RU2782034C2 - Compositions and methods for modulation of hbv and ttr expression - Google Patents

Abstract

FIELD: pharmacology; medicine.

SUBSTANCE: group of inventions relates to the field of pharmacology and medicine, and it is aimed at the treatment of transthyretin amyloidosis. An oligomeric compound is disclosed, while an anionic form of the oligomeric compound is presented by the following chemical structure (SEQ ID NO: 12). In addition, a form of free acid, sodium salt of the above-mentioned oligomeric compound, and an oligomeric compound containing modified oligonucleotide and a conjugating group, characterized by the following formula (SEQ ID NO: 12), are disclosed. A pharmaceutical composition for the treatment of transthyretin amyloidosis, based on the above-mentioned compounds, the use of presented compounds in the production of a drug for the treatment of transthyretin amyloidosis, and, in addition, a method for the treatment of transthyretin amyloidosis in a subject, including administration to the subject of therapeutically effective amount of the oligomeric compound or the pharmaceutical composition containing the specified oligomeric compound, are also disclosed.

EFFECT: group of inventions provides effective treatment of transthyretin amyloidosis.

15 cl, 108 tbl, 113 ex

Description

SEQUENCE LIST

The present invention is registered with a sequence listing in electronic format. The sequence listing is presented as a file called BIOL0248WOSEQ_ST25.txt, created on May 1, 2014, with a size of 16 KB. Sequence listing information in electronic format is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates the amount, activity and/or function of the target nucleic acid. For example, in some cases, antisense compounds lead to a change in transcription or translation of the target. Such modulation of expression can be achieved, for example, by disruption of the target mRNA or by substitutional inhibition. An example of modulating target RNA function by disruption is RNase H disruption of target RNA when hybridized to a DNA-like antisense compound. Another example of modulating gene expression by targeted disruption is RNA interference (RNAi). RNAi refers to antisense gene silencing by a mechanism that uses the RNA-inducible silencing complex (RISC). An additional example of modulating RNA target function is a displacement mechanism, such as miRNA uses in vivo. MicroRNAs are small non-coding RNAs that regulate the expression of RNAs that code for proteins. Binding of the antisense compound to the microRNA prevents the microRNA from binding to its messenger RNA targets, and therefore hinders the function of the microRNA. miRNA mimetics can enhance the natural function of miRNAs. Some antisense compounds alter pre-mRNA splicing. Regardless of the specific mechanism, sequence specificity makes antisense compounds promising as agents for target confirmation and gene functionalization, as well as therapeutic agents for selectively modulating the expression of genes involved in disease pathogenesis.

Antisense technology is an effective way to modulate the expression of one or more specific gene products and, therefore, may be considered exceptionally suitable in a number of therapeutic, diagnostic and research applications. Chemically modified nucleosides can be incorporated into antisense compounds to enhance one or more properties such as nuclease resistance, pharmacokinetics, or affinity for the target nucleic acid. In 1998, the antisense compound Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, California) became the first antisense drug to receive marketing authorization from the US Food and Drug Administration (FDA), and is currently an agent for the treatment of cytomegalovirus (CMV)-induced retinitis in patients with AIDS.

New chemical modifications have improved potency and potency of antisense compounds, opening the potential for oral delivery as well as improved subcutaneous administration, reduced potential side effects, and improved patient comfort. Chemical modifications that enhance the activity of antisense compounds allow lower doses to be administered, reducing the potential for toxicity as well as reducing the overall cost of treatment. Modifications that increase resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Various types of chemical modifications can be combined in a single compound to further optimize compound efficiency.

SUMMARY OF THE INVENTION

In some embodiments of the present disclosure, conjugated antisense compounds are provided. In some embodiments of the present disclosure, conjugated antisense compounds are provided comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In some embodiments of the present disclosure, methods are provided comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In some embodiments of the present disclosure, methods are provided 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.

The asialoglycoprotein receptor (ASGP-R) has previously been described. See, for example, Park et al., PNAS, vol. 102, no. 47, pp. 17125-17129 (2005). Such receptors are expressed on liver cells, in particular hepatocytes. In addition, it has been shown that compounds containing clusters of three N-acetylgalactosamine (GalNAc) ligands are able to bind to ASGP-R, leading to the uptake of said compound into the cell. See, for example, Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, cc. 5216-5231 (May, 2008). Accordingly, conjugates containing such GalNAc clusters have been used to facilitate the uptake of certain compounds into liver cells, in particular hepatocytes. For example, some GalNAc-containing conjugates have been shown to increase the activity of duplex siRNA compounds in liver cells in vivo. In such cases, the GalNAc-containing conjugate is usually attached to the sense helix of the siRNA duplex. Since the sense helix is discarded prior to final hybridization of the antisense helix to the target nucleic acid, it is unlikely that such a conjugate would affect activity. Typically, the conjugate is attached to the 3' end of the siRNA sense helix. See, for example, US Pat. No. 8,106,022. Some of the conjugation groups described herein are more active and/or more easily synthesized than the conjugation groups described previously.

In some embodiments of the present invention, the conjugates are attached to single-stranded antisense compounds, including, but not limited to, RNase H-based antisense compounds and antisense compounds that alter splicing of the target pre-mRNA nucleic acid. In such embodiments, the conjugate must remain attached to the antisense compound long enough to provide the benefit (improved uptake into cells), but then must either cleave or otherwise not interfere with subsequent steps required for activity, such as hybridization to the target nucleic acid. and interaction with RNase H or enzymes associated with splicing or splicing modulation. This balance of properties is more important in the preparation of single-stranded antisense compounds than in siRNA compounds where the conjugate can be simply attached to the sense helix. This document describes single chain antisense compounds having improved activity in liver cells in vivo compared to the same antisense compound without a conjugate. Given the necessary balance of properties for these compounds, this improved activity is unexpected.

In some embodiments of the invention, the conjugating groups of the present document contain a cleavable moiety. As noted, without being limited to any mechanism, it is logical that the conjugate should be retained at the compound long enough to provide increased uptake, but after that it is desirable that some of it, or ideally all of the conjugate, is cleaved, releasing the parent compound (for example, antisense compound) in its most active form. In some embodiments, the cleavable fragment is a cleavable nucleoside. Such embodiments exploit endogenous nucleases in the cell by attaching the remainder of the conjugate (cluster) to the antisense oligonucleotide via the nucleoside via one or more cleavable bonds, such as phosphodiester bonds. In some embodiments, the cluster is linked to the cleavable nucleoside via a phosphodiester bond. In some embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In some embodiments of the invention, the conjugating group may contain two or three cleavable nucleosides. In such embodiments, said cleavable nucleosides are linked to each other, to the antisense compound, and/or to the cassette via cleavable bonds (such as a phosphodiester bond). Some of the conjugates herein do not contain a cleavable nucleoside, but instead contain a cleavable bond. It has been shown that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is easily cleavable in the cell (cleavable bond).

In some embodiments, the conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are eventually metabolized to a more active form. For example, conjugated antisense compounds are cleaved to remove all or part of the conjugate, resulting in an active (or more active) form of the antisense compound that does not contain all or part of the conjugate.

In some embodiments, the conjugates are attached at the 5' end of the oligonucleotide. Some of these 5'-conjugates cleave more efficiently than analogs having the same conjugate group attached at the 3'-end. In some embodiments of the invention, improved activity may correlate with improved digestion. In some embodiments of the invention, oligonucleotides containing the conjugate at the 5' end are more effective than oligonucleotides containing the conjugate at the 3' end (see, for example, Examples 56, 81, 83 and 84). In addition, the 5'-attachment provides an easier synthesis of the oligonucleotide. Typically, oligonucleotides are synthesized on a solid support in the 3' to 5' direction. To obtain a 3'-conjugated oligonucleotide, the pre-conjugated 3'-nucleoside is typically attached to a solid support, and then the oligonucleotide is created in the usual way. However, attaching such a conjugated nucleoside to a solid support complicates the synthesis. In addition, using this approach, the conjugate is then present throughout the synthesis of the oligonucleotide and may be degraded during subsequent steps or may limit the types of reactions and reagents that can be used. Using the structures and techniques described herein for 5'-conjugated oligonucleotides, an oligonucleotide can be synthesized using standard automated techniques and the conjugate can be inserted along with the last (5'-outer) nucleoside or after separation of the oligonucleotide from a solid support.

Given the prior art and the present description, those skilled in the art can readily prepare any of the conjugates and conjugated oligonucleotides described herein. In addition, the synthesis of some of these conjugates and conjugated oligonucleotides described herein is simpler and/or requires fewer steps and therefore less expensive than the synthesis of previously described conjugates, which provides a manufacturing advantage. For example, the synthesis of some conjugate groups consists of fewer synthetic steps, resulting in higher yields than previously described conjugate groups. Conjugate groups such as GalNAc3-10 in Example 46 and GalNAc3-7 in Example 48 are much simpler than previously described conjugates such as those described in U.S. 8106022 or U.S. 7262177, which require assembly of more chemical intermediates. Accordingly, these and other conjugates described herein are advantageous over previously described compounds for use with any oligonucleotide, including single-stranded oligonucleotides and any helix of double-stranded oligonucleotides (eg, siRNA).

Similarly, conjugation groups having exactly one or two GalNAc ligands are described herein. Such conjugated groups have been shown to enhance the activity of antisense compounds. Such compounds are much easier to prepare than conjugates containing three GalNAc ligands. Conjugating groups containing one or two GalNAc ligands can be attached to any antisense compounds, including single stranded oligonucleotides and any helix of double stranded oligonucleotides (eg, siRNA).

In some embodiments of the invention, the conjugates described herein do not significantly change some indicators of tolerability. For example, conjugated antisense compounds are shown herein to be no more immunogenic than unconjugated parent compounds. Since activity improves, embodiments in which tolerability remains the same (or, in fact, if tolerability deteriorates only slightly compared to the increase in activity), have improved characteristics for therapy.

In some embodiments of the invention, conjugation allows you to change the antisense compounds so that they have less pronounced effects in the absence of conjugation. For example, in some embodiments of the invention, the replacement of one or more thiophosphate bonds of a fully thiophosphate antisense compound with phosphodiester bonds results in an improvement in some indicators of tolerability. For example, in some instances, such antisense compounds having one or more phosphodiesters are less immunogenic than the same compounds in which each bond is a thiophosphate. However, in some cases, as shown in Example 26, the same substitution of one or more thiophosate bonds with phosphodiester bonds also results in a decrease in cellular uptake and/or a decrease in activity. In some embodiments of the invention, the conjugated antisense compounds described herein allow such a change in bonds with little or no reduction in uptake and activity, compared with the conjugated all thiophosphate analog. Indeed, in some embodiments, such as in Examples 44, 57, 59, and 86, oligonucleotides containing the conjugate and at least one phosphodiester internucleoside bond actually exhibit enhanced in vivo activity even when compared to the all-thiophosphate analog also containing such or the conjugate. Moreover, since conjugation results in a significant increase in uptake/activity, a small reduction in such a significant increase may be acceptable in order to achieve improved tolerability. Accordingly, in some embodiments, the conjugated antisense compounds contain at least one phosphodiester bond.

In some embodiments of the invention, conjugation of antisense compounds of the present document results in improved delivery, uptake, and activity in hepatocytes. Consequently, more of the compound is delivered to the liver tissue. However, in some embodiments of the invention, such improved delivery alone does not explain the overall increase in activity. In some such embodiments of the invention, a greater amount of the compound enters the hepatocytes. In some embodiments of the invention, even this improved capture of hepatocytes does not in itself explain the overall increase in activity. In such embodiments of the invention, the productive uptake of the conjugated compound is increased. For example, as shown in Example 102, some embodiments of GalNAc-containing conjugates increase the enrichment of antisense oligonucleotides in hepatocytes compared to non-parenchymal cells. This enrichment is advantageous for oligonucleotides that target genes expressed in hepatocytes.

In some embodiments of the invention, the conjugated antisense compounds of the present document result in a reduction in renal exposure. For example, as shown in Example 20, concentrations of antisense oligonucleotides containing certain embodiments of GalNAc-containing conjugates in the kidney are lower than concentrations of antisense oligonucleotides lacking a GalNAc-containing conjugate. This has several beneficial therapeutic effects. For therapeutic indications that do not require activity in the kidneys, exposure to the kidneys exposes them to the risk of toxicity without corresponding benefit. Moreover, the high concentration in the kidneys usually results in the excretion of the compound in the urine, allowing for faster elimination. Accordingly, for extrarenal targets, accumulation in the kidneys is undesirable.

In some embodiments of the present disclosure, conjugated antisense compounds are represented by the formula:

where

A is an antisense oligonucleotide;

B is a cleavable fragment;

C is a conjugate linker;

D is a branch group;

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In the above diagram and in similar diagrams herein, branching group "D" branches as many times as necessary to match the number of groups (E-F) indicated by the number "q". So, if q=1, then the formula is:

if q=2, then the formula is:

if q=3, then the formula is:

if q=4, then the formula is:

if q=5, then the formula is:

In some embodiments of the invention, conjugated antisense compounds are provided having the structure:

In some embodiments of the invention, conjugated antisense compounds are provided having the structure:

In some embodiments of the invention, conjugated antisense compounds are provided having the structure:

In some embodiments of the invention, conjugated antisense compounds are provided having the structure:

In embodiments having more than one particular variable (eg, more than one "m" or "n"), unless otherwise noted, each such particular variable is independently selected. Therefore, for a structure having more than one n, each n is chosen independently, so they may or may not be the same.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory only and do not limit the present description. In this document, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the term "or" means "and/or" unless otherwise indicated. In addition, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" include both elements and components containing a single unit, and elements and components that contain more than one subunit, unless specifically indicated otherwise.

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

A. Definitions

In the absence of specific definitions, the nomenclature used in connection with them, as well as in connection with the techniques and methods of analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry, described in this document, is well known and generally accepted in the art. For chemical synthesis and chemical analysis, standard techniques can be used. Some such techniques and techniques are given, for example, in "Carbohydrate Modifications in Antisense Research" 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, Fla.; and also in Sambrook et al., "Molecular Cloning, A laboratory Manual," ^2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. If applicable, all patents, applications, published applications and other publications, as well as other data mentioned in the text of this description, are incorporated herein by reference in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

As used herein, "nucleoside" means a compound containing a nitrogenous base moiety and a sugar moiety. Nucleosides include, but are not limited to, natural nucleosides (which are found in DNA and RNA) and modified nucleosides. Nucleosides can be linked to a phosphate moiety.

As used herein, "chemical modification" means a chemical difference in a compound from its natural counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nitrogenous base modifications) and modifications of internucleoside bonds. In relation to an oligonucleotide, chemical modification does not only include differences in the sequence of nitrogenous bases.

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

As used herein, "natural sugar moiety" means ribofuranosyl found in natural RNA or deoxyribofuranosyl found in natural DNA.

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

As used herein, "modified sugar moiety" means a substituted sugar moiety or sugar substitute.

As used herein, "substituted sugar moiety" means furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to, furanosyls containing substituents at the 2' position, 3' position, 5' position, and/or 4' position. Some substituted sugar moieties are bicyclic sugar moieties.

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

As used herein, "MY" means -OCH ₂ CH ₂ OCH ₃ .

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

As used herein, the term "sugar substitute" means a structure that does not contain furanosyl and is capable of replacing the natural sugar moiety of a nucleoside such that the resulting nucleoside subunits can bind together and/or bind to other nucleosides to form an oligomeric compound that can hybridize to complementary oligomeric compound. Such structures include rings containing a different number of atoms than furanosyl (eg 4, 6 or 7 membered rings); replacing the oxygen of furanosyl with a non-oxygen atom (eg carbon, sulfur or nitrogen); or the simultaneous change in the number of atoms and the replacement of oxygen. Such structures may also contain substitutions corresponding to those described for substituted sugar moieties (eg, 6-membered carbocyclic bicyclic sugar substitutes, optionally containing additional substituents). Sugar substitutes also include more complex sugar substituents (eg non-circular peptide nucleic acid systems). Sugar substitutes include, without limitation, morpholinos, cyclohexenyls, and cyclohexitols.

As used herein, "bicyclic sugar moiety" means a modified sugar moiety containing a 4- to 7-membered ring (including but not limited to furanosyl) containing a bridge linking two atoms of the 4- to 7-membered ring to form a second ring such that leads to a bicyclic structure. In some embodiments, the 4-7 membered ring is a sugar ring. In some embodiments, the 4-7 membered ring is furanosyl. In some such embodiments, a bridge connects the 2' carbon and the 4' carbon of furanosyl.

As used herein, "nucleotide" means a nucleoside further containing a phosphate linking group. As used herein, "linked nucleosides" may or may not be linked by phosphate bonds and therefore includes, but is not limited to, "linked nucleotides". As used herein, "linked nucleosides" are nucleosides that are linked in a contiguous sequence (ie, there are no additional nucleosides between linked nucleosides).

As used herein, "nucleobase" means a group of atoms that can be bonded to a sugar moiety to form a nucleoside that can be incorporated into an oligonucleotide, and said group of atoms can bind to the complementary natural nitrogenous base of another oligonucleotide or nucleic acid. . The nitrogenous bases may be natural or may be modified.

As used herein, the terms "unmodified nitrogenous base" or "natural nitrogenous base" means the natural heterocyclic nitrogenous bases 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).

As used herein, "modified nitrogenous base" means any nitrogenous base that is not a naturally occurring nitrogenous base.

As used herein, "modified nucleoside" means a nucleoside containing at least one chemical modification compared to natural RNA or DNA nucleosides. Modified nucleosides contain a modified sugar moiety and/or a modified nitrogenous base.

As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside containing a bicyclic sugar moiety.

As used herein, "hindered ethyl nucleoside" or "cEt" means a nucleoside containing a bicyclic sugar moiety that contains a 4'-CH(CH ₃ )-O-2' bridge.

As used herein, "closed nucleic acid nucleoside" or "LNA" means a nucleoside containing a bicyclic sugar moiety that contains a 4'-CH ₂ -O-2' bridge.

As used herein, "2'-substituted nucleoside" means a nucleoside containing a substituent at the 2'-position other than H or OH. Unless otherwise stated, a 2'-substituted nucleoside is not a bicyclic nucleoside.

As used herein, "deoxynucleoside" means a nucleoside containing a 2'-H furanose sugar moiety found in naturally occurring deoxyribonucleosides (DNA). In some embodiments, the 2'-deoxynucleoside may contain a modified nitrogenous base, or may contain an RNA nitrogenous base (eg, uracil).

As used herein, "oligonucleotide" means a compound containing a plurality of linked nucleosides. In some embodiments, the oligonucleotide contains one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNAs), and/or one or more modified nucleosides.

As used herein, "oligonucleoside" means an oligonucleotide in which none of the internucleoside bonds contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.

As used herein, "modified oligonucleotide" means an oligonucleotide containing at least one modified nucleoside and/or at least one modified internucleoside bond.

