embodiments, the present disclosure provides oligonucleotides comprising connected nucleosides. In the embodiments, nucleosides can be linked together using any internucleoside linkage. Two major types of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphodiester (PO), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (PS). Representative phosphorus-free internucleoside linking groups include but are not limited to methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), aminomethylthionate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N, N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphodiester linkages, modified linkages can be used to change (usually increase) the nuclease resistance of oligonucleotides. In certain embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or as individual enantiomers. Representative chiral linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0744] The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), or as for sugar anomers, or as (D) or (L), as for amino acids, etc. All such possible isomers, as well as their racemic and optically pure forms, are included in the antisense compounds provided herein.

[0745] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), methylacetal (3'-O-CH2-O-5'), and thiomethylacetal (3'-S-CH2-O-5'). Additional neutral internucleoside linkages include nonionic linkages comprising siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and P.D. Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S, and CH2 components.

[0746] 4. Certain motifs

[0747] In certain embodiments, antisense oligonucleotides include one or more modified nucleosides (e.g., nucleosides comprising modified sugars and / or modified core bases) and / or one or more modified internucleoside linkages. The modified pattern on the oligonucleotide is referred to herein as a motif. In certain embodiments, sugars, core bases, and linkage motifs are independent of one another.

[0748] a. Certain sugar motifs

[0749] In certain embodiments, the oligonucleotides include one or more types of modified sugar moieties and / or naturally occurring sugar moieties arranged in a defined pattern or sugar modification motif along the oligonucleotide or a region thereof. The motif may include any sugar modification discussed herein and / or other known sugar modifications.

[0750] In certain embodiments, oligonucleotide comprises the region with gapmer sugar motif or is made up of described region, and described gapmer sugar motif comprises two outer regions or " wing " and center or inner region or " gap ".The three regions (5 '-wing, gap and 3 '-wing) of gapmer sugar motif form continuous nucleoside sequence, and wherein at least some sugar moieties of the nucleoside of each wing are different from at least some sugar moieties of the nucleoside of gap.To be precise, the sugar moiety of at least the closest gap of the nucleoside of each wing (the most 3 ' nucleoside of 5 '-wing and the most 5 ' nucleoside of 3 '-wing) is different from the sugar moiety of adjacent gap nucleoside, thereby defining the boundary between wing and gap.In certain embodiments, the sugar moiety in gap is identical to each other.In certain embodiments, gap comprises one or more nucleosides, and described nucleoside has the sugar moiety of the sugar moiety of one or more other nucleosides that is different from gap.In certain embodiments, the sugar motifs of two wings are identical to each other (symmetrical sugar gapmer).In certain embodiments, the sugar motif of 5 '-wing is different from the sugar motif of 3 '-wing (asymmetric sugar gapmer).

[0751] i. Certain 5'-wings

[0752] In certain embodiments, the 5'-wing of a gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 7 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5'-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 5'-wing of a gapmer consists of 6 linked nucleosides.

[0753] In certain embodiments, the 5'-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5'-wing of a gapmer is an LNA nucleoside.

[0754] In certain embodiments, the 5'-wing of the gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside 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.

[0755] In certain embodiments, the 5'-wing of the gapmer comprises 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 comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 5'-wing of the gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5'-wing is an RNA-like nucleoside.

[0756] 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 2'-MOE nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0757] In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0758] ii. Certain 3'-wings

[0759] In certain embodiments, the 3'-wing of a gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 7 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3'-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 3'-wing of a gapmer consists of 6 linked nucleosides.

[0760] In certain embodiments, the 3'-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3'-wing of a gapmer is an LNA nucleoside.

[0761] In certain embodiments, the 3'-wing of the gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of the gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of the gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside 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.

[0762] In certain embodiments, the 3'-wing of the gapmer comprises 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 comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3'-wing of the gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5'-wing is an RNA-like nucleoside.

[0763] 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.

[0764] In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0765] In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3'-wing of a gapmer comprises at least one LNA nucleoside and at least one 2'-deoxynucleoside.

[0766] 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.

[0767] 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.

[0768] 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.

[0769] 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.

[0770] iii. Certain central areas (gaps)

[0771] In certain embodiments, the gap of a gapmer consists of 6 to 20 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 6 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 7 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 8 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 9 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 10 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 11 connected nucleosides. In certain embodiments, the gap of a gapmer consists of 12 connected nucleosides.

[0772] In certain embodiments, each nucleoside in the gap of the gapmer is a 2'-deoxynucleoside. In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, each nucleoside in the gap of the gapmer is a 2'-deoxynucleoside or a "DNA-like" modified nucleoside. In the embodiments, "DNA-like" means that the nucleoside has characteristics similar to DNA, so that the duplex comprising the gapmer and the RNA molecule can activate RNase H. For example, under certain conditions, 2'-(ara)-F has been shown to support RNase H activation and is therefore DNA-like. In certain embodiments, one or more nucleosides in the gap of the gapmer are not 2'-deoxynucleosides and are not DNA-like. In certain embodiments, despite this, the gapmer still supports RNase H activation (e.g., by virtue of the number or position of non-DNA nucleosides).

[0773] In certain embodiments, the gap comprises a stretch of unmodified 2'-deoxynucleosides interrupted by one or more modified nucleosides, thereby producing three sub-regions (two stretches with one or more 2'-deoxynucleosides and one stretch with one or more interrupted modified nucleosides). In certain embodiments, the stretch of unmodified 2'-deoxynucleosides is no longer than 5, 6 or 7 nucleosides. In certain embodiments, the short segment is achieved by using a short gap region. In certain embodiments, the short segment is achieved by interrupting a longer gap region.

[0774] In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, the gap comprises one or more modified nucleosides selected from the group consisting of cEt, FHNA, LNA, and 2-thio-thymidine. In certain embodiments, the gap comprises one modified nucleoside. In certain embodiments, the gap comprises a 5'-substituted sugar moiety selected from the group consisting of 5'-Me and 5'-(R)-Me. In certain embodiments, the gap comprises two modified nucleosides. In certain embodiments, the gap comprises three modified nucleosides. In certain embodiments, the gap comprises four modified nucleosides. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is identical. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is different.

[0775] In certain embodiments, the gap comprises one or more modified linkages. In certain embodiments, the gap comprises one or more methylphosphonate linkages. In certain embodiments, the gap comprises two or more modified linkages. In certain embodiments, the gap comprises one or more modified linkages and one or more modified nucleosides. In certain embodiments, the gap comprises one modified linkage and one modified nucleoside. In certain embodiments, the gap comprises two modified linkages and two or more modified nucleosides.

[0776] b. Certain internucleoside linkage motifs

[0777] In certain embodiments, the oligonucleotide comprises a modified internucleoside linkage arranged along the oligonucleotide or its region with a defined pattern or modified internucleoside linkage motif. In certain embodiments, the oligonucleotide comprises a region with alternating internucleoside linkage motifs. In certain embodiments, the oligonucleotide of the present disclosure comprises a region with a unified modified internucleoside linkage. In certain embodiments, the oligonucleotide comprises a region uniformly connected by thiophosphate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly connected by thiophosphate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and thiophosphate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and thiophosphate and at least one internucleoside linkage is thiophosphate.

[0778] In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 7 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 9 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 12 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 13 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 14 phosphorothioate internucleoside linkages.

[0779] In certain embodiments, the oligonucleotide comprises at least one block having at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block having at least 7 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block having at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block having at least 9 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block having at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block having at least 12 consecutive phosphorothioate internucleoside linkages. In certain embodiments, at least one of the blocks is located at the 3' end of the oligonucleotide. In certain embodiments, at least one of the blocks is located within 3 nucleosides of the 3' end of the oligonucleotide. In certain embodiments, the oligonucleotide comprises less than 15 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 14 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 13 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 12 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 9 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 7 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 5 phosphorothioate internucleoside linkages.

[0780] c. Certain nucleobase modification motifs

[0781] In certain embodiments, oligonucleotides include chemical modifications to the nucleobases arranged along the oligonucleotide or its region in a defined pattern or nucleobase modification motif. In certain embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are chemically modified.

[0782] In certain embodiments, the oligonucleotide comprises a block of modified nucleobases. In certain embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleotides of the 3'-end of the oligonucleotide. In certain embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleotides of the 5'-end of the oligonucleotide.

[0783] In certain embodiments, the nucleobase modification varies with the natural base at the specific position of the oligonucleotide. For example, in certain embodiments, each purine or each 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.

[0784] In certain embodiments, some, all, or none of the cytosine moieties in the oligonucleotide are 5-methylcytosine moieties. In this article, 5-methylcytosine is not a "modified nucleobase." Therefore, unless otherwise indicated, unmodified nucleobases include cytosine residues with 5-methyl groups and those lacking 5 methyl groups. In certain embodiments, the methylation status of all or some cytosine nucleobases is specified.

[0785] In certain embodiments, chemical modification of the nucleobase comprises attaching certain conjugate groups to the nucleobase.In certain embodiments, each purine or each pyrimidine in an oligonucleotide can be optionally modified to comprise a conjugate group.

[0786] d. Certain total lengths

[0787] In certain embodiments, the present disclosure provides oligonucleotides with any one of various length ranges. In certain embodiments, the oligonucleotide is composed of X to Y connected nucleosides, wherein X represents the minimum number of nucleosides in the range and Y represents the maximum number of nucleosides in the range. In some of the embodiments, X and Y are each independently selected from: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50; Prerequisite: X≤Y. For example, in certain embodiments, the oligonucleotides may range from 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 1 1 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 13 to 29, 13 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27,14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides in the oligonucleotide of the compound is limited to a certain range or specific number, the compound can still further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides does not include an oligonucleotide having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may also comprise, for example, one or more conjugate groups, terminal groups, or other substituents.