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

As used herein, "internucleoside bond" means a covalent bond between adjacent nucleosides in an oligonucleotide e.

As used herein, "natural internucleoside bond" means a phosphodiester bond 3' to 5'.

As used herein, "modified internucleoside bond" means any internucleoside bond other than a naturally occurring internucleoside bond.

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

As used herein, "phosphorus linking group" means a linking group containing a phosphorus atom. Phosphorus linking groups include, without limitation, groups having the formula:

where:

R _a and R _d are each independently O, S, CH ₂ , NH or NJ ₁ where J ₁ is C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl;

R _b is O or S;

R _c is OH, SH, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, substituted C ₁ -C ₆ alkoxy, amino, or substituted amino; and

J ₁ is R _b is O or S.

Phosphorus linking groups include, without limitation, phosphodiester, thiophosphate, dithiophosphate, phosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriesters, and boranophosphate.

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

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

As used herein, "neutral linking group" means a linking group that has no charge. Neutral linking groups include, without limitation, phosphotriesters, methylphosphonates, MMI (-CH ₂ -N(CH ₃ )-O-), amide-3 (-CH ₂ -C(=O)-N(H)-), amide -4 (-CH ₂ -N(H)-C(=O)-), forma acetal (-O-CH ₂ -O-) and thioformacetal (-S-CH ₂ -O-). Additional neutral linking groups include non-ionic bonds containing siloxane (dialkylsiloxane), carboxylic ester, carboxamide, sulfide, sulfonic ester and amides (see, for example: Carbohydrate Modifications in Antisense Research; ed. YS Sanghvi and PD Cook, ACS Symposium , series 580; chapters 3 and 4, (pp. 40-65)). Additional neutral linking groups include nonionic bonds containing mixed constituents of N, O, S and CH ₂ .

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

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

As used herein, "oligomeric compound" means a polymeric structure containing two or more substructures. In some embodiments of the invention, the oligomeric compound contains an oligonucleotide. In some embodiments of the invention, the oligomeric compound contains one or more conjugated groups and/or end groups. In some embodiments of the invention, the oligomeric compound consists of an oligonucleotide. Oligomeric compounds also include natural nucleic acids. In some embodiments of the invention, the oligomeric compound contains the backbone of one or more linked monomeric subunits, with each linked monomeric subunit directly or indirectly attached to the heterocyclic main fragment. In some embodiments of the invention, oligomeric compounds may also contain monomeric subunits that are not associated with a heterocyclic basic fragment, thus providing sites with deleted bases. In some embodiments, the bonds connecting the monomeric subunits, sugar moieties or substitutes, and the heterocyclic base moieties can be independently modified. In some embodiments, the sugar bond unit, which may or may not contain a heterocyclic base, may be substituted with a mimetic, such as monomers in peptide nucleic acids.

As used herein, "end group" means one or more atoms attached to either or both of the 3' or 5' ends of an oligonucleotide. In some embodiments of the invention, the end group is a conjugate group. In some embodiments of the invention, the end group contains one or more end group nucleosides.

As used herein, "conjugate" or "conjugating group" means an atom or group of atoms associated with an oligonucleotide or oligomeric compound. Typically, conjugating groups modify one or more properties of the compound to which they are attached, including, but not limited to, pharmacodynamic, pharmacokinetic, binding, uptake, cellular distribution, cellular uptake, charge, and/or excretion properties.

As used herein, "conjugate linker" or "linker" in the context of conjugate groups means a portion of a conjugate group containing any atom or group of atoms and covalently linking (1) an oligonucleotide to another portion of a conjugate group or (2) two or more portions of a conjugate group. groups.

Conjugating groups are shown herein as radicals that provide a bond to form a covalent attachment to an oligomeric compound, such as an antisense oligonucleotide. In some embodiments, the point of attachment at the oligomeric compound is the 3' oxygen atom of the 3'-hydroxyl group of the 3'-terminal nucleoside of the oligomeric compound. In some embodiments, the point of attachment at the oligomeric compound is the 5' oxygen atom of the 5'-hydroxyl group of the 5'-terminal nucleoside of the oligomeric compound. In some embodiments, the bond to form an attachment to the oligomeric compound is a cleavable bond. In some such embodiments, such a cleavable bond constitutes all or part of the cleavable moiety.

In some embodiments, the conjugating groups contain a cleavable moiety (eg, a cleavable bond or a cleavable nucleoside) and a portion of a carbohydrate cassette, such as a portion of a GalNAc cassette. This portion of the carbohydrate cassette contains: a targeting moiety and, optionally, a conjugate linker. In some embodiments of the invention, the portion of the carbohydrate cluster is determined by the amount and nature of the ligand. For example, in some embodiments, the carbohydrate cassette portion contains three GalNAc groups and is designated "GalNAc ₃ ". In some embodiments of the invention, part of the carbohydrate cluster contains 4 GalNAc groups and is designated "GalNAc ₄ ". Specific portions of the carbohydrate cassettes (having a specific linkage, branch groups, and conjugate linker) are described herein and are denoted by a Roman numeral followed by the subscript "a". Accordingly, “GalNac3-1 _a ” refers to a specific portion of the carbohydrate cassette of a conjugating group having 3 GalNac groups and a specifically defined ligament, branching group and linker. Such a carbohydrate cassette moiety is attached to the oligomeric compound via a cleavable moiety, such as a cleavable bond or a cleavable nucleoside.

As used herein, "cleavable moiety" means a bond or group that can be cleaved under physiological conditions. In some embodiments, the cleavable fragment is cleaved within the cell or in intracellular compartments such as the lysosome. In some embodiments, the cleavable fragment is cleaved by endogenous enzymes such as nucleases. In some embodiments, the cleavable moiety contains a group of atoms having one, two, three, four, or more than four cleavable bonds.

As used herein, "cleavable bond" means any chemical bond that can be cleaved. In some embodiments, the cleavable bond is selected from: an amide, a polyamide, an ester, an ester, one or both of a phosphodiester, a phosphate ester, a carbamate, a disulfide, or a peptide.

As used herein, "carbohydrate cluster" means a compound having one or more carbohydrate residues attached to a backbone or linking group (see, for example, Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting," Bioconjugate Chemistry, 2003, (14): 18-29, 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 for examples of carbohydrate conjugated clusters).

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

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

As used herein, "protecting group" means any compound or protecting group known to those skilled in the art. Non-limiting examples of protective groups are provided in the publication "Protective Groups in Organic Chemistry", T.W. Greene, P.G.M. Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, the entire contents of which are hereby incorporated by reference.

As used herein, "single stranded" means an oligomeric compound that is not hybridized to its complement and that does not have sufficient self complementarity to form a stable self duplex.

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

As used herein, "antisense compound" means a compound containing or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid with which it can hybridize, resulting in at least one antisense activity.

As used herein, "antisense activity" means any detectable and/or measurable change due to hybridization of an antisense compound to its target nucleic acid. In some embodiments, antisense activity includes modulating the amount or activity of a target nucleic acid transcript (eg, mRNA). In some embodiments, the antisense activity includes modulating pre-mRNA splicing.

As used herein, "RNase H-based antisense compound" means an antisense compound in which at least a portion of the antisense activity of the antisense compound is due to hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.

As used herein, "RISC-based antisense compound" means an antisense compound in which at least a portion of the antisense activity of the antisense compound is due to an RNA-induced silencing complex (RISC).

As used herein, "detection" or "measurement" means that a test or analysis is performed to detect or measure. Such detection and/or measurement may result in a null value. Therefore, even if the detection or measurement test results in the detection of no activity (no activity), the activity detection or measurement step has been performed.

As used herein, "detectable and/or measurable activity" means a statistically significant activity that is not null.

As used herein, "substantially unchanged" means little or no change in a particular parameter, particularly when compared to another parameter that has changed much more. In some embodiments of the invention, a parameter is substantially unchanged if its change is less than 5%. In some embodiments of the invention, a parameter is essentially unchanged if its change was less than two times, while the change in another parameter was at least ten times. For example, in some embodiments of the invention, the antisense activity is a change in the amount of the target nucleic acid. In some such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the amount of the target nucleic acid changes, but this change need not be zero.

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

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

As used herein, “complementarity of nitrogenous bases” or “complementarity” with respect to nitrogenous bases means a nitrogenous base that is capable of base pairing with another nitrogenous base. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, complementary nucleobase means the nucleobase of an antisense compound that is capable of base pairing with the nucleobase of its target nucleic acid. For example, if a nitrogenous base at a specific position of the antisense compound is capable of hydrogen bonding to a nitrogenous base at a specific position of the target nucleic acid, then that hydrogen bonding position between the oligonucleotide and the target nucleic acid is considered complementary at that nitrogenous base pair. Nitrogenous bases containing certain modifications may retain the ability to pair with the opposite nitrogenous base and, therefore, may still have the complementarity of the nitrogenous bases.

As used herein, "non-complementary" with respect to nitrogenous bases means a pair of nitrogenous bases that do not form hydrogen bonds with each other.

As used herein, "complementary" with respect to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the ability of such oligomeric compounds, or regions thereof, to hybridize with another oligomeric compound, or region thereof, by complementarity of nitrogenous bases. Complementary oligomeric compounds do not have to have the complementarity of the nitrogenous bases in each nucleoside. To some extent, some inconsistencies are acceptable. In some embodiments, complementary oligomeric compounds or regions are 70% complementary to nitrogenous bases (70% complementarity). In some embodiments, complementary oligomeric compounds or regions are 80% complementary. In some embodiments of the invention, complementary oligomeric compounds or regions are 90% complementary. In some embodiments of the invention, complementary oligomeric compounds or regions are 95% complementary. In some embodiments, complementary oligomeric compounds or regions are 100% complementary.

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

As used herein, "hybridization" means the pairing of complementary oligomeric compounds (eg, an antisense compound and its target nucleic acid). Without being limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary nitrogenous bases.

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

As used herein, "fully complementary" in relation to an oligonucleotide or portion thereof means that each nitrogenous base of the oligonucleotide or portion thereof is capable of pairing with the nitrogenous base of a complementary nucleic acid or a continuous portion thereof. Therefore, a fully complementary region contains no mismatches or unhybridized nitrogenous bases in both helices.

As used herein, "percent complementarity" means the percentage of nitrogenous bases of an oligomeric compound that are complementary to an equal length portion of the target nucleic acid. The percent complementarity is calculated by dividing the number of nitrogenous bases of the oligomeric compound that are complementary to the nitrogenous bases at the corresponding positions of the target nucleic acid by the total length of the oligomeric compound.

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

As used herein, "modulating" means changing the amount or quality of a molecule, function, or activity, compared to the amount or quality of the molecule, function, or activity prior to modulation. For example, modulation includes changing, either increasing (stimulating or inducing) or decreasing (inhibiting or decreasing) gene expression. As a further example, modulating expression may include changing the splicing site selection during pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice variant, compared to its amount in the absence of modulation.

As used herein, "chemical motif" means a characteristic site of chemical modifications in an oligonucleotide or region thereof. Motifs can be identified by modifications in certain nucleosides and/or in certain linking groups of the oligonucleotide.

As used herein, "nucleoside motif" means a characteristic site of nucleoside modifications in an oligonucleotide or region thereof. The bonds of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs describing only nucleosides herein are nucleoside motifs. Therefore, in such cases, the connections are not limited.

As used herein, "sugar motif" means a characteristic site of sugar modifications in an oligonucleotide or region thereof.

As used herein, "binding motif" means a characteristic site of binding modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs that describe only links herein are linking motifs. Therefore, in such cases, nucleosides are not limited.

As used herein, "nucleobase modification motif" means a characteristic site of nitrogenous base modifications along an oligonucleotide. Unless otherwise stated, the nitrogenous base modification motif is independent of the sequence of the nitrogenous bases.

As used herein, "sequence motif" means a characteristic region of nitrogenous bases located along an oligonucleotide or portion thereof. Unless otherwise indicated, the sequence motif is independent of chemical modifications and therefore may have any combination of chemical modifications, including no chemical modifications.

As used herein, "type of modification" in relation to a nucleoside or nucleoside of a certain "type" means a chemical modification of the nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, "a nucleoside having a modification of the first type" may be an unmodified nucleoside.

As used herein, "differently modified" means chemical modifications or chemical substituents that are different from each other, including their absence or modifications. Thus, for example, the MOE nucleoside and the 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 the same but contain different nitrogenous bases 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 thymine nitrogenous base are not differently modified.

As used herein, "same type modifications" refers to modifications that are the same with respect to each other, including no modifications. Thus, for example, two unmodified DNA nucleosides have "the same type of modification" even though the DNA nucleoside is unmodified. Such nucleosides having a modification of the same type may contain different nitrogenous bases.

As used herein, "distinct regions" means portions of an oligonucleotide in which the chemical modifications or chemical modification motif of any of the neighboring portions contain at least one difference to enable the regions to be distinguished from each other.

As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use when administered to an animal. In some embodiments of the invention, the pharmaceutically acceptable carrier or diluent is a sterile saline solution. In some embodiments, such sterile saline is pharmaceutical grade saline.

As used herein, the term "metabolic disorder" means a disease or pathological condition characterized primarily by dysregulation of metabolism, a complex set of chemical reactions associated with the breakdown of food to produce energy.

As used herein, the term "cardiovascular disorder" means a disease or pathological condition characterized primarily by impaired function of the heart or blood vessels.

As used herein, the term "mono- or polycyclic ring system" includes all ring systems selected from single or polycyclic radical ring systems in which said rings are fused or linked, and includes single or mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono- and polycyclic structures may contain rings, each of which has the same degree of saturation, or each independently has varying degrees of saturation, including fully saturated, partially saturated or fully unsaturated. Each ring may contain ring atoms selected from C, N, O, and S to form heterocyclic rings, as well as rings containing only C ring atoms, which may be present in a mixed motif, such as in benzimidazole, in which one ring has only ring carbon atoms, while the fused ring has two nitrogen atoms. The mono- or polycyclic ring system may be further substituted with substituent groups, such as phthalimide, which has two ═O groups attached to one of the rings. Mono- or polycyclic ring systems can be attached to parent molecules in a variety of ways, such as directly through a ring atom, by condensation through multiple ring atoms, through a substituent group, or through a bifunctional linking moiety.

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

As used herein, "substituent" and "substituent group" means an atom or group that displaces an atom or group of the specified parent compound. For example, a modified nucleoside substituent is any atom or group that is different from the atom or group found in the naturally occurring nucleoside (e.g., a modified 2' substituent is any atom or group at the 2' position of the nucleoside other than H or OH) . Substituent groups may or may not be protected. In some embodiments of the invention, the compounds of the present description have substituents in one or more than one position of the parent compound. The substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbon group to the parent compound.

Similarly, as used herein, "substituent" in relation to a chemical functional group means an atom or group of atoms that is different from the atom or group of atoms normally contained in the specified functional group. In some embodiments, a substituent displaces a hydrogen atom of a functional group (for example, in some embodiments, a substituent on a substituted methyl group is an atom or group other than hydrogen that displaces one or more hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups which may be used as substituents include, without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)R _aa ), carboxyl (-C(O)OR _aa ), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-OR _aa ), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(R _bb )(R _cc )), imino (=NR _bb ), amido (-C(O)N(R _bb )(R _cc ) or -N(R _bb )C(O)R _aa ), azido (-N ₃ ), nitro (-NO ₂ ), cyano (-CN) , urea (-OC(O)N(R _bb )(R _cc ) or -N(R _bb )C(O)OR _aa ), ureido (-N(R _bb )C(O)-N(R _bb ) (R _cc )), thioureido (-N(R _bb )C(S)N(R _bb )(R _cc )), guanidinyl (-N(R _bb )C(=NR _bb )N(R _bb )(R _cc )), amidinyl (-C(=NR _bb )N(R _bb )(R _cc ) or -N(R _bb )C(=NR _bb )(R _aa )), thiol (-SR _bb ), sulfinyl ( -S(O)R _bb ), sulfonyl (-S(O) ₂ R _bb ) and sulfonamidyl (-S(O) ₂ N(R _bb )(R _cc ) or -N(R _bb )S-(O) _{2Rbb )} . Where each R _aa , R _bb and R _cc is independently H, an optionally linked chemical functional group or an additional substituent group, with the preferred list including, without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents with the compounds described herein are in the recursive degree.

As used herein, "alkyl" as used herein means a saturated straight or branched hydrocarbon radical containing up to twenty-four carbon atoms. Examples of alkyl groups include, without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. Alkyl groups typically contain from 1 to about 24 carbon atoms, more often from 1 to about 12 carbon atoms (C ₁ -C ₁₂ alkyl), with more preferably from 1 to about 6 carbon atoms.

As used herein, "alkenyl" means a straight or branched hydrocarbon radical containing up to twenty-four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups typically contain from 2 to about 24 carbon atoms, more often from 2 to about 12 carbon atoms, with more preferably from 2 to about 6 carbon atoms. The alkenyl groups used herein may optionally contain one or more additional substituent groups.

As used herein, "alkynyl" means a straight or branched hydrocarbon radical containing up to twenty-four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically contain from 2 to about 24 carbon atoms, more often from 2 to about 12 carbon atoms, with more preferably from 2 to about 6 carbon atoms. The alkynyl groups used herein may optionally contain one or more additional substituent groups.

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

As used herein, "alicyclic" means a cyclic ring system in which the ring is aliphatic. The ring system may contain one or more rings, with at least one ring being aliphatic. Preferred alicyclic systems include rings having from about 5 to about 9 ring carbon atoms. The alicyclic groups used herein may optionally contain additional substituent groups.

As used herein, "aliphatic" means a straight or branched hydrocarbon radical of up to twenty-four carbons in which the saturation between any two carbons is a single, double or triple bond. The aliphatic group preferably contains from 1 to about 24 carbon atoms, more often from 1 to about 12 carbon atoms, with more preferably from 1 to about 6 carbon atoms. The straight or branched chain aliphatic group may be interrupted by one or more heteroatoms which include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include, without limitation, polyalkoxy such as polyalkylene glycols, polyamines and polyimines. The aliphatic groups used herein may optionally contain additional substituent groups.

As used herein, "alkoxy" means a radical formed between an alkyl group and an oxygen atom, whereby the oxygen atom is used to attach the alkoxy group to the parent molecule. Examples of alkoxy groups include, without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. The alkoxy groups used herein may optionally contain additional substituent groups.

As used herein, "aminoalkyl" means an amino-substituted C ₁ -C ₁₂ alkyl radical. The alkyl part of the indicated radical 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 an additional substituent group on the alkyl and/or amino moiety.

As used herein, "aralkyl" and "arylalkyl" means an aromatic group that is covalently attached to a C ₁ -C ₁₂ alkyl radical. Part of the alkyl radical of the resulting aralkyl (or arylalkyl) group forms a covalent bond with the parent molecule. Examples include, without limitation, benzyl, phenethyl, and the like. The aralkyl groups used herein may optionally contain additional substituent groups attached to the alkyl, aryl, or both groups that form said radical group.

As used herein, "aryl" and "aromatic" means radicals of a mono- or polycyclic carbocyclic ring system having one or more aromatic rings. Examples of aryl groups include, without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. The aryl groups used herein may optionally contain additional substituent groups.

As used herein, "halogen" and "halogen" means an atom selected from fluorine, chlorine, bromine and iodine.

As used herein, "heteroaryl" and "heteroaromatic" means a radical containing a mono- or polycyclic aromatic ring, ring system, or fused ring system in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl also includes fused ring systems, including systems in which one or more of the fused rings do not contain heteroatoms. Heteroaryl groups typically contain one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. Heteroaryl radicals can be attached to the parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. The heteroaryl groups used herein may optionally contain additional substituent groups.

As used herein, "conjugated compound" means any atoms, groups of atoms, or group of linked atoms suitable for use as a conjugate group. In some embodiments, the conjugate compounds may have or affect one or more properties, including, but not limited to, pharmacodynamic, pharmacokinetic, binding, adsorption, cellular distribution, cellular uptake, charge, and/or excretion properties.

As used herein, unless specified or otherwise modified, "double-stranded" refers to two separate oligomeric compounds that are hybridized to each other. Such double-stranded compounds may have one or more non-hybridized nucleosides at one or both ends of one or both helices (overhangs) and/or one or more internal non-hybridized nucleosides (mismatches), provided that sufficient complementarity exists to maintain hybridization at physiologically relevant conditions.

B. Some compounds

In some embodiments, the invention provides conjugated antisense compounds comprising antisense oligonucleotides and a conjugate.

a. Some antisense oligonucleotides

In some embodiments of the present invention, antisense oligonucleotides are provided. Such antisense oligonucleotides contain linked nucleosides, and each nucleoside contains a sugar moiety and a nitrogenous base. The structure of such antisense oligonucleotides can be viewed in terms of chemical features (eg, modifications and characteristic sites of modifications) and sequence of nitrogenous bases (eg, sequence of the antisense oligonucleotide, entity and sequence of the target nucleic acid).

i. Some chemical features

In some embodiments, the antisense oligonucleotide contains one or more modifications. In some such embodiments, the antisense oligonucleotides contain one or more modified nucleosides and/or modified internucleoside bonds. In some embodiments of the invention, the modified nucleosides contain a modified sugar moiety and/or a modified nitrogenous base.

1. Some sugar fragments

In some embodiments of the invention, the compounds of the present description contain one or more modified nucleosides containing a modified sugar moiety. Such compounds containing one or more sugar-modified nucleosides may have desirable properties, such as improved nuclease stability or increased binding affinity for the target nucleic acid, compared to an oligonucleotide containing only nucleosides that contain naturally occurring sugar moieties. In some embodiments, the modified sugar moieties are substituted sugar moieties. In some embodiments, the modified sugar moieties are sugar substitutes. Such sugar substitutes may contain one or more substitutions corresponding to those of the substituted sugar moieties.

In some embodiments, the modified sugar moieties are substituted sugar moieties containing one or more non-bridging sugar substituents, including, but not limited to, substituents at the 2' and/or 5' positions. Examples of sugar substituents suitable for the 2'-position include, but are not limited to: 2'-F, 2'-OCH ₃ ("OMe" or "O-methyl") and 2'-O(CH ₂ ) ₂ OCH ₃ ("MY"). In some embodiments, the 2' sugar substituent is selected from allyl, amino, azido, thio, O-allyl, OC ₁ -C ₁₀ alkyl, O-C ₁ -C ₁₀ substituted alkyl; OCF ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(Rm)(Rn) and O-CH ₂ -C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C ₁ -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 some embodiments, the substituted sugars contain more than one non-bridging sugar substituent, such as 2'-F-5'-methyl sugar moieties (see, for example, PCT International Application WO 2008/101157 for additional 5', 2' -bis-substituted sugar fragments and nucleosides).

Nucleosides containing 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In some embodiments, the 2'-substituted nucleoside contains a 2'-substituent group selected from halogen, allyl, amino, azido, SH, CN, OCN, CF ₃ , OCF ₃ , O, S, or M(R _m )-alkyl ; O, S or N(R _m )-alkenyl; O, S or N(R _m )-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH ₂ ) ₂ SCH ₃ , O-(CH ₂ ) ₂ -ON(R _m )(R _n ) or O -CH ₂ -C(=O)-N(R _m )(R _n ), where each R _m and R _n independently represents H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl. These 2' substituent groups may be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO ₂ ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In some embodiments, the 2'-substituted nucleoside contains a 2'-substituent group selected from F, NH ₂ , N ₃ , OCF ₃ , O-CH ₃ , O(CH ₂ ) ₃ NH ₂ , CH ₂ -CH=CH ₂ , O-CH ₂ -CH=CH ₂ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O-(CH ₂ ) ₂ -ON(R _m )(R _n ), O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ and N-substituted acetamide (O-CH ₂ -C(=O)-N(R _m )(R _n ), where each R _m and R _n independently represents is H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl.

In some embodiments, the 2'-substituted nucleoside contains a sugar moiety containing a 2' substituent group selected from F, OCF ₃ , O-CH ₃ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O -(CH ₂ ) ₂ -ON(CH ₃ ) ₂ , -O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ and O-CH ₂ -C(=O)-N(H)CH ₃ .

In some embodiments, the 2'-substituted nucleoside contains a sugar moiety containing a 2' substituent group selected from F, O-CH ₃ and OCH ₂ CH ₂ OCH ₃ .

Some modified sugar moieties contain a bridging sugar substituent that forms a second ring, resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety contains a bridge between the 4' and 2' atoms of the furanose ring. Examples of such 4'-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'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' (cEt) and 4'-CH(CH ₂ OCH ₃ )-O-2', and analogs thereof (see, for example, US Pat. of the year); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see, for example, WO 2009/006478 published January 8, 2009); 4'-CH ₂ -N(OCH ₃ )-2' and its analogues (see, for example, WO 2008/150729 published December 11, 2008); 4'-CH ₂ -ON(CH ₃ )-2' (see, for example, US 2004/0171570 published September 2, 2004); 4'-CH ₂ -ON(R)-2' and 4'-CH ₂ -N(R)-O-2'-, where each R is independently H, a protective group or C ₁ -C ₁₂ alkyl; 4'-CH ₂ -N(R)-O-2', where R represents H, C ₁ -C ₁₂ alkyl or a protective group (see, for example, US patent 7427672 issued September 23, 2008); 4'-CH ₂ -C(H)(CH ₃ )-2' (see, for example, Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH ₂ -C(=CH ₂ )-2' and its analogs (see PCT International Application WO 2008/154401 published December 8, 2008).

In some embodiments of the invention such bridges 4'-2' independently contain from 1 to 4 linked groups, independently selected from -[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 )-;

where:

x is 0, 1, or 2;

n is 1, 2, 3 or 4;

each R _a and R _b is independently H, a protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C ₅ -C ₇ alicyclic radical, substituted C ₅ - C ₇ alicyclic radical, halogen, OJ ₁ , NJ ₁ J ₂ , SJ ₁ , N ₃ , COOJ ₁ , acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) ₂ -J ₁ ) or sulfoxyl (S(=O)-J ₁ ); and

each J ₁ and J ₂ is independently H, C ₁ -C ₁₂ alkyl substituted with C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl substituted with C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl substituted with C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, acyl (C (= O) -H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ -C ₁₂ aminoalkyl or a protective group.

Nucleosides containing bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4'-CH ₂ -O-2') BNA, (B) β-D-methyleneoxy (4'-CH ₂ -O-2' ) BNA (also referred to as closed nucleic acid or LNA), (C) ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') BNA, (D) aminooxy (4'-CH ₂ -ON(R)- 2') BNA, (E) hydroxyamino (4'-CH ₂ -N(R)-O-2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH ₃ )-O-2') BNA (also referred to as hindered ethyl or cEt), (G) methylene-thio (4'-CH ₂ -S-2') BNA, (H) methylene-amino (4'-CH ₂ -N(R)- 2') BNA, (I) methyl carbocyclic (4'-CH ₂ -CH(CH ₃ )-2') BNA and (J) propylene carbocyclic (4'-(CH ₂ ) _3-2 ') BNA, as shown below.

where Bx represents a fragment of a nitrogenous base, and R independently represents H, a protective group or C ₁ -C ₁₂ alkyl.

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. U.S.A., 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, 2001, 8, 1-7; Oram et al. Curr. Opinion Mol. Ther., 2001, 3, 239-243; US Pat. WO 2004/106356, WO 1994/14226, WO 2005/021570 and WO 2007/134181; US Patent Publication Nos. US 2004/0171570, US 2007/0287831 and US 2008/0039618; US patents with serial numbers 12/129154 60/989574 61/026995 61/026998 61/056564 61/086231 61/097787 and 61/099844; and PCT International Application Nos. PCT/US 2008/064591, PCT/US 2008/066154 and PCT/US 2008/068922.

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

In some embodiments, the substituted sugar moieties contain one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted and 4'-2'-bridged sugars), (see, for example, PCT International Application WO 2007/134181, published 11/22/07, in which the LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

In some embodiments, the modified sugar moieties are sugar substitutes. In some such embodiments, the natural sugar oxygen atom is substituted, for example, with a sulfur, carbon, or nitrogen atom. In some such embodiments, such a modified sugar moiety also contains bridging and/or non-bridging substituents, as described above. For example, some sugar substitutes contain a 4' sulfur atom and a substitution at the 2' position (see, for example, US Published Application US 2005/0130923 published June 16, 2005) and/or the 5' position. As a further example, carbocyclic bicyclic nucleosides having a 4'-2' bridge 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).

In some embodiments of the invention, sugar substitutes contain rings having more than 5 atoms. For example, in some embodiments of the invention, the sugar substitute contains morpholino. Morpholino compounds and their use in oligomeric compounds have been described in numerous patents and published articles (see, for example: Braasch et at., Biochemistry, 2002, 41, 4503-4510; and US Pat. . As used herein, the term "morpholino" means a sugar substitute having the following structure:

In some embodiments, the morpholino can be modified, for example, by adding or changing various substituent groups in the morpholino structure above. Such sweeteners are referred to herein as "modified morpholinos".

As another example, in some embodiments of the invention, the sugar substitute contains a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitolic nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. & Med. Chem. (2002 ) 10:841-854), fluoro-HNA (F-HNA) and compounds having Formula VI:

where independently for each of the specified at least one tetrahydropyran nucleoside analogue of Formula VI:

Bx is a fragment of a nitrogenous base;

T ₃ and T ₄ are each independently an internucleoside linking group linking a tetrahydropyran nucleoside analog to an antisense compound, or one of T ₃ and T ₄ is an internucleoside linking group linking a tetrahydropyran nucleoside analog to an antisense compound and the other of T ₃ and T ₄ is H, a hydroxyl protecting group, a linked conjugating group, or a 5' or 3' end group;

q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are each independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl; and

each of R ₁ and R ₂ is independently selected from: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ ₁ J ₂ , SJ ₁ , N ₃ , OC(=X)J ₁ , OC(=X)NJ ₁ J ₂ , NJ ₃ C(=X)NJ ₁ J ₂ and CN, where X represents O, S or NJ ₁ and each J ₁ , J ₂ and J ₃ independently represents H or C ₁ -C ₆ alkyl.

In some embodiments, THP-modified nucleosides of Formula VI are provided wherein each q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ is H. In some embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ are other than H. In some embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is methyl. In some embodiments, THP nucleosides of Formula VI are provided wherein one of R ₁ and R ₂ is F. In some embodiments, R ₁ is fluoro and R ₂ is H, R ₁ is methoxy and R ₂ is H and R ₁ is methoxyethoxy and R ₂ is H.

Many other bicyclic and tricyclic ring sugar substitute systems are also known in the art that can be used to modify nucleosides for insertion into antisense compounds (see, for example, the review article: Leumann, J.C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Without limitation, combinations of modifications are also given, such as 2'-F-5'-methyl-substituted nucleosides (see PCT application WO 2008/101157, published 8/21/08, which describes other 5', 2'-bis -substituted nucleosides) and replacement of the oxygen atom of the ribosyl ring with S and additional substitution at the 2'-position (see US Published Application US 2005-0130923 published June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published 11/22/07, in which the 4'-CH ₂ -O-2' bicyclic nucleoside is additionally 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).

In some embodiments of the present description, oligonucleotides containing modified nucleosides are provided. Such modified nucleotides may contain modified sugars, modified nitrogenous bases, and/or modified bonds. Specific modifications are chosen so that the resulting oligonucleotides have the desired characteristics. In some embodiments of the invention, the oligonucleotides contain one or more RNA-like nucleosides. In some embodiments of the invention, the oligonucleotides contain one or more DNA-like nucleotides.

2. Some modifications of nitrogenous bases

In some embodiments of the invention, the nucleosides of the present description contain one or more unmodified nitrogenous bases. In some embodiments of the invention, the nucleosides of the present description contain one or more modified nitrogenous bases.

In some embodiments, the modified nitrogenous bases are selected from: universal bases, hydrophobic bases, displaced bases, enlarged bases, and fluorinated bases described herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl-cytosine, 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-CH ₃ ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8 -amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 2-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, mixed bases, enlarged bases and fluorinated bases described herein. Additional modified nitrogenous bases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1 ,4]benzothiazin-2(3H)-one), the so-called G-clamp, such as substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4] benzoxazin-2(3Н)-one), carbazole cytidine (2Н-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (Н-pyrido[3',2':4,5]pyrrolo[2 ,3-d]pyrimidin-2-one). Modified nitrogenous bases may also include nitrogenous bases in which the purine or pyrimidine base has been replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nitrogenous bases include the nitrogenous bases described in US Pat. No. 3,687,808, described in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, ed., John Wiley & Sons, 1990, 858-859; described by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and described in Sanghvi, YS, chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., eds., CRC Press, 1993, 273-288.

Illustrative US patents that describe the preparation of some of the aforementioned modified nitrogenous bases, as well as other modified nitrogenous bases, include, without limitation, U.S. 3687808; 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121; 5596091; 5614617; 5645985; 5681941; 5750692; 5763588; 5,830,653 and 6,005,096, some of which are pending concurrently with this application, and each of which is incorporated herein by reference in its entirety.

3. Some internucleoside bonds

In some embodiments of the present description, oligonucleotides containing linked nucleosides are provided. In such embodiments, the nucleosides may be linked together via any internucleoside bond. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Exemplary phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters (PO), phosphotriesters, methylphosphonates, phosphoramidates, and thiophosphates (PS). Exemplary phosphorus-free internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH ₂ -N(CH ₃ )-O-CH ₂ -), thiodiether (-OC(O)-S-), thionocarbamate (-OC( O)(NH)-S-); siloxane (-O-Si(H) ₂ -O-); and N,N'-dimethylhydrazine (-CH ₂ -N(CH ₃ )-N(CH ₃ )-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, the nuclease resistance of an oligonucleotide. In some embodiments of the invention, internucleoside bonds having a chiral atom can be obtained as a racemic mixture or as individual enantiomers. Illustrative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. Methods for making phosphorus-containing and phosphorus-free internucleoside bonds are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetric centers and, therefore, can form enantiomers, diastereomers, and other stereoisomeric configurations, which, according to absolute stereochemistry, can be defined as (R) or (S), or, as for anomers sugar, or as (D) or (L), as for amino acids, etc. Antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

Neutral binding linkers include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH ₂ -N(CH ₃ )-O-5'), amide-3 (3'-CH ₂ -C(=O)-N( H)-5'), amide-4 (3'-CH ₂ -N(H)-C(=O)-5'), formaacetal (3'-O-CH ₂ -O-5') and thioformacetal ( 3'-S-CH ₂ -O-5'). Additional neutral internucleoside bonds include non-ionic bonds containing siloxane (dialkylsiloxane), carboxylic ester, carboxamide, sulfide, sulfonic ester, and amides (see, for example: Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, eds., ACS Symposium Series 580; chapters 3 and 4, 40-65). Additional neutral internucleoside bonds include non-ionic bonds containing mixed constituents of N, O, S and CH ₂ .

4. Some motives

In some embodiments, the antisense oligonucleotides contain one or more modified nucleosides (eg, a nucleoside containing a modified sugar and/or a modified nitrogenous base) and/or one or more modified internucleoside linkages. A characteristic site of such modifications in an oligonucleotide is referred to herein as a motif. In some embodiments, the sugar, nitrogenous base, and linker motifs are independent of each other.

a. Some sugar motifs

In some embodiments of the invention, the oligonucleotides contain one or more types of modified sugar fragments and/or natural sugar fragments, located along the oligonucleotide or its region in a certain way or in the form of a sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In some embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar motif that contains two outer regions or "wings" and a central or inner region or "gap". These three gapmer sugar motif regions (5'-wing, gap, and 3'-wing) form a contiguous sequence of nitrogenous bases in which at least some of the nucleoside sugar moieties in each wing differ from at least some of the nucleoside sugar moieties in the gap. In particular, at least those sugar moieties of the nucleosides of each wing that are closest to the gap (3'-nearest nucleoside of the 5'-wing and 5'-nearest nucleoside of the 3'-wing) differ from the sugar moiety of adjacent nucleosides in the gap, thus defining the boundary between the wings and the gap. In some embodiments of the invention, the sugar fragments in the gap are the same among themselves. In some embodiments, the gap contains one or more nucleosides having a sugar moiety that is different from the sugar moiety of one or more other nucleosides in the gap. In some embodiments, the sugar motifs of the two wings are the same (symmetrical sugar gapmer). In some embodiments, the 3'-wing sugar motifs are different from the 3'-wing sugar motif (asymmetric sugar gapmer).

i. Some 5'-wings

In some embodiments, the 5'-wing of the gapmer consists of 1-8 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1-7 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1-6 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1-5 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 2-5 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 3-5 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 4 or 5 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1-4 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1-3 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1 or 2 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 2-4 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 2 or 3 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 3 or 4 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 1 nucleoside. In some embodiments, the 5'-wing of the gapmer consists of 2 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 3 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 4 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 5 linked nucleosides. In some embodiments, the 5'-wing of the gapmer consists of 6 linked nucleosides.

In some embodiments, the 5'-wing of the gapmer contains at least one bicyclic nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least two bicyclic nucleosides. In some embodiments, the 5'-wing of the gapmer contains at least three bicyclic nucleosides. In some embodiments, the 5'-wing of the gapmer contains at least four bicyclic nucleosides. In some embodiments, the 5'-wing of the gapmer contains at least one hindered ethyl nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one LNA nucleoside. In some embodiments, each gapmer 5'-wing nucleoside is a bicyclic nucleoside. In some embodiments, each 5'-wing nucleoside of the gapmer is a hindered ethyl nucleoside. In some embodiments, each gapmer 5'-wing nucleoside is an LNA nucleoside.