[0788] Additionally, when an oligonucleotide is described by a total length range and by regions having specified lengths and when the sum of the specified lengths of the regions is less than the upper limit of the total length range, the oligonucleotide may have additional nucleosides beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the total length range.

[0789] 5. Certain antisense oligonucleotide chemical motifs

[0790] In certain embodiments, the chemical structure characteristics of antisense oligonucleotide are characterized by its sugar motif, internucleoside linkage motif, core base modification motif and total length.In certain embodiments, described parameters are each independent of one another.Therefore, each internucleoside linkage of the oligonucleotide with gapmer sugar motif can be modified or unmodified and can or can not follow the sugar-modified gapmer modification pattern.Therefore, the internucleoside linkage in the wing region of sugar-gapmer can be identical or different from each other, and can be identical or different with the internucleoside linkage in gap region.Equally, described sugar-gapmer oligonucleotide can comprise one or more modified core bases that are independent of the sugar-modified gapmer pattern.It will be appreciated by those skilled in the art that described motif combination can be used to produce various oligonucleotide.

[0791] In certain embodiments, the selection of internucleoside linkages and nucleoside modifications are not independent of each other.

[0792] i. Certain sequences and targets

[0793] In certain embodiments, the invention provides antisense oligonucleotides with a sequence complementary to a target nucleic acid. The antisense compounds can hybridize with target nucleic acid, producing at least one antisense activity. In certain embodiments, antisense compounds and one or more target nucleic acid specific hybridizations. In certain embodiments, the antisense compounds of specific hybridizations have core base sequences, and the core base sequences comprise and have enough complementarity with target nucleic acid to allow hybridization and produce antisense activity and with any non-target, have insufficient complementarity so that under the conditions of wishing specific hybridization (for example, under physiological conditions for in vivo or therapeutic use, and under conditions of measuring in the case of in vitro assay) avoid or reduce the region of non-specific hybridization with non-target nucleic acid sequences. In certain embodiments, oligonucleotides are selective between target and non-target, even if target and non-target all comprise target sequence. In the embodiments, selectivity can be caused by the relative accessibility of the target region of a nucleic acid molecule compared with another nucleic acid molecule.

[0794] In certain embodiments, the disclosure provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. 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, the oligonucleotide is 90% complementary to the target nucleic acid.

[0795] In certain embodiments, the oligonucleotide is 85% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is 80% complementary to the target nucleic acid. In certain embodiments, the antisense compound comprises a region that is fully complementary to the target nucleic acid and at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, the region with full complementarity is 6 to 14 core bases in length.

[0796] In certain embodiments, oligonucleotide comprises hybridization zone and terminal zone.In certain described embodiments, hybridization zone is made up of 12-30 connection nucleosides and is fully complementary to target nucleic acid.In certain embodiments, relative to target nucleic acid, hybridization zone comprises one mispairing.In certain embodiments, relative to target nucleic acid, hybridization zone comprises two mispairings.In certain embodiments, relative to target nucleic acid, hybridization zone comprises three mispairings.In certain embodiments, terminal zone is made up of 1-4 terminal nucleosides.In certain embodiments, terminal nucleosides are at 3 ' end.In certain embodiments, one or more terminal nucleosides are not complementary to target nucleic acid.

[0797] Antisense mechanisms include any mechanism involving hybridization of an oligonucleotide to a target nucleic acid, wherein the hybridization produces a biological effect. In certain embodiments, the hybridization results in degradation or occupancy of the target nucleic acid accompanied by inhibition or stimulation of cellular machinery, such as translation, transcription, or splicing of the target nucleic acid.

[0798] One type of antisense mechanism involving degradation of the target RNA is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Single-stranded antisense compounds known in the art as "DNA-like" trigger RNase H activity in mammalian cells. Consequently, activation of RNase H leads to cleavage of the RNA target, greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

[0799] In certain embodiments, the conjugate group comprises a cleavable moiety. In certain embodiments, the conjugate group comprises one or more cleavable bonds. In certain embodiments, the conjugate group comprises a linker. In certain embodiments, the linker comprises a protein binding moiety. In certain embodiments, the conjugate group comprises a cell targeting moiety (also referred to as a cell targeting group). In certain embodiments, the cell targeting moiety comprises a branched group. In certain embodiments, the cell targeting moiety comprises one or more tethers. In certain embodiments, the cell targeting moiety comprises a carbohydrate or a carbohydrate cluster.

[0800] ii. Certain cleavable parts

[0801] In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain embodiments, the cleavable moiety is connected to the antisense oligonucleotide. In certain embodiments, the cleavable moiety is directly connected to the cell targeting moiety. In certain embodiments, the cleavable moiety is connected to the conjugate linker. In certain embodiments, the cleavable moiety comprises a phosphate or a phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from the group consisting of a purine, a substituted purine, a pyrimidine, or a substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside comprising any protected heterocyclic base selected from the group consisting of uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside that is linked to the 3' position of the antisense oligonucleotide via a phosphodiester linkage and to the linker via a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine that is linked to the 3' position of the antisense oligonucleotide via a phosphodiester linkage and to the linker via a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine linked to the 3' position of the antisense oligonucleotide via a phosphodiester linkage and to a linker via a phosphodiester linkage.

[0802] In certain embodiments, the cleavable moiety is attached to the 3' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 2' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the antisense oligonucleotide via a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached to the linker via a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker via a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.

[0803] In certain embodiments, after the complex is administered to an animal, the cleavable portion is cleaved only after being internalized by the target cell. Inside the cell, the cleavable portion cleaves, thereby releasing the active antisense oligonucleotide. Although not wishing to be bound by theory, it is believed that the cleavable portion is cleaved by one or more nucleases within the cell. In certain embodiments, one or more nucleases cleave the phosphodiester linkage between the cleavable portion and the linker. In certain embodiments, the cleavable portion has a structure selected from the following:

[0804]

[0805] wherein each of Bx, Bx1, Bx2 and Bx3 is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from the group consisting of:

[0806]

[0807] iii. Certain connectors

[0808] In certain embodiments, the conjugate group comprises a linker. In certain embodiments, the linker is covalently bound to a cleavable moiety. In certain embodiments, the linker is covalently bound to an antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell targeting moiety. In certain embodiments, the linker further comprises a covalent connection to a solid support. In certain embodiments, the linker further comprises a covalent connection to a protein binding moiety. In certain embodiments, the linker further comprises a covalent connection to a solid support and further comprises a covalent connection to a protein binding moiety. In certain embodiments, the linker includes a plurality of positions for connecting a tethered ligand. In certain embodiments, the linker includes a plurality of positions for connecting a tethered ligand and is not connected to a branched group. In certain embodiments, the linker further comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not include a linker.

[0809] In certain embodiments, the linker comprises at least a linear group comprising a group selected from the group consisting of an alkyl group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group (-S-), and a hydroxyamino group (-ON(H)-). In certain embodiments, the linear group comprises a group selected from the group consisting of an alkyl group, an amide group, and an ether group. In certain embodiments, the linear group comprises a group selected from the group consisting of an alkyl group and an ether group. In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group comprises at least one neutral linking group. In certain embodiments, the linear group is covalently linked to a cell targeting moiety and a cleavable moiety. In certain embodiments, the linear group is covalently linked to a cell targeting moiety and an antisense oligonucleotide. In certain embodiments, the linear group is covalently linked to a cell targeting moiety, a cleavable moiety, and a solid support. In certain embodiments, the linear group is covalently linked to a cell targeting moiety, a cleavable moiety, a solid support, and a protein binding moiety. In certain embodiments, the linear group comprises one or more cleavable bonds.

[0810] In certain embodiments, the linker comprises a linear group covalently linked to a scaffold group. In certain embodiments, the scaffold comprises a branched aliphatic group comprising a group selected from the group consisting of an alkyl group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group, and a hydroxyamino group. In certain embodiments, the scaffold comprises a branched aliphatic group comprising a group selected from the group consisting of an alkyl group, an amide group, and an ether group. In certain embodiments, the scaffold comprises at least one monocyclic ring system or a polycyclic ring system. In certain embodiments, the scaffold comprises at least two monocyclic ring systems or polycyclic ring systems. In certain embodiments, the linear group is covalently linked to the scaffold group and the scaffold group is covalently linked to a cleavable moiety and a linker. In certain embodiments, the linear group is covalently linked to the scaffold group and the scaffold group is covalently linked to a cleavable moiety, a linker, and a solid support. In certain embodiments, the linear group is covalently linked to the scaffold group and the scaffold group is covalently linked to a cleavable moiety, a linker, and a protein-binding moiety. In certain embodiments, the linear group is covalently linked to the scaffold group and the scaffold group is covalently linked to a cleavable moiety, a linker, a protein-binding moiety, and a solid support. In certain embodiments, the scaffold group comprises one or more cleavable bonds.

[0811] In certain embodiments, the linker comprises a protein-binding moiety. In certain embodiments, the protein-binding moiety is a lipid, such as, for example, but not limited to, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-di-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folic acid, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, a polysaccharide), an endosomal component, a steroid (e.g., ursolic acid, agave sapogenin, diosgenin), a terpene (e.g., a triterpene, such as sarsasapogenin, adiposide, epidiposide-derived lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, bile acid, vitamin E, adamantane, or 1-pentafluoropropyl.