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

In some embodiments, the 5'-wing of the gapmer contains at least one 2'-deoxynucleoside. In some embodiments, each gapmer 5'-wing nucleoside is a 2'-deoxynucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one ribonucleoside. In some embodiments of the invention, each nucleoside of the 5'-wing of the gapmer is a ribonucleoside. In some embodiments, one, more than one, or each of the 5'-wing nucleosides is an RNA-like nucleoside.

In some embodiments, the 5'-wing of the gapmer contains at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

In some embodiments, the 5'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-substituted nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-MOE nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-OMe nucleoside. In some embodiments, the 5'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-deoxynucleoside.

ii. Some 3'-wings

In some embodiments, the 3'-wing of the gapmer consists of 1-8 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1-7 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1-6 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1-5 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 2-5 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 3-5 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 4 or 5 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1-4 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1-3 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1 or 2 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 2-4 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 2 or 3 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 3 or 4 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 1 nucleoside. In some embodiments, the 3'-wing of the gapmer consists of 2 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 3 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 4 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 5 linked nucleosides. In some embodiments, the 3'-wing of the gapmer consists of 6 linked nucleosides.

In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside. In some embodiments, each gapmer 3'-wing nucleoside is a bicyclic nucleoside. In some embodiments of the invention, each nucleoside of the 3'-wing of the gapmer is a hindered ethyl nucleoside. In some embodiments, each gapmer 3'-wing nucleoside is an LNA nucleoside.

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

In some embodiments, the 3'-wing of the gapmer contains at least one 2'-deoxynucleoside. In some embodiments, each gapmer 3'-wing nucleoside is a 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one ribonucleoside. In some embodiments of the invention, each nucleoside of the 3'-wing of the gapmer is a ribonucleoside. In some embodiments, one, more than one, or each of the 5'-wing nucleosides is an RNA-like nucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-substituted nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-MOE nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-OMe nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside and at least one 2'-deoxynucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside and at least one 2'-substituted nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside and at least one 2'-MOE nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside and at least one 2'-OMe nucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside and at least one 2'-deoxynucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2'-deoxynucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside, at least one 2'-substituted nucleoside, and at least one 2'-deoxynucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside, at least one 2'-MOE nucleoside, and at least one 2'-deoxynucleoside.

In some embodiments, the 3'-wing of the gapmer contains at least one bicyclic nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one hindered ethyl nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside. In some embodiments, the 3'-wing of the gapmer contains at least one LNA nucleoside, at least one 2'-OMe nucleoside, and at least one 2'-deoxynucleoside.

iii. Some central regions (gaps)

In some embodiments, the gap gapmer consists of 6-20 linked nucleosides. In some embodiments, the gap gapmer consists of 6-15 linked nucleosides. In some embodiments, the gap gapmer consists of 6-12 linked nucleosides. In some embodiments, the gap gapmer consists of 6-10 linked nucleosides. In some embodiments, the gap gapmer consists of 6-9 linked nucleosides. In some embodiments, the gap gapmer consists of 6-8 linked nucleosides. In some embodiments, the gap gapmer consists of 6 or 7 linked nucleosides. In some embodiments, the gap gapmer consists of 7-10 linked nucleosides. In some embodiments, the gap gapmer consists of 7-9 linked nucleosides. In some embodiments, the gap gapmer consists of 7 or 8 linked nucleosides. In some embodiments, the gap gapmer consists of 8-10 linked nucleosides. In some embodiments, the gap gapmer consists of 8 or 9 linked nucleosides. In some embodiments, the gap gapmer consists of 6 linked nucleosides. In some embodiments, the gap gapmer consists of 7 linked nucleosides. In some embodiments, the gap gapmer consists of 8 linked nucleosides. In some embodiments, the gap gapmer consists of 9 linked nucleosides. In some embodiments, the gap gapmer consists of 10 linked nucleosides. In some embodiments, the gap gapmer consists of 11 linked nucleosides. In some embodiments, the gap gapmer consists of 12 linked nucleosides.

In some embodiments, each nucleoside of the gap gapmer is a 2'-deoxynucleoside. In some embodiments, the gap contains one or more modified nucleosides. In some embodiments, each gap gapmer nucleoside is a 2'-deoxynucleoside or is a modified "DNA-like" nucleoside. In such embodiments, "DNA-like" means that the nucleoside has the same characteristics as DNA, i.e. that a duplex containing a gapmer and an RNA molecule can activate RNase H. For example, it has been shown that, under certain conditions, 2'- (ara)-F supports RNase H activation and is therefore DNA-like. In some embodiments, one or more gap gapmer nucleosides is not a 2'-deoxynucleoside and is not DNA-like. In some such embodiments, the gapmer nevertheless maintains RNase H activation (eg, through the number or position of non-DNA nucleosides).

In some embodiments, the gaps contain a region of an unmodified 2'-deoxynucleoside interrupted by one or more modified nucleosides, resulting in three subregions (two regions of one or more 2'-deoxynucleosides and a region of one or more interrupting modified nucleosides). In some embodiments, no portion of the unmodified 2'-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In some embodiments of the invention, such short sections are formed by using short gap regions. In some embodiments of the invention, short sections are formed by interrupting a longer gap region.

In some embodiments, the gap contains one or more modified nucleosides. In some embodiments, the gap contains one or more modified nucleosides selected from cEt, FHNA, LNA, and 2-thiothymidine. In some embodiments, the gap contains a single modified nucleoside. In some embodiments, the gap contains a 5'-substituted sugar moiety selected from 5'-Me and 5'-(R)-Me. In some embodiments, the gap contains two modified nucleosides. In some embodiments, the gap contains three modified nucleosides. In some embodiments, the gap contains four modified nucleosides. In some embodiments, the gap contains two or more modified nucleosides, and each modified nucleoside is the same. In some embodiments, the gap contains two or more modified nucleosides, and each modified nucleoside is different.

In some embodiments, the gap contains one or more modified bonds. In some embodiments of the invention, the gap contains one or more methylphosphonate linkages. In some embodiments, the gap contains two or more modified bonds. In some embodiments, the gap contains one or more modified bonds and one or more modified nucleosides. In some embodiments, the gap contains one modified bond and one modified nucleoside. In some embodiments, the gap contains two modified bonds and two or more modified nucleosides.

b. Some motifs of internucleoside bonds

In some embodiments of the invention, the oligonucleotides contain modified internucleoside bonds located along the oligonucleotide or its region in a certain way or in the form of a modified internucleoside bond motif. In some embodiments of the invention, the oligonucleotides contain a region having a motif of alternating internucleoside bonds. In some embodiments of the invention, the oligonucleotides according to the present description contain a region of identically modified internucleoside bonds. In some such embodiments, the oligonucleotide contains a region that is uniformly linked by thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide is uniformly linked by thiophosphate internucleoside bonds. In some embodiments of the invention, each internucleoside bond of the oligonucleotide is selected from a phosphodiester and a thiophosphate. In some embodiments, each internucleoside bond of the oligonucleotide is selected from a phosphodiester and a thiophosphate, and at least one internucleoside bond is a thiophosphate.

In some embodiments of the invention, the oligonucleotide contains at least 6 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 7 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 8 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 9 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 10 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 11 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 12 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 13 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least 14 thiophosphate internucleoside bonds.

In some embodiments of the invention, the oligonucleotide contains at least one block of at least 6 adjacent thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least one block of at least 7 adjacent thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least one block of at least 8 adjacent thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least one block of at least 9 adjacent thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least one block of at least 10 adjacent thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains at least one block of at least 12 adjacent thiophosphate internucleoside bonds. In some such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In some such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide. In some embodiments of the invention, the oligonucleotide contains less than 15 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 14 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 13 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 12 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 11 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 10 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 9 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 8 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 7 thiophosphate internucleoside bonds. In some embodiments of the invention, the oligonucleotide contains less than 6 thiophosphate internucleoside bonds. In some embodiments, the oligonucleotide contains less than 5 thiophosphate internucleoside bonds.

With. Some motives for the modification of nitrogenous bases

In some embodiments of the invention, the oligonucleotides contain chemical modifications of nitrogenous bases, located along the oligonucleotide or its region in a certain way or in the form of a modification of nitrogenous bases. In some such embodiments, the nitrogenous base modifications are located in a broken motif. In some embodiments of the invention, modifications of nitrogenous bases are located in an alternating motif. In some embodiments of the invention, each nitrogenous base is modified. In some embodiments of the invention, none of the nitrogenous bases is chemically modified.

In some embodiments of the invention, the oligonucleotides contain a block of modified nitrogenous bases. In some such embodiments, the block is located at the 3' end of the oligonucleotide. In some embodiments, the block is within 3 nucleotides of the 3' end of the oligonucleotide. In some such embodiments, the block is located at the 5' end of the oligonucleotide. In some embodiments, the block is within 3 nucleotides of the 5' end of the oligonucleotide.

In some embodiments of the invention, modifications of nitrogenous bases depend on the natural base at a particular position of the oligonucleotide. For example, in some embodiments of the invention, each purine or each pyrimidine in the oligonucleotide is modified. In some embodiments of the invention, each adenine is modified. In some embodiments of the invention, each guanine is modified. In some embodiments, each thymine is modified. In some embodiments of the invention, each cytosine is modified. In some embodiments of the invention, each uracil is modified.

In some embodiments of the invention, some, all or none of the cytosine fragments in the oligonucleotide are not 5-methylcytosine fragments. As used herein, 5-methylcytosine is not a "modified nitrogenous base". Accordingly, unless otherwise indicated, unmodified nitrogenous bases include both cytosine residues containing 5-methyl and those that do not contain 5-methyl. In some embodiments, the methylation state of all or some of the cytosine nitrogenous bases is specified.

In some embodiments of the invention, chemical modifications of the nitrogenous bases include the addition of certain conjugating groups to the nitrogenous bases. In some embodiments of the invention, each purine or each pyrimidine in the oligonucleotide may be optionally modified to contain a conjugating group.

d. Some common lengths

In some embodiments of the present disclosure, oligonucleotides are provided in any of various length ranges. In some embodiments, the oligonucleotides contain X to Y linked nucleosides, where X is the fewest nucleosides in the range and Y is the most nucleosides in the range. In some such embodiments, each X and Y is independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50; provided that X≤Y. For example, in some embodiments of the invention, the oligonucleotide may consist of 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18 , 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9 -10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22 , 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10 -15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27 , 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11 -21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15 , 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12 -28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23 , 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14 -20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17 , 15-18, 15-1 9, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16- 30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18- 30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21- 25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24- 29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30 or 29-30 linked nucleosides. In those embodiments in which the number of nucleosides in the oligonucleotide of the compound is limited, either to a range or to a certain number, the compound may, however, additionally contain other additional substituents. For example, an oligonucleotide containing 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but unless otherwise indicated, such an oligonucleotide may additionally contain, for example, one or more conjugating groups, end groups, or other substituents.

Moreover, if an oligonucleotide is described by a total length range and regions having a specific length, and if the sum of said region lengths is less than the upper limit of the total length range, then the oligonucleotide may have additional nucleosides in addition to those in the indicated regions, provided that the total number of nucleosides does not exceed the upper limit of the total length range.

5. Some chemical motifs of antisense oligonucleotides

In some embodiments, the chemical structural features of antisense oligonucleotides are described by their sugar motif, internucleoside bond motif, nitrogenous base modification motif, and overall length. In some embodiments of the invention, such parameters are independent of each other. Thus, each internucleoside bond of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not repeat the pattern of modification of the gapmer sugar modifications. Therefore, the internucleoside bonds in the wing regions of the gapmer sugar may be the same or different from each other and may be the same or different from the internucleoside bonds of the gap region. Similarly, such sugar gapmer oligonucleotides may contain one or more modified nitrogenous bases, regardless of the nature of the gapmer sugar modifications. Those skilled in the art will recognize that such motifs can be combined to form multiple oligonucleotides.

In some embodiments of the invention, the choice of internucleoside bond and modification of the nucleoside are independent of each other.

i. Some sequences and targets

In some embodiments of the present invention, antisense oligonucleotides are provided having a sequence complementary to the target nucleic acid. Such antisense compounds can hybridize to a target nucleic acid to produce at least one antisense activity. In some embodiments, the antisense compounds specifically hybridize to one or more target nucleic acids. In some embodiments, a specifically hybridizing antisense compound has a nitrogenous base sequence containing a region with sufficient complementarity to the target nucleic acid to allow hybridization and obtain antisense activity, and insufficient complementarity to any non-target nucleic acid to prevent or reduce non-specific hybridization with non-target nucleic acid sequences in conditions under which specific hybridization is required (eg, under physiological conditions for in vivo or therapeutic applications, and under assay conditions for in vitro assays). In some embodiments of the invention, the oligonucleotides are selective between target and non-target, even if the target and non-target contain the target sequence. In such embodiments, selectivity may result from the relative availability of the target region of one nucleic acid molecule over another.

In some embodiments of the present description, antisense compounds are provided that contain oligonucleotides that are fully complementary to the target nucleic acid along the entire length of the oligonucleotide. In some embodiments, the oligonucleotides are 99% complementary to the target nucleic acid. In some embodiments, the oligonucleotides are 95% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.

In some embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In some embodiments, the antisense compound contains 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 some such embodiments, the region of complete complementarity is 6 to 14 nucleosides in length.

In some embodiments of the invention, the oligonucleotides contain a hybridization region and a terminal region. In some such embodiments, the hybridization region consists of 12-30 linked nucleosides and is fully complementary to the target nucleic acid. In some embodiments, the hybridization region contains one mismatch compared to the target nucleic acid. In some embodiments of the invention, the hybridization region contains two mismatches in comparison with the target nucleic acid. In some embodiments of the invention, the hybridization region contains three mismatches in comparison with the target nucleic acid. In some embodiments of the invention, the terminal region consists of 1-4 terminal nucleosides. In some embodiments, the terminal nucleosides are at the 3' end. In some embodiments of the invention, one or more terminal nucleosides are not complementary to the target nucleic acid.

Antisense mechanisms include any mechanism that involves hybridization of an oligonucleotide to a target nucleic acid such that the hybridization results in a biological effect. In some embodiments of the invention, such hybridization results in either the destruction or use of the target nucleic acid with concomitant suppression or stimulation of a cellular mechanism affecting, for example, translation, transcription, or splicing of the target nucleic acid.

One type of antisense mechanism affecting target RNA degradation is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA helix of an RNA:DNA duplex. Single-stranded antisense compounds that are "DNA-like" are known in the art to induce RNase H activity in mammalian cells. Therefore, activation of RNase H leads to cleavage of the target RNA, thereby greatly enhancing the efficiency of gene expression suppression mediated by the DNA-like oligonucleotide.

In some embodiments of the invention, the conjugating group contains a cleavable fragment. In some embodiments of the invention, the conjugating group contains one or more cleavable bonds. In some embodiments of the invention, the conjugating group contains a linker. In some embodiments of the invention, the linker contains a fragment that binds proteins. In some embodiments, the conjugating group contains a cell-targeting moiety (also referred to as a cell-targeting group). In some embodiments of the invention, the cell-targeting fragment contains a branching group. In some embodiments of the invention, the cell-targeting fragment contains one or more ligaments. In some embodiments of the invention, the fragment targeting the cell contains a carbohydrate or carbohydrate cluster.

ii. Some Split Fragments

In some embodiments of the invention, the cleavable fragment is a cleavable bond. In some embodiments, the cleavable fragment contains a cleavable bond. In some embodiments of the invention, the conjugating group contains a cleavable fragment. In some such embodiments, the cleavage fragment is attached to an antisense oligonucleotide. In some such embodiments, the cleavable fragment is attached directly to the cell-targeting fragment. In some such embodiments, the cleavable moiety is attached to a conjugate linker. In some embodiments, the cleavable fragment contains a phosphate or phosphodiester. In some embodiments, the cleavable fragment is a cleavable nucleoside or nucleoside analogue. In some embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from purine, substituted purine, pyrimidine, or substituted pyrimidine. In some embodiments, the cleavable fragment is a nucleoside containing an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N -benzoyladenine, guanine and 2-N-isobutyrylguanine. In some embodiments, the cleavable moiety is a 2'-deoxynucleoside that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester bond and attached to the linker by a phosphodiester or thiophosphate bond. In some embodiments, the cleavable fragment is a 2'-deoxyadenosine that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester bond and attached to the linker by a phosphodiester or thiophosphate bond. In some embodiments, the cleavable fragment is a 2'-deoxyadenosine that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester bond and attached to the linker by a phosphodiester bond.

In some embodiments, the cleavable fragment is attached to the 3' position of the antisense oligonucleotide. In some embodiments, the cleavable fragment is attached to the 5' position of the antisense oligonucleotide. In some embodiments, the cleavable fragment is attached to the 2' position of the antisense oligonucleotide. In some embodiments, the cleavable fragment is attached to the antisense oligonucleotide by a phosphodiester linkage. In some embodiments of the invention, the cleavable fragment is attached to the specified linker either by a phosphodiester or a thiophosphate bond. In some embodiments of the invention, the cleavable fragment is attached to the specified linker by a phosphodiester bond. In some embodiments of the invention, the conjugating group does not contain a cleavable fragment.

In some embodiments of the invention, the cleavable fragment is cleaved after the introduction of the specified complex in the body of the animal only after its absorption by the target cell. Inside the cell, the cleavage fragment is cleaved, thus releasing the active antisense oligonucleotide. Without being limited by theory, it is believed that the cleavage fragment is cleaved by one or more nucleases within the cell. In some embodiments, one or more nucleases cleave the phosphodiester bond between the cleavable moiety and the linker. In some embodiments of the invention, the cleavable fragment has a structure selected from the following:

where each of Bx, Bx ₁ , Bx ₂ and Bx ₃ independently represents a heterocyclic basic fragment. In some embodiments of the invention, the cleavable fragment has a structure selected from the following:

iii. Some linkers

In some embodiments of the invention, the conjugated groups contain a linker. In some such embodiments, the linker is covalently linked to the cleavable moiety. In some such embodiments, the linker is covalently linked to the antisense oligonucleotide. In some embodiments, the linker is covalently linked to the cell-targeting moiety. In some embodiments of the invention, the linker further comprises a covalent attachment to a solid support. In some embodiments of the invention, the linker further comprises a covalent attachment to the protein binding fragment. In some embodiments of the invention, the linker further comprises a covalent attachment to a solid support and further comprises a covalent attachment to the protein binding moiety. In some embodiments of the invention, the linker contains several positions for attaching associated ligands. In some embodiments of the invention, the linker contains multiple positions for attaching bound ligands and is not attached to a branching group. In some embodiments of the invention, the linker further comprises one or more cleavable bonds. In some embodiments of the invention, the conjugating group does not contain a linker.