[0812] In certain embodiments, the linker has a structure selected from the group consisting of:

[0813]

[0814] wherein each n is independently 1 to 20; and p is 1 to 6.

[0815] In certain embodiments, the linker has a structure selected from the group consisting of:

[0816]

[0817] wherein each n is independently 1 to 20.

[0818] In certain embodiments, the linker has a structure selected from the group consisting of:

[0819]

[0820] Wherein n is 1 to 20.

[0821] In certain embodiments, the linker has a structure selected from the group consisting of:

[0822]

[0823] wherein each L is independently a phosphorus linking group or a neutral linking group; and

[0824] Each n is independently 1 to 20.

[0825] In certain embodiments, the linker has a structure selected from the group consisting of:

[0826]

[0827]

[0828] In certain embodiments, the linker has a structure selected from the group consisting of:

[0829]

[0830] In certain embodiments, the linker has a structure selected from the group consisting of:

[0831]

[0832] In certain embodiments, the linker has a structure selected from the group consisting of:

[0833]

[0834] Wherein n is 1 to 20.

[0835] In certain embodiments, the linker has a structure selected from the group consisting of:

[0836]

[0837] In certain embodiments, the linker has a structure selected from the group consisting of:

[0838]

[0839] In certain embodiments, the linker has a structure selected from the group consisting of:

[0840]

[0841] In certain embodiments, the conjugate linker has the following structure:

[0842]

[0843] In certain embodiments, the conjugate linker has the following structure:

[0844]

[0845] In certain embodiments, the linker has a structure selected from the group consisting of:

[0846]

[0847] In certain embodiments, the linker has a structure selected from the group consisting of:

[0848]

[0849] wherein each n is independently 0, 1, 2, 3, 4, 5, 6 or 7.

[0850] iv. Certain cell targeting moieties

[0851] In certain embodiments, the conjugate group comprises a cell targeting moiety. Certain of the cell targeting moieties increase 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.

[0852] 1. Certain branched groups

[0853] In certain embodiments, the conjugate group comprises a targeting moiety comprising a branched group and at least two tethering ligands. In certain embodiments, the branched group is connected to the conjugate linker. In certain embodiments, the branched group is connected to the cleavable moiety. In certain embodiments, the branched group is connected to the antisense oligonucleotide. In certain embodiments, the branched group is covalently attached to the linker and each tethering ligand. In certain embodiments, the branched group comprises a branched aliphatic group comprising a group selected from the group consisting of an alkyl, an amide, a disulfide, a polyethylene glycol, an ether, a thioether, and a hydroxylamino group. In certain embodiments, the branched group comprises a group selected from the group consisting of an alkyl, an amide, and an ether group. In certain embodiments, the branched group comprises a group selected from the group consisting of an alkyl and an ether group. In certain embodiments, the branched group comprises a monocyclic ring system or a polycyclic ring system. In certain embodiments, the branched group comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not include a branched group.

[0854] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0855]

[0856] wherein each n is independently 1 to 20;

[0857] j is 1 to 3; and

[0858] m is 2 to 6.

[0859] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0860]

[0861] wherein each n is independently 1 to 20; and

[0862] m is 2 to 6.

[0863] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0864]

[0865] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0866]

[0867] wherein each A1 is independently O, S, C=O or NH; and

[0868] Each n is independently 1 to 20.

[0869] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0870]

[0871] wherein each A1 is independently O, S, C=O or NH; and

[0872] Each n is independently 1 to 20.

[0873] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0874]

[0875] wherein A1 is O, S, C=O or NH; and

[0876] Each n is independently 1 to 20.

[0877] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0878]

[0879] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0880]

[0881] In certain embodiments, the branched group has a structure selected from the group consisting of:

[0882]

[0883] 2. Certain tethers

[0884] In certain embodiments, the conjugate group comprises one or more tethers covalently linked to a branched group. In certain embodiments, the conjugate group comprises one or more tethers covalently linked to a linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from the following: 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 the following: 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 the following: 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 the following: alkyl, substituted alkyl, phosphodiester, ether, and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from the following: alkyl, substituted alkyl, phosphodiester, ether, and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from the following: alkyl and phosphodiester in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or a neutral linking group.

[0885] In certain embodiments, the tether includes one or more cleavable bonds. In certain embodiments, the tether is connected to a branched group via an amide or ether group. In certain embodiments, the tether is connected to a branched group via a phosphodiester group. In certain embodiments, the tether is connected to a branched group via a phosphorus linking group or a neutral linking group. In certain embodiments, the tether is connected to a branched group via an ether group. In certain embodiments, the tether is connected to a part via an amide or ether group. In certain embodiments, the tether is connected to a part via an ether group. In certain embodiments, the tether is connected to a part via an amide or ether group. In certain embodiments, the tether is connected to a part via an ether group.

[0886] In certain embodiments, each tether comprises a chain length of about 8 to about 20 atoms between the ligand and the side-chain group. In certain embodiments, each tether comprises a chain length of about 10 to about 18 atoms between the ligand and the side-chain group. In certain embodiments, each tether comprises a chain length of about 13 atoms.

[0887] In certain embodiments, the tether has a structure selected from the group consisting of:

[0888]

[0889] wherein each n is independently 1 to 20; and

[0890] Each p is from 1 to about 6.

[0891] In certain embodiments, the tether has a structure selected from the group consisting of:

[0892]

[0893] In certain embodiments, the tether has a structure selected from the group consisting of:

[0894]

[0895] wherein each n is independently 1 to 20.

[0896] In certain embodiments, the tether has a structure selected from the group consisting of:

[0897]

[0898] wherein L is a phosphorus linking group or a neutral linking group;

[0899] Z1 is C(=O)O-R2;

[0900] Z2 is H, C1-C6 alkyl or substituted C1-C6 alkyl;

[0901] R2 is H, C1-C6 alkyl or substituted C1-C6 alkyl; and

[0902] Each ml is independently from 0 to 20, wherein for each tether, at least one ml is greater than 0.

[0903] In certain embodiments, the tether has a structure selected from the group consisting of:

[0904]

[0905] In certain embodiments, the tether has a structure selected from the group consisting of:

[0906]

[0907] wherein Z2 is H or CH3; and

[0908] Each ml is independently from 0 to 20, wherein for each tether, at least one ml is greater than 0.

[0909] In certain embodiments, the tether has a structure selected from the group consisting of:

[0910] wherein each n is independently 0, 1, 2, 3, 4, 5, 6 or 7.

[0911] In certain embodiments, the tether comprises a phosphorus linking group. In certain embodiments, the tether does not comprise any amide bonds. In certain embodiments, the tether comprises a phosphorus linking group and does not comprise any amide bonds.

[0912] 3. Certain ligands

[0913] In certain embodiments, the present disclosure provides ligands, wherein each ligand is covalently linked to a tether. In certain embodiments, each ligand is selected to have affinity for at least one type of receptor on a target cell. In certain embodiments, a ligand is selected to have affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, a ligand is selected to have affinity for the liver asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is independently selected from galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose. In certain embodiments, each ligand is N-acetylgalactosamine (GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In certain embodiments, the targeting moiety comprises 3 N-acetylgalactosamine ligands.

[0914] In certain embodiments, the ligand is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a multivalent carbohydrate cluster, a polysaccharide, a modified polysaccharide or a polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thiosugar. For example, 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-acetylamino-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-formylamino-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine and N-glycolyl-α-neuraminic acid. For example, the thiosaccharide can be selected from the group consisting of 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, and 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester.

[0915] In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. In certain embodiments, "N-acetylgalactosamine" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, including the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-D-galactopyranose are used interchangeably. Thus, in structures where one form is depicted, these structures are also intended to include the other form. For example, when a structure is shown for the α form: 2-(acetylamino)-2-deoxy-D-galactopyranose, the structure is also intended to include the other form. In certain embodiments, in certain preferred embodiments, the β form of 2-(acetylamino)-2-deoxy-D-galactopyranose is a preferred embodiment.

[0916]

[0917]

[0918] In certain embodiments, one or more ligands have a structure selected from the group consisting of:

[0919]

[0920] wherein each R1 is selected from OH and NHCOOH.

[0921] In certain embodiments, one or more ligands have a structure selected from the group consisting of:

[0922]

[0923] In certain embodiments, one or more ligands have a structure selected from the group consisting of:

[0924]

[0925] In certain embodiments, one or more ligands have a structure selected from the group consisting of:

[0926]

[0927] i. Certain conjugates

[0928] In certain embodiments, the conjugate group comprises the above structural features. In certain of these embodiments, the conjugate group has the following structure:

[0929]

[0930] wherein each n is independently 1 to 20.

[0931] In certain such embodiments, the conjugate group has the structure:

[0932]

[0933] In certain such embodiments, the conjugate group has the structure:

[0934]

[0935] wherein each n is independently 1 to 20;

[0936] Z is H or a linked solid support;

[0937] Q is an antisense compound;

[0938] X is O or S; and

[0939] Bx is a heterocyclic base moiety.

[0940] In certain such embodiments, the conjugate group has the structure:

[0941]

[0942] In certain such embodiments, the conjugate group has the structure:

[0943]

[0944] In certain such embodiments, the conjugate group has the structure:

[0945]

[0946] In certain such embodiments, the conjugate group has the structure:

[0947]

[0948] In certain such embodiments, the conjugate group has the structure:

[0949]

[0950] In certain such embodiments, the conjugate group has the structure:

[0951]

[0952] In certain such embodiments, the conjugate group has the structure:

[0953]

[0954] In certain such embodiments, the conjugate group has the structure:

[0955]

[0956] In certain embodiments, the conjugate does not comprise pyrrolidine.

[0957] In certain such embodiments, the conjugate group has the structure:

[0958]

[0959] In certain such embodiments, the conjugate group has the structure:

[0960]

[0961] In certain such embodiments, the conjugate group has the structure:

[0962]

[0963] In certain such embodiments, the conjugate group has the structure:

[0964]

[0965] In certain such embodiments, the conjugate group has the structure:

[0966]

[0967] In certain such embodiments, the conjugate group has the structure:

[0968]

[0969] In certain such embodiments, the conjugate group has the structure:

[0970]

[0971] In certain such embodiments, the conjugate group has the structure:

[0972]

[0973] In certain such embodiments, the conjugate group has the structure:

[0974]

[0975] In certain such embodiments, the conjugate group has the structure:

[0976]

[0977] In certain such embodiments, the conjugate group has the structure:

[0978]

[0979] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[0980]

[0981] wherein X is a substituted or unsubstituted tether having six to eleven consecutively bonded atoms.

[0982] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[0983]

[0984] wherein X is a substituted or unsubstituted tether having ten consecutively bonded atoms.

[0985] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[0986]

[0987] wherein X is a substituted or unsubstituted tether having four to eleven consecutively bonded atoms and wherein the tether comprises exactly one amide bond.

[0988] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[0989]

[0990] wherein Y and Z are independently selected from C1-C 12 Substituted or unsubstituted alkyl, alkenyl or alkynyl groups, or groups containing ethers, ketones, amides, esters, carbamates, amines, piperidines, phosphates, phosphodiesters, phosphorothioates, triazoles, pyrrolidines, disulfides or thioethers.

[0991] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[0992]

[0993] wherein Y and Z are independently selected from C1-C 12 Substituted or unsubstituted alkyl, or a group containing exactly one ether or exactly two ethers, amides, amines, piperidines, phosphates, sulfate diesters or thiophosphates.

[0994] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[0995]

[0996] wherein Y and Z are independently selected from C1-C 12 a substituted or unsubstituted alkyl group.

[0997] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[0998]

[0999] wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

[1000] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[1001]

[1002] wherein m is 4, 5, 6, 7 or 8, and n is 1, 2, 3 or 4.

[1003] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[1004]

[1005] wherein X is a substituted or unsubstituted tether having four to thirteen consecutively bonded atoms, and wherein X does not comprise an ether group.

[1006] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[1007]

[1008] wherein X is a substituted or unsubstituted tether having eight consecutively bonded atoms, and wherein X does not comprise an ether group.

[1009] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[1010]

[1011] wherein X is a substituted or unsubstituted tether having four to thirteen consecutively bonded atoms, and wherein the tether comprises exactly one amide bond, and wherein X does not comprise an ether group.

[1012] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[1013]

[1014] wherein X is a substituted or unsubstituted tether having four to thirteen consecutively bonded atoms and wherein the tether consists of an amide bond and a substituted or unsubstituted C2-C 11 Alkyl composition.

[1015] In certain embodiments, the cell targeting portion of the conjugate group has the following structure:

[1016]

[1017] Wherein Y is selected from C1-C 12 Substituted or unsubstituted alkyl, alkenyl or alkynyl groups, or groups containing ethers, ketones, amides, esters, carbamates, amines, piperidines, phosphates, phosphodiesters, phosphorothioates, triazoles, pyrrolidines, disulfides or thioethers.

[1018] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[1019]

[1020] Wherein Y is selected from C1-C 12 Substituted or unsubstituted alkyl groups, or groups containing ethers, amines, piperidines, phosphates, phosphodiesters, or phosphorothioates.

[1021] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[1022]

[1023] Wherein Y is selected from C1-C 12 a substituted or unsubstituted alkyl group.

[1024] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[1025]

[1026] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[1027] In certain such embodiments, the cell targeting portion of the conjugate group has the following structure:

[1028]

[1029] Where n is 4, 5, 6, 7 or 8.

[1030] b. Certain conjugated antisense compounds

[1031] In certain embodiments, the conjugate is bound 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:

[1032]

[1033] in

[1034] A is an antisense oligonucleotide;

[1035] B is the cleavable part

[1036] C is the conjugate linker

[1037] D is a branched group

[1038] Each E is a tether;

[1039] Each F is a ligand; and

[1040] q is an integer between 1 and 5.

[1041] In certain embodiments, the conjugated antisense compound has the following structure:

[1042]

[1043] in

[1044] A is an antisense oligonucleotide;

[1045] C is the conjugate linker

[1046] D is a branched group

[1047] Each E is a tether;

[1048] Each F is a ligand; and

[1049] q is an integer between 1 and 5.

[1050] In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

[1051] In certain such embodiments, the branched group comprises at least one cleavable bond.

[1052] In certain embodiments, each tether comprises at least one cleavable bond.

[1053] In certain embodiments, the conjugate is bound to the nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside.

[1054] In certain embodiments, the conjugated antisense compound has the following structure:

[1055]

[1056] in

[1057] A is an antisense oligonucleotide;

[1058] B is the cleavable part

[1059] C is the conjugate linker

[1060] Each E is a tether;

[1061] Each F is a ligand; and

[1062] q is an integer between 1 and 5.

[1063] In certain embodiments, the conjugate is bound 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:

[1064]

[1065] in

[1066] A is an antisense oligonucleotide;

[1067] C is the conjugate linker

[1068] Each E is a tether;

[1069] Each F is a ligand; and

[1070] q is an integer between 1 and 5.

[1071] In certain embodiments, the conjugated antisense compound has the following structure:

[1072]

[1073] in

[1074] A is an antisense oligonucleotide;

[1075] B is the cleavable part

[1076] D is a branched group

[1077] Each E is a tether;

[1078] Each F is a ligand; and

[1079] q is an integer between 1 and 5.

[1080] In certain embodiments, the conjugated antisense compound has the following structure:

[1081]

[1082] in

[1083] A is an antisense oligonucleotide;

[1084] D is a branched group

[1085] Each E is a tether;

[1086] Each F is a ligand; and

[1087] q is an integer between 1 and 5.

[1088] In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

[1089] In certain embodiments, each tether comprises at least one cleavable bond.

[1090] In certain embodiments, the conjugated antisense compound has a structure selected from the group consisting of:

[1091] In certain embodiments, the conjugated antisense compound has a structure selected from the group consisting of:

[1092]

[1093] In certain embodiments, the conjugated antisense compound has a structure selected from the group consisting of:

[1094]

[1095] In certain embodiments, the conjugated antisense compound has the following structure:

[1096]

[1097] Representative U.S. patents, U.S. patent application publications, and international patent application publications that teach the preparation of certain of the above-described conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties, and other modifications include, but are not limited to, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, 2006 / 0148740, 2011 / 0123520, WO 2013 / 033230, and WO 2012 / 037254, each of which is incorporated herein by reference in its entirety.

[1098] Representative publications that teach certain of the above-described conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties, and other modifications include, but are not limited to, BIES SEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent" J. Med. Chem. (1995) 38:1846-1852; BIESSEN et al., "Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor" J. Med. Chem. (1995) 38:1538-1546; LEE et al., "New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes" s"Bioorganic&MedicinalChemistry(2011)19:2494-2500; RENSE N et al.,"Determination of the Upper Size Limit for Uptake and Proc essing of Ligands by the Asialoglycoprotein Receptor onHepatocyte s in Vitro and in Vivo" J. Biol. Chem. (2001) 276(40): 37577-37584; RENSEN et al., "Design and Synthesis of Novel N-Acetylgalactosamine-TerminatedGlycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein 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., "Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor" Tetrahedron, 1997, 53 (2), 759-770, each of which is incorporated herein by reference in its entirety.

[1099] In certain embodiments, conjugated antisense compounds comprise an RNase H-based oligonucleotide (eg, a gapmer) or a splice-modifying oligonucleotide (eg, a fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain embodiments, the conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bio org Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med 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;Sliedregt et al., J Med 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., Gene 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., BioconjugChem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Dru Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998 / 013381; WO2011 / 038356; WO1997 / 046098; WO2008 / 098788; WO2004 / 101619; WO2012 / 037254; WO2011 / 120053; WO2011 / 100131; WO2011 / 163121; WO2012 / 177947; WO2013 / 033230; WO2013 / 075035; WO2012 / 083185; WO2012 / 083046; WO2009 / 082607; WO2009 / 134487; WO2010 / 144740; WO2010 / 148013; WO1997 / 020563; WO2010 / 088537; WO2002 / 043771; WO2010 / 129709; WO2012 / 068187; WO2009 / 126933; WO2004 / 024757; WO2010 / 054406; WO2012 / 089352; WO2012 / 089602; WO2013 / 166121; WO2013 / 165816;U.S. Patents 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; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Applications Nos. US2011 / 0097264; US2011 / 0097265; US2013 / 0004427; US2005 / 0164235; US2006 / 0148740; US2008 / 0281044; US 2010 / 0240730; US2003 / 0119724; US2006 / 0183886; US2008 / 0206869; US2011 / 0269814; US2009 / 0286973; US2011 / 0207 799; US2012 / 0136042; US2012 / 0165393; US2008 / 0281041; US2009 / 0203135; US2012 / 0035115; US2012 / 0095075; US201 2 / 0101148; US2012 / 0128760; US2012 / 0157509; US2012 / 0230938; US2013 / 0109817; US2013 / 0121954; US2013 / 0178512; US2013 / 0236968; US2011 / 0123520; US2003 / 0077829; US2008 / 0108801 and US2009 / 0203132; each of which is incorporated by reference in its entirety.