In some embodiments of the invention, the linker contains at least one linear group containing groups selected from alkyl, amide, disulfide, polyethylene glycol, thioether (-S-) and hydroxylamino (-O-N(H)-) groups. In some embodiments of the invention, the linear group contains groups selected from alkyl, amide and ether groups. In some embodiments of the invention, the linear group contains groups selected from alkyl and ether groups. In some embodiments of the invention, the linear group contains at least one phosphorus linking group. In some embodiments of the invention, the linear group contains at least one phosphodiester group. In some embodiments of the invention, the linear group contains at least one neutral linking group. In some embodiments, the linear group is covalently attached to the cell-targeting moiety and to the cleavable moiety. In some embodiments, the linear group is covalently attached to the cell-targeting moiety and to the antisense oligonucleotide. In some embodiments, the linear group is covalently attached to the cell-targeting moiety, to the cleavable moiety, and to the solid support. In some embodiments, the linear group is covalently attached to the cell-targeting moiety, to the cleavable moiety, to the solid support, and to the protein binding moiety. In some embodiments of the invention, the linear group contains one or more cleavable bonds.

In some embodiments of the invention, the linker contains a linear group covalently attached to the backbone group. In some embodiments, the backbone contains a branched aliphatic group that contains groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In some embodiments, the backbone contains a branched aliphatic group that contains groups selected from alkyl, amide, and ether groups. In some embodiments of the invention, the backbone contains at least one mono- or polycyclic ring system. In some embodiments, the backbone contains at least two mono- or polycyclic ring systems. In some embodiments, the linear group is covalently attached to the backbone group and the backbone group is covalently attached to the cleavable moiety and the linker. In some embodiments, the linear group is covalently attached to the backbone group and the backbone group is covalently attached to the cleavable moiety, the linker, and the solid support. In some embodiments, the linear group is covalently attached to the backbone group, and the backbone group is covalently attached to the cleavable moiety, the linker, and the protein binding moiety. In some embodiments, the linear group is covalently attached to the backbone group and the backbone group is covalently attached to the cleavable moiety, the linker, the protein binding moiety, and the solid support. In some embodiments, the backbone group contains one or more cleavable bonds.

In some embodiments of the invention, the linker contains a fragment that binds proteins. In some embodiments, the protein-binding moiety is a lipid, such as, for example, including but not limited to, cholesterol, cholic acid, adamantane-acetic acid, 1-pyrene-butyric acid, dihydrotestosterone, 1,3-bis -O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine , vitamin (eg, folate, vitamin A, vitamin E, biotin, pyridoxal), peptide, carbohydrate (eg, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), endosomolytic component, steroid (eg, uvaol, hecigenin, diosgenin) , a terpene (eg, a triterpene, eg, sarsasapogenin, friedelin, lithocholic acid derivatized with epifridelanol), or a cationic lipid. In some embodiments, the protein-binding moiety is a C16 to C22 saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane, or 1-pentafluoropropyl.

In some embodiments of the invention, the linker has a structure selected from:

where each n is independently 1 to 20; and p is between 1 and 6.

In some embodiments of the invention, the linker has a structure selected from:

where each n is independently between 1 and 20.

In some embodiments of the invention, the linker has a structure selected from:

where n is between 1 and 20.

In some embodiments of the invention, the linker has a structure selected from:

where each L is independently a phosphorus linking group

or a neutral linking group; and

each n is independently 1 to 20.

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments of the invention, the linker has a structure selected from:

where n is between 1 and 20.

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments, the conjugate linker has the structure:

In some embodiments, the conjugate linker has the structure:

In some embodiments of the invention, the linker has a structure selected from:

In some embodiments of the invention, the linker has a structure selected from:

where each n is independently 0, 1, 2, 3, 4, 5, 6, or 7.

iv. Some fragments targeting a cell

In some embodiments of the invention, the conjugating groups contain fragments that target the cell. Some of these cell-targeting moieties increase cellular uptake of antisense compounds. In some embodiments, cell-targeting moieties comprise a branch group, one or more linkages, and one or more ligands. In some embodiments, cell-targeting moieties comprise a branch group, one or more ligaments, one or more ligands, and one or more cleavable bonds.

1. Some branch groups

In some embodiments of the invention, the conjugating groups contain a targeting fragment containing a branching group and at least two linked ligands. In some embodiments, a branching group attaches a conjugate linker. In some embodiments, the branching group attaches a cleavable fragment. In some embodiments, the branching group attaches an antisense oligonucleotide. In some embodiments of the invention, the branch group is covalently attached to the linker and each of the associated ligands. In some embodiments, the branching group contains a branched aliphatic group that contains groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In some embodiments, the branching group contains groups selected from alkyl, amide, and ether groups. In some embodiments, the branch group contains groups selected from alkyl and ether groups. In some embodiments of the invention, the branch group contains a mono- or polycyclic ring system. In some embodiments, the branch group contains one or more cleavable bonds. In some embodiments of the invention, the conjugating group does not contain a branching group.

In some embodiments, the branch group has a structure selected from:

where each n is independently 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In some embodiments, the branch group has a structure selected from:

where each n is independently 1 to 20; and

m is from 2 to 6.

In some embodiments, the branch group has a structure selected from:

In some embodiments, the branch group has a structure selected from:

where each A ₁ independently represents O, S, C=O or NH; and

each n is independently 1 to 20.

In some embodiments, the branch group has a structure selected from:

where each A ₁ independently represents O, S, C=O or NH; and

each n is independently 1 to 20.

In some embodiments, the branch group has a structure selected from:

where A ₁ represents O, S, C=O or NH; and

each n is independently 1 to 20.

In some embodiments, the branch group has a structure selected from:

In some embodiments, the branch group has a structure selected from:

In some embodiments, the branch group has a structure selected from:

2. Some links

In some embodiments of the invention, the conjugating groups contain one or more ligaments covalently attached to the branching group. In some embodiments of the invention, the conjugating groups contain one or more ligaments covalently attached to the linking group. In some embodiments of the invention, each linkage is a linear aliphatic group containing one or more groups selected from alkyl, ether, thioether, disulfide, amide, and polyethylene glycol groups in any combination. In some embodiments, each linkage is a linear aliphatic group containing one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester, and polyethylene glycol groups in any combination. In some embodiments of the invention, each linkage is a linear aliphatic group containing one or more groups selected from alkyl, ether and amide groups in any combination. In some embodiments, each linkage is a linear aliphatic group containing one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether, and amide groups in any combination. In some embodiments of the invention, each linkage is a linear aliphatic group containing one or more groups selected from alkyl and phosphodiester in any combination. In some embodiments of the invention, each link contains at least one phosphorus linking group or a neutral linking group.

In some embodiments, the bond contains one or more cleavable bonds. In some embodiments, the linkage is attached to the branching group through an amide or ether group. In some embodiments, the linkage is attached to the branching group via a phosphorodiester group. In some embodiments, the linkage is attached to the branching group via a phosphorus linking group or via a neutral linking group. In some embodiments, the linkage is attached to the branching group via an ether group. In some embodiments, the linkage is attached to the ligand through either an amide or an ether group. In some embodiments, the linkage is attached to the ligand through an ether group. In some embodiments, the linkage is attached to the ligand through either an amide or an ether group. In some embodiments, the linkage is attached to the ligand through an ether group.

In some embodiments of the invention, each bond has a length of from about 8 to about 20 atoms in the chain between the ligand and the branch group. In some embodiments of the invention, each bond has a length of from about 10 to about 18 atoms in the chain between the ligand and the branch group. In some embodiments of the invention, each bond has a length of about 13 atoms in the chain.

In some embodiments of the invention, the ligament has a structure selected from:

where each n is independently 1 to 20; and

each p is between 1 and about 6.

In some embodiments of the invention, the ligament has a structure selected from:

In some embodiments of the invention, the ligament has a structure selected from:

where each n is independently between 1 and 20.

In some embodiments of the invention, the ligament has a structure selected from:

where L is either a phosphorus linking group or a neutral linking group;

Z ₁ is C(=O)OR ₂ ;

Z ₂ represents H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl;

R ₂ is H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl; and

each m ₁ is independently 0 to 20, with at least one m ₁ greater than 0 for each bundle.

In some embodiments of the invention, the ligament has a structure selected from:

In some embodiments of the invention, the ligament has a structure selected from:

where Z ₂ represents H or CH ₃ ; and

each m ₁ is independently 0 to 20, with at least one m ₁

greater than 0 for each bundle.

In some embodiments of the invention, the ligament has a structure selected from:

или

где каждый n независимо равен 0, 1, 2, 3, 4, 5, 6 или 7

or

where each n is independently 0, 1, 2, 3, 4, 5, 6, or 7

In some embodiments of the invention, the ligament contains a phosphorus linking group. In some embodiments of the invention, the ligament does not contain any amide bond. In some embodiments of the invention, the ligament contains a phosphorus linking group and does not contain any amide bond.

3. Some ligands

In some embodiments of the present disclosure, ligands are provided, with each ligand covalently attached to the ligament. In some embodiments, each ligand is selected to have affinity for at least one type of receptor on the target cell. In some embodiments, the ligands are selected to have affinity for at least one type of receptor on the surface of a mammalian liver cell. In some embodiments, the ligands are selected to have affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In some embodiments of the invention, each ligand is a carbohydrate. In some embodiments, each ligand is independently selected from galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose. In some embodiments of the invention, each ligand is N-acetylgalactosamine (GalNAc). In some embodiments of the invention, the targeting fragment contains 2-6 ligands. In some embodiments of the invention, the targeting fragment contains 3 ligands. In some embodiments of the invention, the targeting fragment contains 3 N-acetylgalactosamine ligands.

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

In some embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. In some embodiments of the invention "N-acetylgalactosamine" refers to 2-(acetylamino)-2-deoxy-B-galactopyranose. In some embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. In some embodiments, "GalNac" or "Gal-NAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, which includes the β form: 2-(acetylamino)-2-deoxy-β-D -galactopyranose, and α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose. In some embodiments, both β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose can be used interchangeably. Accordingly, structures in which one form is depicted are intended to include the other form as well. For example, if a structure is shown for the α-form: 2-(acetylamino)-2-deoxy-D-galactopyranose, this structure is understood to include the other form as well. In some preferred embodiments, the β-form of 2-(acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

2-(Acetyl amino)-2-deoxy-D-galactopyranose

2-(Acetylamino)-2-deoxy-β-D-galactopyranose

2-(Acetylamino)-2-deoxy-α-D-galactopyranose

In some embodiments, one or more ligands have a structure selected from:

where each R ₁ is selected from OH and NHCOOH.

In some embodiments, one or more ligands have a structure selected from:

In some embodiments, one or more ligands have a structure selected from:

In some embodiments, one or more ligands have a structure selected from:

i. Some conjugates

In some embodiments of the invention, the conjugating groups contain the structural features presented above. In some such embodiments, the conjugating groups have the following structure:

where each n is independently between 1 and 20.

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

where each n is independently 1 to 20;

Z is H or a bonded solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic basic fragment.

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some embodiments of the invention, the conjugates do not contain pyrrolidine.

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some such embodiments, the conjugating groups have the following structure:

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted bond of six to eleven consecutively bonded atoms.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted bond of ten consecutively bonded atoms.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted linkage of four x-eleven successively linked atoms, and while the specified link contains only one amide bond.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where Y and Z are independently selected from a C ₁ -C ₁₂ substituted or unsubstituted alkyl, alkenyl or alkynyl group or a group containing an ester, ketone, amide, ester, carbamate, amine, piperidine, phosphate, phosphodiester, thiophosphate, triazole, pyrrolidine, disulfide or thioether.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where Y and Z are independently selected from C ₁ -C ₁₂ substituted or unsubstituted alkyl group or a group containing exactly one ester or exactly two esters, amide, amine, piperidine, phosphate, phosphodiester or thiophosphate.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where Y and Z are independently selected from C ₁ -C ₁₂ substituted or unsubstituted alkyl group.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where the type is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where m is 4, 5, 6, 7 or 8 and n is 1, 2, 3 or 4.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted bunch of four to thirteen consecutive atoms, and while X does not contain an ester group.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted bunch of eight consecutive atoms, and while X does not contain an ester group.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted linkage of four to thirteen successively linked atoms, and while the specified linkage contains only one amide bond, and X does not contain an ester group.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where X is a substituted or unsubstituted linkage of four x-thirteen successively linked atoms, and while the specified linkage consists of an amide bond and a substituted or unsubstituted C ₂ -C ₁₁ alkyl group.

In some embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where Y is selected from a C ₁ -C ₁₂ substituted or unsubstituted alkyl, alkenyl or alkynyl group or a group containing an ether, ketone, amide, ester, carbamate, amine, piperidine, phosphate, phosphodiester, thiophosphate, triazole, pyrrolidine, disulfide or thioether .

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where Y is selected from C ₁ -C ₁₂ substituted or unsubstituted alkyl group or a group containing an ether, amine, piperidine, phosphate, phosphodiester or thiophosphate.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where Y is selected from C ₁ -C ₁₂ substituted or unsubstituted alkyl group.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In some such embodiments, the cell-targeting moiety of the conjugating group has the following structure:

where n is 4, 5, 6, 7 or 8.

b. Certain conjugated antisense compounds

C is a conjugate linker D is a branch group

In some embodiments, the conjugates are linked to the nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside. In some embodiments, the conjugated antisense compound has the following structure:

where

A is an antisense oligonucleotide;

B is a cleavable fragment

C is the conjugate linker

D is a branch group

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In some embodiments, the conjugated antisense compound has the following structure:

where

A is an antisense oligonucleotide;

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In some such embodiments, the conjugate linker contains at least one cleavable bond.

In some such embodiments, the branch group contains at least one cleavable bond.

In some embodiments of the invention, each ligament contains at least one cleavable bond.

In some embodiments, the conjugates are linked to the nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside.

In some embodiments, the conjugated antisense compound has the following structure:

where

A is an antisense oligonucleotide;

B is a cleavable fragment

C is the conjugate linker

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In some embodiments, the conjugates are linked to the nucleoside of the antisense oligonucleotide at the 2', 3', or 5' position of the nucleoside. In some embodiments, the conjugated antisense compound has the following structure:

where

A is an antisense oligonucleotide;

C is the linker of the conjugate

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In some embodiments, the conjugated antisense compound has the following structure:

where

A is an antisense oligonucleotide;

B is a cleavable fragment

D is a branch group

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In some embodiments, the conjugated antisense compound has the following structure:

where

A is an antisense oligonucleotide;

D is a branch group

each E is a bunch;

each F is a ligand; and

q is an integer from 1 to 5.

In some such embodiments, the conjugate linker contains at least one cleavable bond.

In some embodiments of the invention, each ligament contains at least one cleavable bond.

In some embodiments, the conjugated antisense compound has a structure selected from the following:

In some embodiments, the conjugated antisense compound has a structure selected from the following:

In some embodiments, the conjugated antisense compound has a structure selected from the following:

Illustrative United States Patents, United States Patent Application Publications, and International Patent Application Publications that describe the preparation of some of the above conjugates, conjugated antisense compounds, linkages, linkers, branching groups, ligands, cleavable moieties, and other modifications, include , without limitation, US 5994517, US 6300319, US 6660720, US 6906182, US 7262177, US 7491805, US 8106022, US 7723509, US 2006/0148740, US 2011/0123520, WO 2013/02032 each of WO 2013/02032 which is incorporated herein by reference in its entirety.

Illustrative publications that describe the preparation of some of the above conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties, and other modifications include, without limitation, BIESSEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent" J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., "Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor" J. Med. Chem. (1995) 38:1538-1546, LEE et al., "New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes" Bioorganic & Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo" J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of 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), 159-110, each of which is included herein by reference in its entirety.

In some embodiments, the conjugated antisense compounds comprise an RNase H-based oligonucleotide (such as a gapmer) or a splice-modulating oligonucleotide (such as a fully modified oligonucleotide) and any conjugation group containing at least one, two, or three GalNAc groups. In some embodiments, the conjugated antisense compound contains any of the conjugating groups listed in 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., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al, Bioorg Med Chem, 200J, 16, 5216-5231; Lee et al., BioorgMed 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 Vase 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., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al, Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO 1998/013381; W02011/038356; WO 1997/046098; W02008/098788; WO2004/101619; W02012/037254; WO 2011/120053; WO 2011/100131; WO 2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; W02012/083046; W02009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; W02010/088537; W02002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; W02012/089352; W02012/089602; WO 2013/166121; WO 2013/165816; US Patents 4,751,219; 8552163; 6908903; 7262177; 5994517; 6300319; 8106022; 7491805; 7491805; 7582744; 8137695; 6383812; 6525031; 6660720; 7723509; 8541548; 8344125; 8313772; 8349308; 8450467; 8501930; 8158601; 7262177; 6906182; 6620916; 8435491; 8404862; 7851615; published US patent applications US 2011/0097264; US 2011/0097265; US 2013/0004427; US 2005/0164235; US 2006/0148740; US 2008/0281044; US 2010/0240730; US 2003/0119724; US 2006/0183886; US 2008/0206869; US 2011/0269814; US 2009/0286973; US 2011/0207799; US 2012/0136042; US 2012/0165393; US 2008/0281041; US 2009/0203135; US 2012/0035115; US 2012/0095075; US 2012/0101148; US 2012/0128760; US 2012/0157509; US 2012/0230938; US 2013/0109817; US 2013/0121954; US 2013/0178512; US 2013/0236968; US 2011/0123520; US 2003/0077829; US 2008/0108801; and US 2009/0203132; each of which is incorporated by reference in its entirety.

C.Some applications and features

In some embodiments, the conjugated antisense compounds exhibit effective reduction of the target RNA in vivo. In some embodiments of the invention, unconjugated antisense compounds accumulate in the kidney. In some embodiments, the conjugated antisense compounds accumulate in the liver. In some 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 involved in metabolic, cardiovascular, and other diseases, disorders, or pathological conditions. Thus, provided herein are methods of treating such diseases, disorders, or conditions by contacting liver tissues with conjugated antisense compounds that target RNAs associated with such diseases, disorders, or conditions. Therefore, methods for improving any of a variety of metabolic, cardiovascular, and other diseases, disorders, or conditions using the conjugated antisense compounds of the present invention are also provided.

In some embodiments of the invention, conjugated antisense compounds are more effective than unconjugated analogs at a certain tissue concentration. Without being limited by any theory or mechanism, in some embodiments of the invention, the conjugate may allow more efficient entry of the conjugated antisense compound into the cell, or the possibility of more productive entry into the cell. For example, in some embodiments, the conjugated antisense compounds may exhibit greater target reduction than the unconjugated counterpart, with both the conjugated antisense compound and its unconjugated analogue present in tissue at the same concentrations. For example, in some embodiments, conjugated antisense compounds may exhibit greater target reduction than their unconjugated counterparts, with both the conjugated antisense compound and its unconjugated analogue present in the liver at the same concentrations.

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

In some embodiments, the conjugating groups described herein may further enhance efficacy by increasing the affinity of the conjugated antisense compound for a particular cell or tissue type. In some embodiments, the conjugating groups described herein may further enhance efficacy by increasing recognition of the conjugated antisense compound by one or more cell surface receptors. In some embodiments, the conjugating groups described herein may further enhance efficacy by facilitating endocytosis of the conjugated antisense compound.