[1100] C. Certain uses and features

[1101] In certain embodiments, the conjugated antisense compounds show effective reduction of target RNA in vivo. In certain embodiments, unconjugated antisense compounds accumulate in the kidney. In certain embodiments, conjugated antisense compounds accumulate in the liver. In certain embodiments, the conjugated antisense compounds are well tolerated. Such properties make conjugated antisense compounds particularly useful for inhibiting many target RNAs, including but not limited to those involving metabolic, cardiovascular and other diseases, disorders or conditions. Therefore, provided herein are methods for treating the disease, disorder or condition by contacting liver tissue with a conjugated antisense compound targeting an RNA associated with the disease, disorder or condition. Therefore, also provided are methods for using the conjugated antisense compounds of the present invention to improve any of various metabolic, cardiovascular and other diseases, disorders or conditions.

[1102] In certain embodiments, a conjugated antisense compound is more effective than its unconjugated counterpart at a particular tissue concentration. Without wishing to be bound by any theory or mechanism, in certain embodiments, the conjugate may allow the conjugated antisense compound to enter cells more efficiently or more effectively. For example, in certain embodiments, a conjugated antisense compound may exhibit greater target reduction than its unconjugated counterpart, where the conjugated antisense compound and its unconjugated counterpart are both present at the same concentration in a tissue. For example, in certain embodiments, a conjugated antisense compound may exhibit greater target reduction than its unconjugated counterpart, where the conjugated antisense compound and its unconjugated counterpart are both present at the same concentration in the liver.

[1103] The productive and non-productive uptake of oligonucleotides has been discussed previously (see, for example, Geary, RS, E. Wancewicz et al. (2009). "Effect of Dose and Plasma Concentration on Liver Uptake and Pharmacologic Activity of a2'-Methoxyethyl Modified Chimeric Antisense Oligonucleotide Targeting PTEN." Biochem. Pharmacol. 78(3):284-91; & Koller, E., T. M. Vincent et al. (2011). "Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes." Nucleic Acids Res. 39(11):4795-807). The conjugate groups described herein can improve productive uptake.

[1104] In certain embodiments, the conjugate groups described herein can further improve efficacy by increasing the affinity of the conjugated antisense compound for a particular type of cell or tissue. In certain embodiments, the conjugate groups described herein can further improve efficacy by increasing recognition of the conjugated antisense compound by one or more cell surface receptors. In certain embodiments, the conjugate groups described herein can further improve efficacy by promoting endocytosis of the conjugated antisense compound.

[1105] In certain embodiments, the cleavable moiety can further improve efficacy by allowing the conjugate to be cleaved from the antisense oligonucleotide after the conjugated antisense compound enters the cell. Thus, in certain embodiments, the conjugated antisense compound can be administered at a lower dose than would be necessary for an unconjugated antisense oligonucleotide.

[1106] Previously, phosphorothioate linkage has been incorporated into antisense oligonucleotide. The phosphorothioate linkage has resistance to nuclease and therefore improves the stability of oligonucleotide. In addition, phosphorothioate linkage is also in conjunction with certain proteins, which causes the accumulation of antisense oligonucleotide in the liver. Oligonucleotide with less phosphorothioate linkage accumulates less in the liver and accumulates more in the kidney (see, for example, Geary, R., " Pharmacokinetic Properties of 2'-O-(2-Methoxyethyl)-Modified Oligonucleotide Analogs in Rats," Journal of Pharmacology and Experimental Therapeutics, Vol. 296, No. 3, 890-897; & Pharmacological Properties of 2'-O-Methoxyethyl Modified Oligonucleotides in Antisense a Drug Technology, Chapter 10, Crooke, ST ed., 2008). In certain embodiments, oligonucleotides with fewer phosphorothioate internucleoside linkages and more phosphodiester internucleoside linkages accumulate less in the liver and more in the kidneys. This is undesirable when treating diseases in the liver for several reasons: (1) less drug reaches the desired site of action (the liver); (2) the drug escapes into the urine; and (3) the kidneys are exposed to relatively high concentrations of the drug, which can lead to nephrotoxicity. Therefore, for liver diseases, phosphorothioate linkages provide important benefits.

[1107] However, in certain embodiments, administration of oligonucleotides uniformly linked by phosphorothioate internucleoside linkages induces one or more proinflammatory responses. (See, e.g., J Lab Clin Med. 1996 Sep; 128(3): 329-38. "Amplification of antibody production by phosphorothioate oligodeoxynucleotides". Branda et al.; and also see, e.g., Toxicologic Properties in Antisense a Drug Technology, Chapter 12, pp. 342-351, Crooke, ST ed., 2008). In certain embodiments, administration of oligonucleotides in which the majority of internucleoside linkages comprise phosphorothioate internucleoside linkages induces one or more proinflammatory responses.

[1108] In certain embodiments, the extent of the proinflammatory effect may depend on several variables (e.g., backbone modifications, off-target effects, nucleobase modifications, and / or nucleoside modifications), see, for example, Toxicologic Properties in Antisense a Drug Technology, Chapter 12, pages 342-351, Crooke, ST, ed., 2008). In certain embodiments, the extent of the proinflammatory effect may be mitigated by adjusting one or more variables. For example, the extent of the proinflammatory effect of a given oligonucleotide may be mitigated by replacing any number of phosphorothioate nucleoside linkages with phosphodiester nucleoside linkages and thereby reducing the total number of phosphorothioate nucleoside linkages.

[1109] In certain embodiments, it is desirable to reduce the number of phosphorothioate linkages, if it is possible to achieve this without losing stability and without distributing the linkages from the liver to the kidney. For example, in certain embodiments, the number of phosphorothioate linkages can be reduced by replacing phosphorothioate linkages with phosphodiester linkages. In such an embodiment, the antisense compounds with fewer phosphorothioate linkages and more phosphodiester linkages can induce fewer proinflammatory reactions or do not induce proinflammatory reactions. Although the antisense compounds with fewer phosphorothioate linkages and more phosphodiester linkages can induce fewer proinflammatory reactions, the antisense compounds with fewer phosphorothioate linkages and more phosphodiester linkages can not accumulate in the liver and can be less effective than the antisense compounds with more phosphorothioate linkages at the same or similar dosage. In certain embodiments, it is therefore desirable to design an antisense compound with multiple phosphodiester linkages and multiple phosphorothioate linkages but also with stability and good distribution to the liver.

[1110] In certain embodiments, conjugated antisense compounds accumulate more in the liver and less in the kidney than unconjugated counterparts, even when some thiophosphate linkages are replaced by less proinflammatory phosphodiester nucleoside linkages. In certain embodiments, conjugated antisense compounds accumulate more in the liver and are not excreted into the urine as much as their unconjugated counterparts, even when some thiophosphate linkages are replaced by less proinflammatory phosphodiester nucleoside linkages. In certain embodiments, the use of conjugates allows people to design more effective and better tolerated antisense drugs. In fact, in certain embodiments, conjugated antisense compounds have a therapeutic index greater than unconjugated counterparts. This allows conjugated antisense compounds to be administered at higher absolute doses because there is a smaller risk of proinflammatory response and a smaller risk of nephrotoxicity. This higher dose allows people to administer less frequently because it is expected that clearance (metabolism) is similar. In addition, because the compound is more effective, as described above, it is allowed that the concentration becomes lower before the next dose without losing therapeutic activity, thereby allowing even longer time periods between administrations.

[1111] In certain embodiments, it is still desirable to include some thiophosphate linkages. For example, terminal linkages are susceptible to destruction by exonucleases, so in certain embodiments, those linkages are thiophosphates or other modified linkages. The internucleoside linkages connecting two deoxynucleosides are susceptible to destruction by endonucleases, so in certain embodiments, those linkages are thiophosphates or other modified linkages. The internucleoside linkages between a modified nucleoside and a deoxynucleoside (wherein the deoxynucleoside is on the 5' side of the linkage deoxynucleoside) are susceptible to destruction by endonucleases, so in certain embodiments, those linkages are thiophosphates or other modified linkages. The internucleoside linkages between two modified nucleosides of certain types and between a deoxynucleoside and a certain type of modified nucleoside (wherein the modified nucleoside is on the 5' side of the linkage) have sufficient resistance to nuclease digestion, and the linkages can be phosphodiester.

[1112] In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 16 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 15 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 14 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 13 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 12 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 11 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 10 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 9 phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide conjugated to an antisense compound comprises less than 8 phosphorothioate linkages.