In some embodiments, the cleavable fragment may further enhance efficiency by allowing the conjugate from the antisense oligonucleotide to be cleaved after the conjugated antisense compound enters the cell. Accordingly, in some embodiments, the conjugated antisense compounds may be administered at lower doses than needed for the unconjugated antisense oligonucleotides.

Previously, thiophosphate bonds have already been introduced into antisense oligonucleotides. Such thiophosphate bonds are nuclease resistant and thereby improve the stability of the oligonucleotide. In addition, thiophosphate bonds also bind certain proteins, leading to accumulation of the antisense oligonucleotide in the liver. Oligonucleotides with fewer thiophosphate bonds accumulate less in the liver and more in the kidneys (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; and Pharmacological Properties of 2'-O-Methoxyethyl Modified Oligonucleotides in Antisense a Drug Technology, chapter 10, Crooke, S.T., ed., 2008). In some embodiments of the invention, oligonucleotides with fewer thiophosate internucleoside bonds and with more phosphodiester internucleoside bonds accumulate less in the liver and more in the kidneys. This is undesirable in the treatment of liver disease for several reasons: (1) less drug reaches the site of desired action (liver); (2) the drug is excreted in the urine; (3) the kidneys are exposed to a relatively high concentration of the drug, which can lead to toxicity in the kidneys. Therefore, for liver diseases, thiophosate bonds provide an important advantage.

However, in some embodiments of the invention, the introduction of oligonucleotides, uniformly linked by thiophosate internucleoside bonds, causes one or more pro-inflammatory reactions. (See, for example: J Lab Clin Med. 1996 Sep; 128(3):329-38. "Amplification of antibody production by phosphorothioate oligodeoxynucleotides", Branda et al.; and see also, for example: Toxicologic Properties in Antisense a Drag Technology, chapter 12, pages 342-351, Crooke, S.T., ed., 2008). In some embodiments of the invention, the introduction of oligonucleotides, in which most of the internucleoside bonds contains thiophosphate internucleoside bonds, causes one or more pro-inflammatory reactions.

In some embodiments, the degree of pro-inflammatory effect may depend on several variables (e.g., skeletal modification, off-target, nitrogenous base modifications, and/or nucleoside modifications), see, for example: Toxicologic Properties in Antisense a Drug Technology, chapter 12, page 342 -351, Crooke, S.T., ed., 2008). In some embodiments of the invention, the degree of pro-inflammatory effect can be attenuated by the selection of one or more variables. For example, the extent of the pro-inflammatory effect of a given oligonucleotide can be attenuated by replacing any number of thiophosphate internucleoside bonds with phosphodiester internucleoside bonds, thereby reducing the total number of thiophosphate internucleoside bonds.

In some embodiments of the invention, it may be desirable to reduce the number of thiophosphate bonds, if this can be done without loss of stability and without shifting the distribution from the liver to the kidneys. For example, in some embodiments of the invention, the number of thiophosphate bonds can be reduced by replacing thiophosphate bonds with phosphodiester bonds. In such an embodiment, an antisense compound having fewer thiophosphate bonds and more phosphodiester bonds may elicit fewer or no pro-inflammatory responses. Although an antisense compound having fewer thiophosphate bonds and more phosphodiester bonds may cause fewer pro-inflammatory reactions, an antisense compound having fewer thiophosphate bonds and more phosphodiester bonds may not accumulate in the liver and may be less effective in the same or a similar dose compared to an antisense compound having more thiophosphate bonds. Therefore, in some embodiments of the invention, it is desirable to develop an antisense compound that has many phosphodiester bonds and many thiophosphate bonds, but which also has stability and good distribution in the liver.

In some embodiments, conjugated antisense compounds accumulate more in the liver and less in the kidneys than unconjugated analogs, even if some of the thiophosphate bonds are replaced by less pro-inflammatory phosphodiester internucleoside bonds. In some embodiments, conjugated antisense compounds accumulate more in the liver and are not as highly excreted in the urine as their unconjugated counterparts, even if some of the thiophosphate bonds are replaced by less pro-inflammatory phosphodiester internucleoside bonds. In some embodiments of the invention, the use of a conjugate allows the development of more effective and better tolerated antisense drugs. Indeed, in some embodiments of the invention, conjugated antisense compounds have a broader therapeutic index than unconjugated analogs. This allows the conjugated antisense compound to be administered at a higher absolute dose due to less risk of a pro-inflammatory response and less risk of renal toxicity. This higher dose makes it possible to administer the dose less frequently, since the same clearance ratio (metabolism) is expected. In addition, since the compound is more effective as described above, a decrease in concentration before the next dose can be allowed without loss of therapeutic activity, which allows for even longer periods between doses.

In some embodiments of the invention, it remains necessary to introduce a certain amount of thiophosphate bonds. For example, terminal bonds are easily attacked by exonucleases, and therefore, in some embodiments of the invention, these bonds are thiophosphate or other modified bonds. Internucleoside bonds connecting two deoxynucleosides are readily attacked by endonucleases and therefore, in some embodiments, these bonds are thiophosphate or other modified bonds. Internucleoside bonds between a modified nucleoside and a deoxynucleoside, where the deoxynucleoside is located on the 5' side of the binding deoxynucleoside, are readily attacked by endonucleases, and therefore, in some embodiments, these bonds are thiophosphate or other modified bonds. Internucleoside bonds between two specific types of modified nucleosides and between a deoxynucleoside and a specific type of modified nucleoside, where the modified nucleoside is located on the 5' side of the linker, are sufficiently resistant to nuclease cleavage that the bond may be a phosphodiester.

In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 16 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 15 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 14 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 13 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 12 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 11 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 10 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 9 thiophosphate bonds. In some embodiments, the antisense oligonucleotide of the conjugated antisense compound contains less than 8 thiophosphate bonds.

In some embodiments, antisense compounds containing one or more of the conjugation groups described herein have increased potency and/or efficacy and/or tolerability compared to the parent antisense compound lacking such one or more conjugation groups. Accordingly, in some embodiments of the invention, the attachment of such conjugating groups to the oligonucleotide is desirable. Such conjugating groups may be attached at the 5' and/or 3' end of the oligonucleotide. In some cases, the addition at the 5' end is synthetically desirable. Typically, oligonucleotides are synthesized by attaching a 3'-terminal nucleoside to a solid support, followed by linking the nucleosides 3' to 5' using techniques well known in the art. Accordingly, if a conjugation group is needed at the 3' end, then one can (1) attach the conjugation group to the 3' end of the nucleoside and attach this conjugated nucleoside to a solid support to subsequently obtain an oligonucleotide, or (2) attach the conjugation group to the 3' end nucleoside of the finished oligonucleotide after synthesis. Neither of these approaches is particularly efficient, and therefore both are costly. In particular, attachment of a conjugated nucleoside to a solid support, although described in the Examples section of this document, is not an efficient method. In some embodiments of the invention, the addition of conjugating groups to the 5'-terminal nucleoside is synthetically simpler than attachment to the 3'-terminus. It is possible to attach an unconjugated 3'-terminal nucleoside to a solid support and prepare an oligonucleotide according to standard and well-documented reactions. Then it is only necessary to attach the 5'-nucleoside having a conjugating group in the last coupling step. In some embodiments, this is more efficient than attaching the conjugated nucleoside directly to a solid support, as is commonly done to produce a 3'-conjugated oligonucleotide. The Examples provided herein show attachment to the 5' end. In addition, some conjugating groups have synthetic advantages. For example, some conjugate groups containing phosphorus linking groups are synthetically easier and more efficient than other conjugate groups, including the conjugate groups previously described (eg, WO/2012/037254).

In some embodiments, the conjugated antisense compounds are administered to a subject. In such embodiments, antisense compounds containing one or more of the conjugation groups described herein have increased potency and/or efficacy and/or tolerability compared to the parent antisense compound lacking such one or more conjugation groups. Without being limited to the mechanism, it is assumed that the conjugating group facilitates distribution, delivery and/or uptake in the target cell or tissue. In some embodiments, after entering the target cell or tissue, it is desirable that all or part of the conjugating groups are cleaved to release the active oligonucleotide. In some embodiments of the invention, it is not necessary that the entire conjugating group be cleaved from the oligonucleotide. For example, in Example 20a, the conjugated oligonucleotide was injected into mice and many different chemical species were found, each containing different parts of the conjugating group remaining on the oligonucleotide (Table 10a). This conjugated antisense compound showed good efficacy (Table 10). Thus, in some embodiments of the invention, such a metabolite profile of multiple partial cleavage of the conjugating groups does not affect the activity/efficiency. However, in some embodiments of the invention it is desirable that the prodrug (conjugated oligonucleotide) gives one active connection. In some cases, when multiple forms of the active compound are found, it may be necessary to determine the relative amounts and effects for each of them. In some embodiments, where regulatory testing is required (eg, by the US FDA or other authority), it is desirable to have one (or predominantly one) active particle. In some such embodiments of the invention, it is desirable that such a single active particle was an antisense oligonucleotide that does not contain any parts of the conjugating group. In some embodiments, conjugation groups at the 5' end are more likely to result in complete metabolism of the conjugation groups. Without being limited by mechanism, it may be that endogenous enzymes responsible for metabolism at the 5' end (eg, 5' nucleases) are more active/efficient than their 3' counterparts. In some embodiments of the invention, certain conjugating groups are more susceptible to metabolism to a single active species. In some embodiments of the invention, some conjugating groups are more susceptible to metabolism to the oligonucleotide.

D. Antisense

In some embodiments, the oligomeric compounds of the present invention are antisense compounds. In such embodiments, the oligomeric compound is complementary to the target nucleic acid. In some embodiments, the target nucleic acid is RNA. In some embodiments of the invention, the target nucleic acid is a non-coding RNA. In some embodiments, the target nucleic acid encodes a protein. In some embodiments, the target nucleic acid is selected from mRNA, pre-mRNA, miRNA, non-coding RNA, including small non-coding RNA, and promoter-targeted RNA. In some embodiments, the oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, the oligomeric compounds of the present invention may be microRNA mimetics that typically bind to multiple targets.

In some embodiments, the antisense compounds comprise a moiety having a nitrogen base sequence that is at least 70% complementary to the nitrogen base sequence of the target nucleic acid. In some embodiments, the antisense compounds comprise a moiety having a nitrogen base sequence that is at least 80% complementary to the nitrogen base sequence of the target nucleic acid. In some embodiments, the antisense compounds comprise a moiety having a nitrogen base sequence that is at least 90% complementary to the nitrogen base sequence of the target nucleic acid. In some embodiments, the antisense compounds comprise a moiety having a nitrogen base sequence that is at least 95% complementary to the nitrogen base sequence of the target nucleic acid. In some embodiments, the antisense compounds comprise a moiety having a nitrogen base sequence that is at least 98% complementary to the nitrogen base sequence of the target nucleic acid. In some embodiments, the antisense compounds comprise a moiety having a nitrogen base sequence that is 100% complementary to the nitrogen base sequence of the target nucleic acid. In some embodiments, the antisense compounds are at least 70%, 80%, 90%, 95%, 98%, or 100% complementary to the nucleic acid sequence of the target nucleic acid over the entire length of the antisense compound.

Antisense mechanisms include any mechanism involving the hybridization of an oligomeric compound with a target nucleic acid such that the hybridization results in a biological effect. In some embodiments of the invention, such hybridization results in either the destruction or use of the target nucleic acid with concomitant suppression or stimulation of a cellular mechanism affecting, for example, translation, transcription, or polyadenylation of the target nucleic acid or a nucleic acid with which the target acid may otherwise interact. .

One type of antisense mechanism affecting target RNA degradation is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA helix of an RNA:DNA duplex. Single-stranded antisense compounds that are "DNA-like" are known in the art to induce RNase H activity in mammalian cells. Therefore, activation of RNase H leads to cleavage of the target RNA, thereby greatly enhancing the efficiency of gene expression suppression mediated by the DNA-like oligonucleotide.

Antisense mechanisms also include, without limitation, RNAi mechanisms that use the RISC pathway. Such RNAi mechanisms include, without limitation, miRNA, ssRNA, and miRNA mechanisms. Such mechanisms include the creation of miRNA mimetic and/or anti-miRNA.

Antisense mechanisms also include, without limitation, mechanisms that hybridize or mimic non-coding RNA other than miRNA or mRNA. Such non-coding RNAs include, but are not limited to, promoter-targeted RNA and small and long RNAs that affect transcription or translation of one or more nucleic acids.

In some embodiments of the invention, the oligonucleotides containing the conjugates described herein are RNAi compounds. In some embodiments, the oligomeric oligonucleotides containing the conjugates described herein are ssRNA compounds. In some embodiments of the invention, oligonucleotides containing the conjugates described herein are paired with a second oligomeric compound to form an siRNA. In some such embodiments, the second oligomeric compound also contains a conjugate. In some embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In some embodiments of the invention, oligonucleotides containing the conjugates described herein are an antisense strand in an siRNA compound. In some embodiments of the invention, oligonucleotides containing the conjugates described herein represent the sense strand in an siRNA compound. In those embodiments in which the conjugated oligomeric compound is a double-stranded siRNA, the conjugate may be on the sense strand, the antisense strand, or both the sense strand and the antisense strand.

D. Target nucleic acids, regions and segments

In some embodiments, the conjugated antisense compounds target any nucleic acid. In some embodiments, the target nucleic acid encodes a target protein that is clinically relevant. In such embodiments, the modulation of the target nucleic acid results in a beneficial clinical effect. Some target nucleic acids include, but are not limited to, the target nucleic acids shown in Table 1.

The targeting process typically includes identifying at least one target region, segment, or site in a target nucleic acid for antisense interaction to result in that desired effect.

In some embodiments, the target region is a structurally defined region of a nucleic acid. For example, in some 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 another defined region or target nucleic acid segment.

In some embodiments, the target segment is the portion of the target region of at least about 8 bases targeted by the conjugated antisense compound. Target segments may contain DNA or RNA sequences that contain at least 8 contiguous nitrogen bases from the 5' end of one of the target segments (the remaining nitrogen bases extend one after the other from the same DNA or RNA, starting just before the 5' end of the target segment). segment and continuing until the DNA or RNA contains from about 8 to about 30 nitrogenous bases). Target segments are also represented by DNA or RNA sequences that contain at least 8 adjacent nitrogenous bases from the 3'-end of one of the target segments (the remaining nitrogenous bases extend one after another from the same DNA or RNA, starting immediately after the 3'-end of the target segment). segment and continuing until the DNA or RNA contains from about 8 to about 30 nitrogenous bases). Target segments can also be DNA or RNA sequences that contain at least 8 contiguous nitrogenous bases from the interior of the target segment sequence, and can extend in either or both directions until the conjugated antisense compound contains from about 8 to about 30 nitrogenous bases.

In some embodiments, antisense compounds that target the nucleic acids listed in Table 1 may be modified as described herein. In some embodiments, antisense compounds may have a modified sugar moiety, an unmodified sugar moiety, or a mixture of modified and unmodified sugar moieties, as described herein. In some embodiments, antisense compounds may have a modified internucleoside bond, an unmodified internucleoside bond, or a mixture of modified and unmodified internucleoside bonds, as described herein. In some embodiments, antisense compounds may have a modified nucleobase, an unmodified nucleobase, or a mixture of modified and unmodified nucleobases, as described herein. In some embodiments, antisense compounds may have the motif described herein.

In some embodiments, antisense compounds that target the nucleic acids listed in Table 1 may be conjugated as described herein.

1. Hepatitis B (HBV)

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

The HBV virus is a double-stranded hepatotropic virus that only infects humans and primates. Viral replication occurs predominantly in the liver and, to a lesser extent, in the kidneys, pancreas, bone marrow and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64: 2000; 51-68.). Viral and immune markers can be detected in the blood and are characteristic antigen-antibody profiles that develop over time. The first detectable viral marker is HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA. During the incubation period, titers may be high, but HBV DNA and HBeAg levels begin to decline rapidly at the onset of the disease and may not be detectable at the peak of clinical illness (Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med. 350: 2004; 1118-1129). HBeAg is a viral marker found in the blood that correlates with active viral replication and hence high viral load and infectivity (Hepatitis B e antigen-the dangerous end game of hepatitis B. N Engl J Med. 347: 2002; 208- 210). The presence of anti-HBsAg and anti-HBcAg (IgG) indicates recovery and immunity in a previously infected individual.

Currently, therapies for chronic HBV infection recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon-alpha (INFa), pegylated interferon-alpha-2a (Peg-IFN2a), entecavir and tenofovir. The nucleoside and nitrogenous base therapies, entecavir and tenofovir, are successful in reducing viral load, but rates of HBeAg seroconversion and HBsAg reduction are even lower than those achievable with IFNa therapy. Other similar therapies have also been used, including lamivudine (3TC), telbivudine (LdT), and adefovir, but the therapeutic efficacy of nucleoside/nitrous base therapies is generally limited by the emergence of resistance.

Therefore, there is a need in the art for the discovery and development of new antiviral therapies. In addition, there is a need for new anti-HBV therapies capable of increasing HBeAg and HBsAg seroconversion rates. In recent clinical studies, a correlation has been found between seroconversion and reduction in HBeAg (Fried et al (2008) Hepatology 47:428), and reduction in HBsAg (Moucari et al (2009) Hepatology 49:1151). Decreased levels of antigens may allow immunological control of HBV infection, as high levels of antigens are expected to induce immunological tolerance. Existing nucleoside therapies for HBV can significantly reduce serum HBV levels, but have little effect on HBeAg and HBsAg levels.

Antisense compounds targeting HBV have been described previously in WO 2011/047312, WO 2012/145674 and WO 2012/145697, the entire content of each of which is incorporated herein by reference. Clinical studies are planned to evaluate the effect of HBV-targeting antisense compounds on patients. However, there is still a need to provide patients with additional and more effective treatment options.

Certain Conjugated Antisense Compounds Targeting the HBV Nucleic Acid

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

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of the sequence of SEQ ID NO: 3. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 3.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 4. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 1 comprises sequence of nitrogenous bases SEQ ID NO: 4.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of the sequence of SEQ ID NO: 5. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 5.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of the sequence of SEQ ID NO: 6. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 6.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of the sequence of SEQ ID NO: 7. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 7.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 8. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 8.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogen bases of SEQ ID NO: 9. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 9.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 1 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 10. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 1 comprises sequence of nitrogenous bases SEQ ID NO: 10.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains at least 8 contiguous nitrogen bases of the sequence of SEQ ID NO: 11. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 contains sequence of nitrogenous bases SEQ ID NO: 11.