[1113] In certain embodiments, the antisense compounds comprising one or more conjugate groups described herein have increased activity and / or efficacy and / or tolerance compared to the parent antisense compounds lacking the one or more conjugate groups. Therefore, in certain embodiments, it is desirable for the conjugate group to be connected to an oligonucleotide. The conjugate group may be connected to the 5'-end and / or 3'-end of an oligonucleotide. In some cases, the connection of the 5'-end is desirable in synthesis. Typically, oligonucleotides are synthesized by coupling nucleosides sequentially from 3' to 5' using technology well known in the art. Therefore, if it is desired that the conjugate group be located at the 3'-end, then (1) the conjugate group may be connected to the 3'-end nucleoside and the conjugated nucleoside may be connected to the solid support for subsequent preparation of oligonucleotides or (2) the conjugate group may be connected to the 3'-end nucleoside of a complete oligonucleotide after synthesis. None of these methods are very effective and therefore are expensive. Specifically, although shown in the examples herein, the connection of conjugated nucleosides to a solid support is an inefficient process. In certain embodiments, the conjugate group is connected to the 5'-terminal nucleoside than being connected at the 3'-end in synthesis more easily. People can be connected to the 3'-terminal nucleoside of non-conjugated solid support and use standard and well-characterized reaction to prepare oligonucleotide. Then people only need to connect the 5' nucleoside with the conjugate group in the last coupling step. In certain embodiments, this is more effective than directly connecting the conjugated nucleoside to the solid support (such as usually carried out to prepare the 3'-conjugated oligonucleotide). The embodiments herein show the connection at the 5'-end. In addition, some conjugate groups have synthetic advantages. For example, some conjugate groups comprising a phosphorus linking group are simpler in synthesis and prepare more effectively (for example, WO / 2012 / 037254) than other conjugate groups comprising previously reported conjugate groups.

[1114] In certain embodiments, conjugated antisense compounds are administered to a subject. In the embodiments, the antisense compounds comprising one or more conjugate groups described herein have increased activity and / or efficacy and / or tolerance compared to the parent antisense compounds lacking the one or more conjugate groups. Unbound by mechanism, it is believed that the conjugate groups contribute to distribution, delivery and / or uptake into target cells or tissues. In certain embodiments, once inside the target cell or tissue, it is desired that all or part of the conjugate groups be cracked to release active oligonucleotides. In certain embodiments, it is unnecessary for the entire conjugate group to be cracked from the oligonucleotide. For example, in Example 20, conjugated oligonucleotides were administered to mice and many different chemicals were detected, each of which contained different parts of the conjugate groups remaining on the oligonucleotides (Table 23a). This conjugated antisense compound exhibits good efficacy (Table 23). Therefore, in certain embodiments, the metabolic characteristics of the multiple partial crackings of the conjugate groups do not interfere with activity / efficacy. Nevertheless, in certain embodiments, it is desired that prodrugs (conjugated oligonucleotides) produce single active compounds. In some cases, if multiple forms of active compounds are found, it is necessary to determine the relative amount and activity of each. In certain embodiments where regulatory review (e.g., USFDA or corresponding agencies) is required, it is desirable to have a single (or primarily single) active substance. In certain embodiments, it is desirable that the single active substance be an antisense oligonucleotide lacking any portion of a conjugate group. In certain embodiments, the conjugate group at the 5'-end is more likely to result in complete metabolism of the conjugate group. Independent of the mechanism, the endogenous enzyme responsible for the metabolism at the 5' end (e.g., 5' nucleoside) may be more active / more effective than the 3' counterpart. In certain embodiments, a specific conjugate group is more suitable for the metabolism of a single active substance. In certain embodiments, some conjugate groups are more suitable for the metabolism of oligonucleotides.

[1115] D.Antonym

[1116] In certain embodiments, the oligomeric compound of the present invention is an antisense compound. In such embodiments, the oligomeric compound is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is RNA. In certain embodiments, the target nucleic acid is non-coding RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain embodiments, the target nucleic acid is selected from mRNA, pre-mRNA, microRNA, non-coding RNA (including small non-coding RNA) and promoter-directed RNA. In certain embodiments, the oligomeric compound is at least partially complementary to more than one target nucleic acid. For example, the oligomeric compound of the present invention can be a microRNA mimic, which typically binds to multiple targets.

[1117] In certain embodiments, the antisense compound comprises a portion having a core base sequence that is at least 70% complementary to the core base sequence of the target nucleic acid. In certain embodiments, the antisense compound comprises a portion having a core base sequence that is at least 80% complementary to the core base sequence of the target nucleic acid. In certain embodiments, the antisense compound comprises a portion having a core base sequence that is at least 90% complementary to the core base sequence of the target nucleic acid. In certain embodiments, the antisense compound comprises a portion having a core base sequence that is at least 95% complementary to the core base sequence of the target nucleic acid. In certain embodiments, the antisense compound comprises a portion having a core base sequence that is at least 98% complementary to the core base sequence of the target nucleic acid. In certain embodiments, the antisense compound comprises a portion having a core base sequence that is 100% complementary to the core base 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 core base sequence of the target nucleic acid over the entire length of the antisense compound.

[1118] Antisense mechanisms include any mechanism involving hybridization of an oligomeric compound to a target nucleic acid, wherein the hybridization produces a biological effect. In certain embodiments, the hybridization results in degradation or occupancy of the target nucleic acid accompanied by inhibition or stimulation of cellular machinery, such as translation, transcription, or polyadenylation of nucleic acids with which the target nucleic acid or target nucleotides may otherwise interact.

[1119] One type of antisense mechanism involving degradation of the target RNA is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Single-stranded antisense compounds known in the art as "DNA-like" trigger RNase H activity in mammalian cells. Consequently, activation of RNase H leads to cleavage of the RNA target, greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

[1120] Antisense mechanisms also include but are not limited to RNAi mechanisms utilizing RISC pathways. Said RNAi mechanisms include but are not limited to siRNA, ssRNA and microRNA mechanisms. Said mechanisms include the generation of microRNA mimics and / or anti-microRNAs.

[1121] Antisense mechanisms also include, but are not limited to, mechanisms that hybridize or mimic non-coding RNAs rather than microRNAs or mRNAs. The non-coding RNAs include, but are not limited to, promoter-guided RNAs and short and long RNAs that affect the transcription or translation of one or more nucleic acids.

[1122] In certain embodiments, the oligonucleotide comprising the conjugate described herein is an RNAi compound. In certain embodiments, the oligonucleotide comprising the conjugate described herein is an ssRNA compound. In certain embodiments, the oligonucleotide comprising the conjugate described herein is paired with a second oligomeric compound to form siRNA. In certain embodiments, the second oligomeric compound also comprises a conjugate. In certain embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In certain embodiments, the oligonucleotide comprising the conjugate described herein is the antisense strand in the siRNA compound. In certain embodiments, the oligonucleotide comprising 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 located on the sense strand, the antisense strand, or both the sense strand and the antisense strand.

[1123] D. Apolipoprotein C-III (apoCIII)

[1124] In certain embodiments, the conjugated antisense compounds target any In certain embodiments, the target nucleic acid encodes a clinically relevant Target Protein. In such embodiments, modulation of the target nucleic acid results in a clinical benefit.

[1125] The targeting process generally involves identifying at least one target region, segment, or site within the target nucleic acid for antisense interaction to occur such that the desired effect will occur.

[1126] 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 a 3'UTR, a 5'UTR, an exon, an intron, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid region or target segment.

[1127] In certain embodiments, the target segment is at least about 8 core base portions of the target region targeted by the conjugated antisense compound. The target segment may include a DNA or RNA sequence comprising at least 8 continuous core bases from the 5'-end of one of the target segments (the remaining core bases are immediately adjacent to the 5'-end upstream of the target segment and continue until the DNA or RNA comprises a continuous segment of the same DNA or RNA from about 8 to about 30 core bases). The target segment is also represented by a DNA or RNA sequence comprising at least 8 continuous core bases from the 3'-end of one of the target segments (the remaining core bases are immediately adjacent to the 3'-end downstream of the target segment and continue until the DNA or RNA comprises a continuous segment of the same DNA or RNA from about 8 to about 30 core bases). The target segment may also be represented by a DNA or RNA sequence comprising at least 8 continuous core bases from the interior of the sequence of the target segment, and may extend in either or both directions until the conjugated antisense compound comprises about 8 to about 30 core bases.

[1128] In certain embodiments, targeting The antisense compounds of nucleic acids can be modified as described herein. In certain embodiments, the antisense compounds can 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 compounds can have a modified internucleoside linkage, an unmodified internucleoside linkage, or a mixture of modified and unmodified internucleoside linkages as described herein. In certain embodiments, the antisense compounds can have a modified core base, an unmodified core base, or a mixture of modified and unmodified core bases as described herein. In certain embodiments, the antisense compounds can have a motif as described herein.

[1129] In certain embodiments, targeting Antisense compounds to nucleic acids can be conjugated as described herein.

[1130] ApoCIII is a component of HDL and triglyceride (TG)-rich lipoproteins. Elevated ApoCIII levels are associated with elevated TG levels and diseases such as cardiovascular disease, metabolic syndrome, obesity, and diabetes. Elevated TG levels are associated with pancreatitis. ApoCIII has been shown to clear TG-rich lipoproteins by inhibiting lipoprotein lipase (LPL) and by interfering with lipoproteins that bind to the cell surface glycosaminoglycan matrix, inhibiting lipolysis. Antisense compounds targeting ApoCIII have previously been disclosed in WO2004 / 093783 and WO2012 / 149495, each of which is incorporated herein by reference in its entirety.