In some embodiments, a compound having the following chemical structure contains or consists of ISIS 505358 with 5'-X, where X is the conjugation group described herein:

In some embodiments, the compound contains or consists of ISIS 712408 having the following chemical structure:

In some embodiments, the compound contains or consists of ISIS 695324 having the following chemical structure:

In some embodiments of the invention, the compound contains or consists of SEQ ID NO: 3, 5'-GalNAc and chemical modifications and is represented by the following chemical structure:

where any R ¹ is OCH ₂ CH ₂ OCH ₃ (MOE) and R ² is H; or R ¹ and R ² together form a bridge, wherein R ¹ is -O- and R ² is -CH ₂ -, -CH(CH ₃ )- or -CH ₂ CH ₂ -, and R ¹ and R ² are directly connected so that the resulting bridge is selected from: -O-CH ₂ -, -O-CH(CH ₃ )- and -O-CH ₂ CH ₂ -;

and for each pair of R ³ and R ⁴ on one ring, independently for each ring: any R ³ is selected from H and —OCH ₂ CH ₂ OCH ₃ and R ⁴ is H; or R ³ and R ⁴ together form a bridge, wherein R ³ is —O— and R ⁴ is —CH ₂ —, —CH(CH ₃ )—or —CH ₂ CH ₂ —, and R ³ and R ⁴ are directly connected so that the resulting bridge is selected from: -O-CH ₂ -, -O-CH(CH ₃ )- and O-CH ₂ CH ₂ -;

and R ⁵ is selected from H and -CH ₃ ;

a Z is selected from S' and O'.

In some embodiments, the compound contains an antisense oligonucleotide as described in WO 2012/145697, the entire content of which is incorporated herein by reference, and a conjugation group as described herein. In some embodiments, the compound contains an antisense oligonucleotide having the nitrogenous base sequence of any of SEQ ID NOs 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379 described in WO 2012/ 145697, and the conjugating group described in this document. In some embodiments, the compound contains an antisense oligonucleotide as described in WO 2011/047312, the entire content of which is incorporated herein by reference, and a conjugation group as described herein. In some embodiments, the compound contains an antisense oligonucleotide having the nitrogenous base sequence of any of SEQ ID NOs 14-22 described in WO 2011/047312 and a conjugation group as described herein. In some embodiments, the compound contains an antisense oligonucleotide as described in WO 2012/145674, the entire content of which is incorporated herein by reference, and a conjugation group as described herein. In some embodiments, the compound comprises an antisense oligonucleotide having the nitrogenous base sequence of any of SEQ ID NOs 18-35 described in WO 2012/145674. In some embodiments, the compound contains a double-stranded oligonucleotide as described in WO 2013/159109, the entire content of which is incorporated herein by reference, and a conjugation group as described herein. In some embodiments, the compound comprises a double-stranded oligonucleotide wherein one helix has the sequence of nitrogenous bases of any of SEQ ID NOs 30-125 described in WO 2013/159109. The nitrogenous base sequences of all of the aforementioned reference SEQ ID NOs are incorporated herein by reference.

Therapeutic indications for HBV

In some embodiments of the present invention, methods are provided for using a conjugated antisense compound that targets an HBV nucleic acid to modulate HBV expression in a subject. In some embodiments of the invention, the expression of HBV is reduced.

In some embodiments of the present invention, methods are provided for using a conjugated antisense compound that targets an HBV nucleic acid in a pharmaceutical composition for treating a subject. In some embodiments of the invention, the subject suffers from a pathological condition associated with HBV. In some embodiments of the invention, the pathological condition associated with HBV includes, but is not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, bile effusion, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis and HBV viremia. In some embodiments of the invention, the pathological condition associated with hBV may be characterized by symptoms, which may include any or all of the following signs: flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, bile effusion, nausea and vomiting, pain in the liver, clay-colored or gray stools, general itching and dark-colored urine, in combination with a positive test for the presence of hepatitis B virus, hepatitis B viral antigen, or a positive test for the presence of an antibody specific for hepatitis B viral antigen. In some embodiments carrying out the invention, the subject is at risk of a pathological condition associated with HBV. This includes subjects who have one or more risk factors for developing an HBV-related condition, including sexual contact with an individual infected with hepatitis B virus, living in the same household with an individual with lifelong hepatitis B virus infection, exposure to the blood of a person infected with hepatitis B virus B, Injection of Prohibited Substances by a Subject with Hemophilia and Visiting Hepatitis B Sites. In some embodiments, the subject is identified as being in need of treatment for an HBV-related condition.

In some embodiments, a method is provided for reducing levels of HBV DNA and/or HBV antigens in an HBV-infected animal, comprising administering to said animal a conjugated antisense compound that targets an HBV nucleic acid. In some embodiments of the invention, the antigen is HBsAG or HBeAG. In some embodiments of the invention, the amount of HBV antigen can be significantly reduced, resulting in seroconversion.

In some embodiments of the present invention, methods are provided for using a conjugated antisense compound targeted to an HBV nucleic acid for the manufacture of a drug.

In some embodiments, the present invention provides a conjugated antisense compound that targets an HBV nucleic acid, or a pharmaceutically acceptable salt thereof, for use in therapy.

In some embodiments, a conjugated antisense compound targeted to an HBV nucleic acid is provided for use in the treatment of an HBV-associated condition. The pathological condition associated with HBV includes, but is not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, bile effusion, liver cancer, liver inflammation, liver fibrosis, cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis and HBV viremia.

In some embodiments, a conjugated antisense compound targeted to an HBV nucleic acid is provided for use in reducing levels of HBV DNA and/or HBV antigen in an HBV infected animal, comprising administering to said animal a conjugated antisense compound targeted to the HBV nucleic acid. In some embodiments of the invention, the antigen is HBsAG or HBeAG. In some embodiments of the invention, the amount of HBV antigen can be significantly reduced, resulting in seroconversion.

It should be understood that any of the compounds described herein can be used in the above methods and applications. For example, in some embodiments, a conjugated antisense compound targeted to an HBV nucleic acid in the above methods and uses may include, but is not limited to, a conjugated antisense compound targeted to SEQ ID NO: 1 that contains at least 8 contiguous nitrogenous bases of the sequence SEQ ID NO: 3-11; a conjugated antisense compound targeting SEQ ID NO: 1, which contains a nitrogenous base sequence of any of SEQ ID NO: 3-11; a compound containing or consisting of ISIS 505358, ISIS 509934, ISIS 510100, ISIS 552023, ISIS 552024, ISIS 552032, ISIS 552859, ISIS 552925, or ISIS 577119 and conjugating groups; a compound containing an antisense oligonucleotide as described in WO 2012/145697, the entire disclosure of which is incorporated herein by reference, and a conjugating group; a compound containing an antisense oligonucleotide having the sequence of nitrogenous bases of any of SEQ ID NOs 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376 and 1379 described in WO 2012/145697, and conjugating the group described in this document; a compound containing an antisense oligonucleotide having a nitrogenous base sequence of any of SEQ ID NOs 14-22 described in WO 2011/047312 and a conjugating group as described herein; a compound containing an antisense oligonucleotide having a nitrogenous base sequence of any of SEQ ID NOs 18-35 described in WO 2012/145674; or a compound containing a double-stranded oligonucleotide in which one helix has the sequence of nitrogenous bases of any of SEQ ID NO 30-125 described in WO 2013/159109.

2. Transthyretin (TTR)

TTR (also known as prealbumin, hyperthyroxinemia, dysprealbuminaemic, thyroxine; senile systemic amyloidosis, amyloid polyneuropathy, amyloidosis I, PALB; distransthyretinemic, HST2651; TVPA; dysprealbuminemic euthyroid hyperthyroxinemia) is a serum/plasma and cerebrospinal fluid protein responsible for the transport of thyroxine and retinol (Sakaki et al, Mol Biol Med. 1989, 6:161-8). Structurally, TTR is a homotetramer; point mutations and misfolding of the protein leads to the deposition of amyloid fibrils and is associated with disorders such as senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and familial amyloid cardiopathy (FAC).

TTR is synthesized primarily by the liver and choroid plexus of the brain, and to a lesser extent by the human retina (Pallia, Clin Chem Lab Med, 2002, 40, 1292-1300). Transthyretin, which is synthesized in the liver, is secreted into the blood, while transthyretin from the choroid plexus is destined for cerebrospinal fluid (CSF). Synthesis of transthyretin in the choroid plexus accounts for about 20% of total local protein synthesis and as much as 25% of total CSF protein (Dickson et al., J Biol Chem, 1986, 261, 3475-3478).

Thanks to the ability to perform genetic and immunohistochemical diagnostic tests, patients with TTR amyloidosis have been found in many peoples of the world. Recent studies have shown that TTR amyloidosis is not a rare endemic disease, as previously thought, and may affect at least 25% of the adult population (Tanskanen et al, Ann Med. 2008;40(3):232-9).

On a biochemical level, TTR was identified as the major protein component in the amyloid deposits of FAP patients (Costa et al, Proc. Natl. Acad. Sci. USA 1978, 75:4499 4503) 30 of this protein is the most common molecular defect causing this disease (Saraiva et al, J. Clin. Invest. 1984, 74: 104-119). In FAP, ubiquitous systemic extracellular deposition of TTR aggregates occurs and amyloid fibrils occur throughout connective tissue, especially in the peripheral nervous system (Sousa and Saraiva, Prog. Neurobiol. 2003, 71: 385^-00). After TTR deposition, axonal degeneration occurs, originating in unmyelinated and myelinated small diameter fibers, which ultimately leads to the loss of neurons in the ganglion centers.

TTR-targeting antisense compounds have been previously described in US 2005/0244869, WO 2010/017509 and WO 2011/139917, the entire contents of each of which are incorporated herein by reference. An antisense oligonuclear base targeting TTR, ISIS-TTRrx, is currently undergoing phase 2/3 clinical trials to investigate its efficacy in the treatment of subjects suffering from familial amyloid polyneuropathy. However, there is still a need to provide patients with additional and more effective treatment options.

Some Conjugated Antisense Compounds Targeting the TTR Nucleic Acid

In some embodiments, conjugated antisense compounds target a TTR nucleic acid having the sequence GENBANK® accession number NM 000371.3, which is incorporated herein as SEQ ID NO: 2. In certain such embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

In some embodiments, the conjugated antisense compound targeted to SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of any of SEQ ID NO: 12-19. In some embodiments, the conjugated antisense compound targeted to SEQ ID NO: 2 contains a nitrogenous base sequence of any of SEQ ID NO: 12-19.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 12. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 comprises sequence of nitrogenous bases SEQ ID NO: 12.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 13. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 comprises sequence of nitrogenous bases SEQ ID NO: 13.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 14. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 comprises sequence of nitrogenous bases SEQ ID NO: 14.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 15. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 contains sequence of nitrogenous bases SEQ ID NO: 15.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 16 contains at least 8 contiguous nitrogen bases of SEQ ID NO: 78. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 16 contains sequence of nitrogenous bases SEQ ID NO: 78.

In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 17. In some embodiments, a conjugated antisense compound targeted at SEQ ID NO: 2 comprises sequence of nitrogenous bases SEQ ID NO: 17.

In some embodiments, a conjugated antisense compound targeting SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 18. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 contains sequence of nitrogenous bases SEQ ID NO: 18.

In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 contains at least 8 contiguous nitrogenous bases of SEQ ID NO: 19. In some embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 contains sequence of nitrogenous bases SEQ ID NO: 19.

In some embodiments of the invention, a compound having the following chemical structure contains or consists of ISIS 420915 with 5'-X, where X is the conjugate group described herein:

In some embodiments, the compound comprises or consists of ISIS 682877 having the following chemical structure:

In some embodiments, the compound comprises or consists of ISIS 682884 having the following chemical structure:

In some embodiments, the compound contains or consists of SEQ ID NO: 12, 5'-GalNAc and chemical modifications, and is represented by the following chemical structure:

where any R ¹ represents-OCH ₂ CH ₂ OCH ₃ (MOE), and R ² represents H; or R ¹ and R ² together form a bridge, wherein R ¹ is -O- and R ² is -CH ₂ -, -CH(CH ₃ )- or -CH ₂ CH ₂ -, and R ¹ and R ² are directly connected so that the resulting bridge is selected from: -O-CH ₂ -, -O-CH(CH ₃ )- and O-CH ₂ CH ₂ -;

and for each pair of R ³ and R ⁴ on one ring, independently for each ring: any R ³ is selected from H and —OCH ₂ CH ₂ OCH ₃ and R ⁴ is H; or R ³ and R ⁴ together form a bridge, wherein R ³ is —O— and R ⁴ is —CH ₂ —, —CH(CH ₃ )—or —CH ₂ CH ₂ —, and R ³ and R ⁴ are directly connected so that the resulting bridge is selected from: -O-CH ₂ -, -O-CH(CH ₃ )- and -O-CH ₂ CH ₂ -;

and R ⁵ is selected from H and -CH ₃ ;

a Z is selected from S' and O'.

In some embodiments, the compound contains an antisense oligonucleotide as described in WO 2011/139917 or US 8101743, the entire content of which is incorporated herein by reference, and a conjugating group. In some embodiments, the compound comprises an antisense oligonucleotide having the nitrogenous base sequence of any of SEQ ID NOs 8-160, 170-177 described in WO 2011/139917 and a conjugation group as described herein. In some embodiments, the compound contains an antisense oligonucleotide having the nitrogenous base sequence of any of SEQ ID NOs 12-89 described in US 8 101 743 and a conjugation group as described herein. In some embodiments, the compound contains an antisense oligonucleotide having a nitrogenous base sequence complementary to the preferred target segment of any of SEQ ID NOs 90-133 described in US 8101743 and a conjugation group described herein. The nitrogenous base sequences of all of the aforementioned reference SEQ ID NOs are incorporated herein by reference.

Therapeutic indications for TTR

In some embodiments of the present invention, methods are provided for using a conjugated antisense compound that targets a TTR nucleic acid to modulate TTR expression in a subject. In some embodiments of the invention, TTR expression is reduced.

In some embodiments of the present invention, methods are provided for using a conjugated antisense compound that targets a TTR nucleic acid in a pharmaceutical composition for treating a subject. In some embodiments of the invention, the subject suffers from a disease, disorder or pathological condition associated with transthyretin, or its symptom. In some embodiments of the invention, the disease, disorder or pathological condition associated with transthyretin is transthyretin amyloidosis. "Transthyretin-associated amyloidosis" or "transthyretin amyloidosis" or "transthyretin amyloid disease", as used herein, is any pathology or disease associated with transthyretin dysfunction or dysregulation that results in the formation of transthyretin-containing amyloid fibrils. Transthyretin amyloidosis includes, but is not limited to, hereditary TTR amyloidosis, leptomeningeal amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy, familial oculoleptomeningeal amyloidosis, senile cardiac amyloidosis, or senile systemic amyloidosis.

In some embodiments of the present invention, methods are provided for using a conjugated antisense compound targeted to a TTR nucleic acid for the manufacture of a drug.

In some embodiments, the present invention provides a conjugated antisense compound that targets a TTR nucleic acid, or a pharmaceutically acceptable salt thereof, for use in therapy.

In some embodiments, a conjugated antisense compound targeted to a TTR nucleic acid is provided for use in the treatment of a transthyretin-associated disease, disorder, condition, or symptom thereof. In some embodiments of the invention, the disease, disorder or pathological condition associated with transthyretin is transthyretin amyloidosis.

It should be understood that any of the compounds described herein can be used in the above methods and applications. For example, in some embodiments, a conjugated antisense compound targeting a TTR nucleic acid in the above methods and uses may include, but is not limited to, a conjugated antisense compound targeted to SEQ ID NO: 2 that contains at least 8 contiguous nitrogenous bases of any of SEQ ID NOs: 12-19; a conjugated antisense compound targeting SEQ ID NO: 2, which contains a nitrogenous base sequence of any of SEQ ID NO: 12-19; a compound containing or consisting of ISIS 420915, ISIS 304299, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957 or ISIS 420959 and conjugating groups; a compound containing an antisense oligonucleotide described in WO 2011/139917 or US 8101743, the entire content of each of which is incorporated herein by reference, and a conjugating group; a compound containing an antisense oligonucleotide having a nitrogenous base sequence of any of SEQ ID NOs 8-160, 170-177 described in WO 2011/139917 and a conjugating group as described herein; an antisense oligonucleotide having a nitrogenous base sequence of any of SEQ ID NOs 12-89 described in US 8101743 and a conjugating group as described herein; or a compound containing an antisense oligonucleotide having a nitrogenous base sequence complementary to the preferred target segment of any of SEQ ID NOs 90-133 described in US 8101743 and a conjugating group as described herein. The nitrogenous base sequences of all of the aforementioned reference SEQ ID NOs are incorporated herein by reference.

E. Certain Pharmaceutical Compositions

In some embodiments of the present disclosure, pharmaceutical compositions are provided that contain one or more antisense compounds. In some embodiments of the invention, such a pharmaceutical composition contains a suitable pharmaceutically acceptable diluent or carrier. In some embodiments of the invention, the pharmaceutical composition contains a sterile saline solution and one or more antisense compounds. In some embodiments of the invention, such a pharmaceutical composition consists of a sterile saline solution and one or more antisense compounds. In some embodiments, the sterile saline solution is pharmaceutical grade saline. In some embodiments of the invention, the pharmaceutical composition contains one or more antisense compounds and sterile water. In some embodiments of the invention, the pharmaceutical composition consists of one or more antisense compounds and sterile water. In some embodiments, the sterile saline solution is pharmaceutical grade water. In some embodiments of the invention, the pharmaceutical composition contains one or more antisense compounds and phosphate-buffered saline (PBS). In some embodiments of the invention, the pharmaceutical composition consists of one or more antisense compounds and phosphate buffered saline (PBS). In some embodiments, the sterile saline solution is pharmaceutical grade PBS.

In some embodiments of the invention, antisense compounds can be mixed with pharmaceutically acceptable active and/or inert substances to obtain pharmaceutical compositions or formulations. Compositions and methods of formulating pharmaceutical compositions depend on a variety of criteria, including, but not limited to, the route of administration, the severity of the disease, or the dose to be administered.

Pharmaceutical compositions containing antisense compounds encompass any pharmaceutically acceptable salts, esters or salts of such esters. In some embodiments of the invention, pharmaceutical compositions containing antisense compounds contain one or more oligonucleotides that, when administered to a mammal, including a human, can provide (directly or indirectly) its biologically active metabolite or residue. Accordingly, for example, the present disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, the sodium and potassium salts.

The prodrug may include the introduction of additional nucleosides at one or both ends of the oligonucleotide a, which are cleaved in the body by endogenous nucleases to form an active antisense oligonucleotide.

Lipid moieties have been used in numerous methods in nucleic acid therapies. In some such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes prepared from mixtures of cationic lipids and neutral lipids. In some methods, DNA complexes with mono- or polycationic lipids are obtained without the participation of a neutral lipid. In some embodiments, the lipid moiety is selected to improve the distribution of the pharmaceutical agent in a particular cell or tissue. In some embodiments, the lipid moiety is selected to improve the distribution of the pharmaceutical agent in adipose tissue. In some embodiments of the invention, the lipid fragment is selected to improve the distribution of the pharmaceutical agent in muscle tissue.