[1131] Certain conjugated antisense compounds targeting ApoCIII nucleic acids

[1132] In certain embodiments, the conjugated antisense compound targets an ApoCIII nucleic acid having a sequence of any one of the following: Accession No. NM_000040.1 (incorporated herein as SEQ ID NO: 1); GENBANK Accession No. NT_033899.8 truncated from nucleobases 20262640 to 20266603 (incorporated herein as SEQ ID NO: 2); and GENBANK Accession No. NT_035088.1 truncated from nucleotides 6238608 to 6242565 (incorporated herein as SEQ ID NO: 3). In certain such embodiments, the conjugated antisense compound is at least 90%, at least 95%, or 100% complementary to any one of SEQ ID NOs: 1-3.

[1133] In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises at least 8 contiguous nucleobases of SEQ ID NO: 87. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises the nucleobase sequence of SEQ ID NO: 87.

[1134] Table A: Antisense compounds targeting ApoCIII SEQ ID NO: 1

[1135]

[1136] ApoCIII therapeutic indications

[1137] In certain embodiments, the present invention provides methods for modulating the expression of ApoCIII in a subject using conjugated antisense compounds targeted to ApoCIII nucleic acids.In certain embodiments, the expression of ApoCIII is reduced.

[1138] In certain embodiments, the present invention provides methods for treating subjects using conjugated antisense compounds targeting ApoCIII nucleic acids in pharmaceutical compositions. In certain embodiments, the subject has cardiovascular and / or metabolic diseases, disorders, or conditions. In certain embodiments, the subject has hypertriglyceridemia, non-familial hypertriglyceridemia, familial hypertriglyceridemia, heterozygous familial hypertriglyceridemia, homozygous familial hypertriglyceridemia, mixed dyslipidemia, atherosclerosis, the risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hyperlipidemia, hypercholesterolemia, hyperfatty acidemia, hepatic steatosis, non-alcoholic steatohepatitis, pancreatitis, and / or non-alcoholic fatty liver disease.

[1139] In certain embodiments, the present invention provides methods for preparing a pharmaceutical product using conjugated antisense compounds targeted to ApoCIII nucleic acids.

[1140] E. Certain Nucleic Acid GalNAc Conjugates

[1141] In certain embodiments, the conjugated antisense compound comprises a core base sequence of an antisense compound in the following table and a modified antisense compound connected to a GalNAc conjugate. In certain embodiments, the conjugated antisense compound comprises a core base sequence of an antisense compound in the following table and a chemically modified antisense compound connected to a GalNAc conjugate. Unless otherwise indicated, all internucleoside linkages are phosphorothioate internucleoside linkages. The subscript "l" indicates an LNA bicyclic nucleoside. The subscript "d" indicates a 2'-deoxynucleoside. The subscript "e" indicates a 2'-MOE modified nucleoside. The subscript "v" indicates 2-amino-2'-deoxyadenosine.

[1142] Table B

[1143]

[1144]

[1145] F. Certain pharmaceutical compositions

[1146] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising one or more antisense compounds. In certain embodiments, the 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, the pharmaceutical composition consists of a sterile saline solution and one or more antisense compounds. In certain embodiments, 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, 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, sterile saline is pharmaceutical grade PBS.

[1147] In certain embodiments, antisense compounds can be mixed with pharmaceutically acceptable active substances and / or inert substances for the preparation of pharmaceutical compositions or formulations. The composition and the method for preparing the pharmaceutical composition depend on many criteria, including but not limited to the route of administration, the extent of the disease or the dose to be administered.

[1148] The pharmaceutical composition comprising antisense compounds encompasses any pharmaceutically acceptable salt, ester or salt of the ester. In certain embodiments, the pharmaceutical composition comprising antisense compounds comprises one or more oligonucleotides, which, when administered to an animal (including a human), can (directly or indirectly) provide a bioactive metabolite or its residue. Therefore, for example, the disclosure further relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of the prodrugs, and other bioequivalents of antisense compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[1149] Prodrugs may include the incorporation of additional nucleosides at one or both termini of the oligonucleotide, which are cleaved by endogenous nucleases in vivo to form the active antisense oligonucleotide.

[1150] Lipid moieties have been used in nucleic acid therapies in various methods. In some of the methods, the nucleic acid is introduced into a preformed liposome or lipid complex made from a mixture of a cationic lipid and a neutral lipid. In some methods, a DNA complex with a monocationic lipid or a polycationic lipid is formed in the absence of a neutral lipid. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to a specific cell or tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to muscle tissue.

[1151] In certain embodiments, provided herein are pharmaceutical compositions comprising one or more modified oligonucleotides and one or more excipients. In certain of the embodiments, the excipient is selected from water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose and polyvinyl pyrrolidone.

[1152] In certain embodiments, the pharmaceutical compositions provided herein comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful in preparing certain pharmaceutical compositions, including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethyl sulfoxide are used.

[1153] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more tissue-specific delivery molecules designed to deliver one or more agents of the present disclosure to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical compositions comprise liposomes coated with tissue-specific antibodies.

[1154] In certain embodiments, the pharmaceutical compositions provided herein comprise a cosolvent system. Certain of the cosolvent systems comprise, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, the cosolvent system is used for hydrophobic compounds. A non-limiting example of a cosolvent system is a VPD cosolvent system comprising 3% w / v benzyl alcohol, 8% w / v non-polar surfactant polysorbate 80, and 1% w / v PEG-100. TM and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of the cosolvent system can be varied significantly without significantly changing its solubility and toxicity characteristics. In addition, the identity of the cosolvent components can be varied: for example, other surfactants can be used instead of polysorbate 80. TM The fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, such as polyvinyl pyrrolidone; and other sugars or polysaccharides can replace dextrose.

[1155] In certain embodiments, the pharmaceutical compositions provided herein are prepared for oral administration. In certain embodiments, the pharmaceutical compositions are prepared for buccal administration.

[1156] In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer such as Hanks solution, Ringer's solution or physiological saline buffer. In certain embodiments, other ingredients (e.g., ingredients that help dissolve or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. Some pharmaceutical compositions for injection are presented in unit dosage form, such as in ampoules or in multi-dose containers. Some pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles and may contain preparatons, such as suspending agents, stabilizers and / or dispersants. Some solvents suitable for pharmaceutical compositions for injection include but are not limited to lipophilic solvents and fatty oils (e.g., sesame oil), synthetic fatty acid esters (e.g., ethyl oleate or triglycerides) and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the agent to allow for the preparation of highly concentrated solutions.

[1157] In certain embodiments, the pharmaceutical compositions are prepared for transmucosal administration. In certain such embodiments, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[1158] In certain embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of oligonucleotides. 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 a subject being treated. Determining a therapeutically effective amount is fully within the capabilities of those skilled in the art.

[1159] In certain embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In certain embodiments, when administered in vivo, the prodrug is chemically converted into a more active form biologically, pharmaceutically or therapeutically of the oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than corresponding active forms. For example, in some cases, prodrugs have higher bioavailability than corresponding active forms (e.g., by oral administration). In some cases, compared with corresponding active forms, prodrugs may have improved solubility. In certain embodiments, prodrugs are less water-soluble than corresponding active forms. In some cases, the prodrug has superior ability to transmit across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, prodrugs are esters. In certain embodiments, esters are metabolically hydrolyzed to carboxylic acids when administered. In some cases, compounds containing carboxylic acids are corresponding active forms. In certain embodiments, prodrugs include short peptides (polyamino acids) bonded to acid groups. In certain embodiments, peptide cleavage is used to form corresponding active forms when administered.

[1160] In certain embodiments, the present disclosure provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.

[1161] In certain embodiments, the present disclosure provides a method for administering a pharmaceutical composition comprising an oligonucleotide of the present disclosure to an animal. Suitable routes of administration include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppositories, by inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, intrathecal drugs are administered to achieve local exposure rather than systemic exposure. For example, pharmaceutical compositions can be injected directly into the desired region (e.g., into the liver) to be administered.

[1162] Non-limiting disclosure and incorporated by reference

[1163] Although certain compounds, compositions and methods described herein have been specifically described according to certain embodiments, the following examples are merely illustrative of the compounds described herein and are not intended to limit the compounds. Each reference, GenBank accession number and analog cited in this application is incorporated herein by reference in its entirety.

[1164] Although the sequence listing accompanying this document identifies each sequence as "RNA" or "DNA" when required, in practice, those sequences may be modified with any combination of chemical modifications. One skilled in the art will readily appreciate that, in some cases, the designation "RNA" or "DNA" to describe a modified oligonucleotide is arbitrary. For example, an oligonucleotide comprising a nucleoside containing a 2'-OH sugar moiety and a thymidine base may be described as a DNA having a modified sugar (2'-OH for the natural 2'-H of DNA) or an RNA having a modified base (thymidine (methylated uracil) for the natural uracil of RNA).

[1165] Thus, the nucleic acid sequences provided herein (including but not limited to those in the sequence listing) are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to those with modified core bases. By way of further example and not limitation, an oligonucleotide with the core base sequence "ATCGATCG" encompasses any oligonucleotide with that core base sequence (whether modified or unmodified), including but not limited to those comprising RNA bases, such as those with the sequence "AUCGAUCG" and those with some DNA bases and some RNA bases such as "AUCGATCG" and those with other modified bases such as "ATCGATCG." me CGAUCG" oligonucleotide, wherein me C indicates a cytosine base containing a methyl group at the 5-position.

[1166] The present invention also includes the following embodiments:

[1167] Embodiment 1. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8 consecutive nucleobases that are complementary to an equal length portion of nucleobases 3533 to 3552 of SEQ ID NO: 3, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 3.

[1168] Embodiment 2. A compound as described in embodiment 1, wherein the modified oligonucleotide comprises a nucleobase sequence comprising a portion of at least 10, at least 12, at least 14, at least 16, at least 18, at least 19 or at least 20 consecutive nucleobases that are complementary to an equal length portion of SEQ ID NO: 3.

[1169] Embodiment 3. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8, at least 10, at least 12, at least 14, at least 15 or at least 16 consecutive nucleobases that are complementary to an equal length portion of nucleobases 3514 to 3558 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 3.

[1170] Embodiment 4. The compound of any preceding embodiment, wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95% or 100% complementary to SEQ ID NO:3.

[1171] Embodiment 5. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleobases comprising the nucleobase sequence of SEQ ID NO: 87.

[1172] Embodiment 6. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221.

[1173] Embodiment 7. The compound of any preceding embodiment, wherein the modified oligonucleotide is single-stranded.

[1174] Embodiment 8. The compound of any one of embodiments 1-6, wherein the modified oligonucleotide is double-stranded.

[1175] Embodiment 9. The compound of any preceding embodiment, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

[1176] Embodiment 10. The compound of embodiment 9, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[1177] Embodiment 11. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.

[1178] Embodiment 12. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least two phosphodiester internucleoside linkages.

[1179] Embodiment 13. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least 3 phosphodiester internucleoside linkages.

[1180] Embodiment 14. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least 4 phosphodiester internucleoside linkages.

[1181] Embodiment 15. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least 5 phosphodiester internucleoside linkages.

[1182] Embodiment 16. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least 6 phosphodiester internucleoside linkages.

[1183] Embodiment 17. The compound of embodiment 10, wherein the modified oligonucleotide comprises at least 7 phosphodiester internucleoside linkages.

[1184] Embodiment 18. The compound of any one of embodiments 11 to 17, wherein each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

[1185] Embodiment 19. The compound of any one of embodiments 1 to 8, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[1186] Embodiment 20. A compound consisting of ISIS 304801 and a conjugate group.

[1187] Embodiment 21. The compound of any preceding embodiment, wherein the modified oligonucleotide comprises at least one modified sugar.

[1188] Embodiment 22. The compound of embodiment 21, wherein at least one modified sugar is a bicyclic sugar.

[1189] Embodiment 23. The compound of embodiment 21, wherein at least one modified sugar comprises 2'-O-methoxyethyl, constrained ethyl, 3'-fluoro-HNA, or 4'-(CH2) n -O-2' bridge, where n is 1 or 2.

[1190] Embodiment 24. The compound of any preceding embodiment, wherein at least one nucleoside comprises a modified nucleobase.

[1191] Embodiment 25. The compound of embodiment 24, wherein the modified nucleobase is 5-methylcytosine.

[1192] Embodiment 26. The compound of any preceding embodiment, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises:

[1193] a gap segment consisting of linked deoxynucleosides;

[1194] a 5' wing segment consisting of linked nucleosides;

[1195] a 3' wing segment consisting of linked nucleosides;

[1196] wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

[1197] Embodiment 27. The compound of any preceding embodiment, wherein the modified oligonucleotide consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25, 16, or 20 linked nucleosides.

[1198] Embodiment 28. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 consecutive nucleobases that are complementary to an equal length portion of SEQ ID NO: 87, wherein the modified oligonucleotide comprises:

[1199] a gap segment consisting of ten linked deoxynucleosides;

[1200] a 5' wing segment consisting of five linked nucleosides;

[1201] a 3' wing segment consisting of five linked nucleosides;

[1202] wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is 5-methylcytosine.

[1203] Embodiment 29. The compound of any one of embodiments 1 to 28, wherein the conjugate group is attached to the modified oligonucleotide at the 5' end of the modified oligonucleotide.

[1204] Embodiment 30. The compound of any one of embodiments 1 to 28, wherein the conjugate group is attached to the modified oligonucleotide at the 3' end of the modified oligonucleotide.

[1205] Embodiment 31. The compound of any one of embodiments 1-30, wherein the conjugate group comprises exactly one ligand.

[1206] Embodiment 32. The compound of any one of embodiments 1-30, wherein the conjugate group comprises exactly two ligands.

[1207] Embodiment 33. The compound of any one of embodiments 1-30, wherein the conjugate group comprises three or more ligands.

[1208] Embodiment 34. The compound of any one of embodiments 1-30, wherein the conjugate group comprises exactly three ligands.

[1209] Embodiment 35. A compound as described in any of embodiments 31-34, wherein each ligand is selected from the following: polysaccharides, modified polysaccharides, mannose, galactose, mannose derivatives, galactose derivatives, D-pyranose mannoside, L-pyranose mannoside, D-arabinose, L-galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-furanose mannoside , β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-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-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose Glucose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose.

[1210] Embodiment 36. The compound of embodiment 35, wherein each ligand is N-acetylgalactosamine.

[1211] Embodiment 37. The compound of any one of Embodiments 1 to 30, wherein the conjugate group comprises:

[1212]

[1213] Embodiment 38. The compound of any one of embodiments 1 to 30, wherein the conjugate group comprises:

[1214]

[1215] Embodiment 39. The compound of any one of embodiments 1 to 30, wherein the conjugate group comprises:

[1216]

[1217] Embodimen...

Claims

1. An oligomeric compound comprising a modified oligonucleotide having the formula: A es G es m C es T es T es m C ds T ds T ds G ds T ds m C ds m C ds A ds G ds m C ds T es T es T es A es T e (SEQ ID NO:87); wherein, A = adenine nucleobase, m C=5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e =2'-O(CH2)2-OCH3ribofuranosyl sugar moiety, d =β-D-2'-deoxyfuranosyl sugar moiety, and s =phosphorothioate internucleoside linkages, Wherein, the compound further comprises a conjugate linked to the 5' terminal nucleoside of the oligonucleotide, wherein the conjugate has the following formula: wherein the modified oligonucleotide is linked to the conjugate via a cleavable moiety (CM), and wherein the cleavable moiety is 5'-P(OH)(=O)-O-3'.

2. An oligomeric compound according to the following chemical structure, or a salt thereof. The oligomeric compound according to claim 2 , which is a sodium salt or a potassium salt.

4. An oligomeric compound according to the following chemical structure:

5. A composition comprising a compound according to any one of claims 1 to 4 and at least one pharmaceutically acceptable carrier.

6. Use of a compound according to any one of claims 1 to 4 or a composition according to claim 5 in the preparation of a medicament for administration to an animal, wherein administration of the medicament to an animal reduces the triglyceride level and / or increases the HDL level of the animal.

7. The use according to claim 6, wherein the animal is a human.

8. The use of claim 6, wherein the administration comprises co-administering the compound or composition with a second agent.

9. The use according to claim 8, wherein the compound or composition and the second agent are administered concomitantly.

10. The use according to claim 6, wherein the administration is parenteral.

11. The use according to claim 6, wherein the administration is subcutaneous or intravenous.

12. The use according to claim 6, wherein the animal suffers from or is at risk of suffering from any one or more of hypertriglyceridemia, Fredrickson type I dyslipidemia, FCS, LPLD, pancreatitis, diabetes, insulin insensitivity and chylomicronemia.

13. The use according to claim 6, wherein the animal suffers from or is at risk of suffering from dyslipidemia.

14. An oligomeric compound comprising a modified oligonucleotide having the formula: A es G ex m C ex T ex T ex m C ds T ds T ds G ds T ds m C ds m C ds A ds G ds m C ds T ex T ex T es A es T e (SEQ ID NO:87); wherein, A = adenine nucleobase, m C=5-methylcytosine nucleobase, G = guanine nucleobase, T = thymine nucleobase, e =2'-O(CH2)2-OCH3ribofuranosyl sugar moiety, d =β-D-2'-deoxyfuranosyl sugar moiety, and s =phosphorothioate internucleoside linkages, x = phosphodiester or phosphorothioate internucleoside linkages, The compound further comprises a conjugate group attached to the 5' terminal nucleoside or the 3' terminal nucleoside of the oligonucleotide, wherein the conjugate group comprises three N-acetylgalactosamine ligands.

15. The compound of claim 14, wherein the conjugate group comprises a conjugate linker having a structure selected from the group consisting of: wherein each L is independently a phosphorus linking group or a neutral linking group; and each n is independently 1 to 20.

16. The compound according to claim 14 or 15, wherein the conjugate group comprises a conjugate linker having a structure selected from the group consisting of: (a) (b) (c) (d) (e) (f) (g) wherein each n is independently 1 to 20; p is 1 to 6; (h) wherein each n is independently 1 to 20, (i) (j) Where n is 1 to 20, (k) (l) wherein each n is independently 0, 1, 2, 3, 4, 5, 6 or 7, and (m) 17. The compound according to any one of claims 14-16, wherein the conjugate group further comprises a conjugate linker, wherein the conjugate linker has the following structure:

18. The compound according to any one of claims 14 to 17, wherein the compound is in the form of a salt, wherein optionally the compound is in the form of a sodium salt and / or a potassium salt.

19. A pharmaceutical composition comprising a compound according to any one of claims 14 to 18 and a pharmaceutically acceptable diluent or carrier.

20. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence containing a portion of at least 8, at least 10, at least 12, at least 14, at least 15 or at least 16 consecutive nucleobases that are complementary to an equal length portion of nucleobases 3514 to 3558 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 3.

Images