In some embodiments of the invention, the pharmaceutical compositions provided herein contain one or more modified oligonucleotides and one or more excipients. In some such embodiments, the excipients are selected from water, saline solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, and pouring inyl pyrrolidone.

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

In some embodiments of the invention, the pharmaceutical composition provided herein contains one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the present description in a specific tissue or cell types. For example, in some embodiments of the invention, the pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In some embodiments of the invention, the pharmaceutical composition provided herein contains a co-solvent system. Some such co-solvent systems contain, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is an absolute ethanol solution containing 3% wt./vol. benzyl alcohol, 8% w/v. non-polar surfactant Polysorbate 80™ and 65% w/v. polyethylene glycol 300. The proportions of such co-solvent systems can vary substantially without significantly altering their solubility and toxicity characteristics. In addition, the nature of the components of the co-solvents may vary: for example, other surfactants may be used instead of Polysorbate 80™; the size of the polyethylene glycol fraction may vary; instead of polyethylene glycol, other biocompatible polymers can be used, for example, polyvinylpyrrolidone; and other sugars or polysaccharides may be used instead of dextrose.

In some embodiments of the invention, the pharmaceutical composition provided herein is prepared for oral administration. In some embodiments of the invention, pharmaceutical compositions are prepared for buccal administration.

In some embodiments of the invention, the pharmaceutical composition is prepared for administration by injection (eg, intravenous, subcutaneous, intramuscular, etc.). In some such embodiments, the pharmaceutical composition contains a carrier and is formulated in an aqueous solution such as water or physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. In some embodiments of the invention, other ingredients are included (for example, ingredients that improve solubility or serve as preservatives). In some embodiments, injection suspensions are prepared using suitable liquid carriers, suspending agents, and the like. Some pharmaceutical compositions for injection are presented in unit dose form, for example in ampoules, or in multi-dose containers. Some injectable pharmaceutical compositions are suspensions, solutions or emulsions in oily or aqueous liquid vehicles and may contain adjuvants such as suspending, stabilizing and/or dispersing agents. Some solvents suitable for use in injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Such suspensions may also optionally contain suitable stabilizers or agents which increase the solubility of the pharmaceutical agents so that highly concentrated solutions can be obtained.

In some embodiments of the invention, the pharmaceutical composition is prepared for transmucosal administration. In some such embodiments, penetrants are used in the formulation to match the barrier to be penetrated. Such penetrants are well known in the art.

In some embodiments of the invention, the pharmaceutical composition provided herein contains the oligonucleotide in a therapeutically effective amount. In some embodiments, the therapeutically effective amount is sufficient to prevent, alleviate, or alleviate the symptoms of a disease or to increase the lifespan of the subject being treated. Determining a therapeutically effective amount is within the ability of those skilled in the art.

In some embodiments, one or more of the modified oligonucleotides provided herein are formulated as a prodrug. In some embodiments, when administered in vivo, the prodrug is chemically converted to a biologically, pharmaceutically, or therapeutically more active form of the oligonucleotide. In some embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in some cases, the prodrug may be more bioavailable (eg, when administered orally) than the corresponding active form. In some cases, the prodrug may have improved solubility as compared to the corresponding active form. In some embodiments, the prodrugs are less water soluble than the corresponding active form. In some cases, such prodrugs have excellent transmission across cell membranes, while water solubility impairs their mobility. In some embodiments, the prodrug is an ester. In some such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In some cases, the compound containing the carboxylic acid is the corresponding active form. In some embodiments, the prodrug contains a short peptide (polyamino acid) linked to an acid group. In some such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In some embodiments of the present disclosure, compositions and methods for reducing the amount or activity of a target nucleic acid in a cell are provided. In some embodiments of the invention, the cell is in the body of an animal. In some embodiments of the invention, the animal is a mammal. In some embodiments of the invention, the animal is a rodent. In some embodiments of the invention, the animal is a primate. In some embodiments, the animal is a non-human primate. In some embodiments of the invention, the animal is a human.

In some embodiments of the present disclosure, methods are provided for administering to an animal a pharmaceutical composition comprising an oligonucleotide of the present disclosure. Suitable routes of administration include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppositories, inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous ). In some embodiments of the invention, pharmaceutical intrathecal agents are administered to achieve local rather than systemic effects. For example, pharmaceutical compositions may be injected directly into the area of desired effect (eg, the liver).

Non-limiting description and inclusion by reference

While some of the compounds, compositions, and methods described herein have been described in detail in accordance with certain embodiments of the invention, the following examples are merely illustrative of the compounds described herein and should not be considered limiting. Each of the references, GenBank access numbers and like references referred to in this application are incorporated herein by reference in their entirety.

Certain compounds, compositions, and methods are described herein as "exactly" or "containing only" a certain number of specific elements or characteristics. Such descriptions are used to indicate that while a compound, composition, or method may include other optional elements, the amount of a particular element or characteristic is a certain number. For example, "conjugate containing exactly one GalNAc" is a conjugate that contains one and exactly one GalNAc, although it may contain elements other than the specified one GalNAc.

Although the sequence listing that accompanies this file specifies each sequence as either "RNA" or "DNA", as needed, these sequences can actually be modified with any combination of chemical modifications. Those skilled in the art will recognize that such designation as "RNA" or "DNA" to describe modified oligonucleotides is arbitrary in some cases. For example, an oligonucleotide containing a nucleoside containing a 2'-OH sugar moiety and a thymine base can be described as DNA having a modified sugar (2'-OH instead of the natural 2'-H in DNA) or as RNA having a modified base (thymine (methylated uracil) instead of natural uracil in RNA).

Accordingly, 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 naturally occurring or modified RNA and/or DNA, including but not limited to such nucleic acids. acids that contain modified nitrogenous bases. As a further example and without limitation, an oligonucleotide containing the nitrogenous base sequence "ATCGATCG" encompasses any oligonucleotides having such a nitrogenous base sequence, modified or unmodified, including, but not limited to, those compounds that contain an RNA base, such as those that have the sequence "AUCGAUCG" and those that have some DNA bases and some RNA bases such as "AUCGATCG", as well as oligonucleotides having other modified bases such as "AT ^me CGAUCG", where ^me C stands for cytosine base containing a methyl group at the 5-position.

EXAMPLES

The following examples illustrate some embodiments of the present disclosure and are non-limiting. Moreover, when specific embodiments are given, the inventors intend the general application of said specific embodiments. For example, a description of an oligonucleotide containing a particular motif provides a valid basis for additional oligonucleotides containing the same or similar motif. And, for example, if a particular high affinity modification occurs at a certain position, then other high affinity modifications are considered appropriate at that position, unless otherwise indicated.

Example 1. General method for obtaining phosphoramidites. Compounds 1, 1a and 2

Bx is a heterocyclic base

Compounds 1, 1a and 2 were prepared according to procedures well known in the art as described herein (see Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and see also published PCT International Applications (WO 2011/115818, WO 2010/077578, WO 2010/036698, WO 2009/143369, WO 2009/006478 and WO 2007/090071) and US Pat.

Example 2 Preparation of Compound 7

Compound 3 (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem., 1991, 34, 2692).

Example 3 Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4 Preparation of Compound 18

Compound 11 was prepared according to the methods described in Example 3. Compound 14 is commercially available. Compound 17 was prepared in the same manner as described in Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 5 Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6 Preparation of Compound 24

Compounds 18 and 23 were prepared as described in the methods in Examples 4 and 5.

Example 7 Preparation of Compound 25

Compound 24 was obtained according to the methods presented in Example 6.

Example 8 Preparation of Compound 26

Compound 24 was obtained according to the methods presented in Example 6.

Example 9 General preparation of conjugated ASOs containing GalNAc ₃ -1 at the 3' end, Compounds 29

Where protected GalNAc ₃ -1 has the structure:

The GalNAc _{3 cluster portion of the GalNAc 3 -1 conjugating group (GalNAc 3 -1 a )} can be combined with any cleavable moiety to form various conjugating groups. In this case, GalNAc ₃ -1 _a has the formula:

A solid support binding the protected GalNAc ₃ -1, Compound 25, was prepared according to the methods presented in Example 7. An oligomeric Compound 29 containing GalNAc ₃ -1 at the 3' end was prepared according to standard procedures in an automated DNA/RNA synthesizer (see Dupouy et al., Angew Chem Int ed 2006, 45, 3623-3627). Phosphoramide building blocks. Compounds 1 and 1a were prepared according to the methods presented in Example 1. The phosphoramidites depicted are illustrative and are not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds having the desired sequence and composition. The order and amount of phosphoramidites added to the solid support can be adjusted to produce the broken oligomeric compounds described herein. Such broken oligomeric compounds may have the desired composition and base sequence dictated by any given target.

Example 10 General preparation of conjugated ASOs containing GalNAc ₃ -1 at the 5' end, Compounds 34

Unylinker™ 30 is commercially available. Oligomeric compound 34 containing a GalNAc ₃ -1 cassette at the 5' end was prepared by standard methods in an automatic DNA/RNA synthesizer (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627 ). Phosphoramide building blocks. Compounds 1 and 1a were prepared according to the methods presented in Example 1. The phosphoramidites depicted are illustrative and are not intended to be limiting as other phosphoramidite building blocks can be used to produce an oligomeric compound having the desired sequence and composition. The order and amount of phosphoramidites added to the solid support can be adjusted to produce the broken oligomeric compounds described herein. Such broken oligomeric compounds may have the desired composition and base sequence dictated by any given target.

Example 11 Preparation of Compound 39

Compounds 4, 13 and 23 were prepared according to the methods described in Examples 2, 4 and 5. Compound 35 was prepared according to the same methods as described in Rouchaud et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

Example 12 Preparation of Compound 40

Compound 38 was obtained according to the methods presented in Example 11.

Example 13 Preparation of Compound 44

Compounds 23 and 36 were prepared according to the methods presented in Examples 5 and 11. Compound 41 was prepared according to the same methods as described in WO 2009082607.

Example 14 Preparation of Compound 45

Compound 43 was obtained according to the methods presented in Example 13.

Example 15 Preparation of Compound 47

Compound 46 is commercially available.

Example 16 Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 were prepared as described in the methods in Examples 4 and 15.

Example 17 Preparation of Compound 54

Compound 53 was obtained according to the methods presented in Example 16.

Example 18 Preparation of Compound 55

Compound 53 was obtained according to the methods presented in Example 16.

Example 19 General Method for the Preparation of Conjugated ASOs Containing GalNAc ₃ -1 at the 3' Position Using Solid Phase Methods (preparation of ISIS 647535, 647536 and 651900)

Unless otherwise noted, all reagents and solutions used for the synthesis of oligomeric compounds were purchased from commercial suppliers. Standard phosphoramidite building blocks and a solid support were used to introduce nucleoside residues, which include, for example, T, A, G, and ^m C residues. -MY.

Synthesis of antisense oligonucleotides (ASOs) was performed on an ABI 394 synthesizer (1-2 µmol scale) or an AKTA Oligopilot synthesizer manufactured by GE Healthcare Bioscience (40-200 µmol scale) by the phosphoramidite binding method on a VIMAD solid support filled with GalNAc ₃ -1 (110 µmol/g, Guzaev et al., 2003) packed into a column. For the binding step, the phosphoramidites were added in a 4-fold excess compared to the loading on a solid support, and the condensation of the phosphoramidite was carried out for 10 minutes. All other steps were performed according to protocols provided by the manufacturer. A 6% solution of dichloroacetic acid in toluene was used to remove the dimethoxytrityl (DMT) group from the 5'-hydroxyl group of the nucleotide. In the coupling step, 4,5-dicyanoimidazole (0.7 M) in anhydrous CH ₃ CN was used as an activator. Thiophosphate bonds were introduced by sulfonation with a 0.1 M solution of xanthan hydride in a 1:1 mixture of pyridine/CH ₃ CN for 3 minutes of contact time. A 20% solution of tert-butyl hydroperoxide in CH ₃ CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside bonds for 12 minutes of contact time.

After assembling the desired sequence, the cyanoethyl phosphate protecting groups were removed with a 1:1 (v/v) mixture of triethylamine and acetonitrile over a 45 minute contact time. Associated with a solid support ASO suspended in an aqueous solution of ammonia (28-30 wt.%) and heated at 55°C for 6 hours.

The unbound ASO was then filtered off and the ammonia was evaporated by boiling. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54 x 8 cm, A=100 mM ammonium acetate in 30% aqueous CH ₃ CN, B=1.5 M NaBr in A, 0-40% B in 60 min, flow rate 14 ml min-1, λ=260 nm). The residue was desalted by HPLC on a reversed phase column to give the desired ASOs in an isolated yield of 15-30% relative to the initial load on a solid support. ASO was characterized by ion pair HPLC combined with MS analysis on an Agilent 1100 MSD system.

Conjugate-free antisense oligonucleotides were synthesized according to standard oligonucleotide synthesis methods well known in the art.

Using these methods, three separate antisense compounds targeted to ApoC III were obtained. As shown in Table 4 below, each of the three ApoC III antisense compounds has the same nitrogenous base sequence. ISIS 304801 is a 5-10-5 MOE gapmer containing only thiophosphate linkages; ISIS 647535 is the same as ISIS 304801 except that it contains GalNAc ₃ -1 conjugated at its 3'end; and ISIS 647536 is the same as ISIS 647535 except that some of the internucleoside bonds of this compound are phosphodiester bonds. As further shown in Table 4, two separate antisense compounds were synthesized that targeted SRB-1. ISIS 440762 is a 2-10-2 cEt gapmer containing only thiophosphate internucleoside bonds; ISIS 651900 is the same as ISIS 440762 except that it contains GalNAc ₃ -1 at its 3' end.

Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"k" is a 6'-(S)-CH ₃ bicyclic nucleoside (eg cEt); "s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO); and "o'" means -O-P(=O)(OH)-. The superscript "m" stands for 5-methylcytosines. "GalNAc ₃ -1" means a conjugate group having the structure shown earlier in Example 9. It should be noted that GalNAc ₃ -1 contains cleavable adenosine, which binds ASO to the rest of the conjugate, which is designated as "GalNAc ₃ -1 _a ". The nomenclature used in the table above shows the complete sequence of nitrogenous bases, including adenosine, which is part of the conjugate. Therefore, in the table above, sequences can also be listed with the ending "GalNAc ₃ -1", without "A _do ". This conditional use of the subscript "a" to denote the part of the conjugating group that does not contain a cleavable nucleoside or a cleavable fragment is used in all of the Examples presented. This part of the conjugating group that does not contain a cleavable fragment is referred to herein as a "cluster" or "conjugate cluster" or "GalNAc ₃ cluster". In some cases, the conjugating group is described for convenience by separately representing its cluster and its cleavable moiety.

Example 20 Dose-Dependent Antisense Inhibition of Human ApoC III in huApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoC III transgenic mice were maintained on a 12 hour light/dark cycle and allowed free access to Teklad lab food. Prior to the start of the experiment, the animals were acclimatized for at least 7 days in the research laboratory. Prepared ASO in PBS and sterilized by filtration through a 0.2 micron filter. ASO was dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were intraperitoneally injected once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25, 0.75, 2.25, or 6.75 µmol/kg or PBS as control . Each experimental group consisted of 4 animals. Forty-eight hours after the last dose, each mouse was bled and sacrificed, and tissues were collected.

ApoC III mRNA analysis

ApoC III mRNA levels in mouse liver were determined using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc. Eugene, Oregon) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), then normalized to a PBS treated control. The results shown below are presented as the average percentage of ApoC III mRNA levels for each experimental group, normalized to the PBS-treated control, and are labeled "% PBS". Table 5 below also shows the half maximum effective dose (ED ₅₀ ) for each ASO.

Both antisense compounds were shown to reduce ApoC III RNA compared to a control sample treated with PBS. In addition, the antisense compound conjugated to GalNAc ₃ -1 (ISIS 647535) was significantly more effective than the antisense compound not containing the GalNAc ₃ -1 conjugate (ISIS 304801).

ApoC III protein assay (turbidimetric assay)

Plasma ApoC III protein analysis was performed according to the methods described by Graham et al. Circulation Research, prior to print, published online March 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed without dilution using an Olympus clinical analyzer and a commercially available ApoC III turbidimetric assay kit (Kamiya, cat. no. KAI-006, Kamiya Biomedical, Seattle, WA). The analysis protocol was performed according to the supplier's description.

As shown in Table 6 below, both antisense compounds reduced the ApoC III protein compared to the PBS control. In addition, the antisense compound conjugated to GalNAc ₃ -1 (ISIS 647535) was significantly more effective than the antisense compound not containing the GalNAc ₃ -1 conjugate (ISIS 304801).

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

Triglyceride levels were measured relative to mice injected with PBS and expressed as "% PBS". The results are shown in Table 7. Both antisense compounds were shown to reduce triglyceride levels. In addition, the antisense compound conjugated to GalNAc ₃ -1 (ISIS 647535) was significantly more effective than the antisense compound not containing the GalNAc ₃ -1 conjugate (ISIS 304801).

Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and various fractions of cholesterol (HDL and LDL). The results are presented in Tables 8 and 9. Both antisense compounds were shown to reduce total cholesterol levels; both lower LDL; and both increase HDL. In addition, the antisense compound conjugated to GalNAc ₃ -1 (ISIS 647535) was significantly more effective than the antisense compound not containing the GalNAc ₃ -1 conjugate (ISIS 304801). An increase in HDL levels and a decrease in LDL levels represent the beneficial cardiovascular effect of antisense inhibition of ApoC III.

Pharmacokinetic analysis (PK)

We also appreciated FC ASO. Liver and kidney samples were crushed and extracted according to standard protocols. Samples were analyzed for MSD1 using IP-HPLC-MS. The tissue content (µg/g) of full length ISIS 304801 and 647535 was determined and the results are shown in Table 10. Liver concentrations of full length antisense compounds were shown to be similar for the two antisense compounds. Therefore, even though the CalNAc ₃ -1 conjugated antisense compound is more active in the liver (as shown by the RNA and protein data presented above), its liver content is not much higher. Indeed, the calculated EC50 value (shown in Table 10) confirms that the observed increase in conjugate potency cannot be attributed solely to increased uptake. This result suggests that the conjugate improves performance by a different mechanism than simple hepatic accumulation, possibly by increasing the productive uptake of the antisense compound into cells.

These results also show that the concentration of the GalNAc ₃ -1-conjugated antisense compound in the kidneys is lower than the concentration of the antisense compound not containing the GalNAc conjugate. This has several beneficial therapeutic effects. For therapeutic indications that do not require activity in the kidneys, exposure to the kidneys exposes them to the risk of toxicity without corresponding benefit. Moreover, the high concentration in the kidneys usually results in the excretion of the compound in the urine, allowing for faster elimination. Accordingly, for extrarenal targets, accumulation in the kidneys is undesirable. These data suggest that conjugation with GalNAc ₃ -1 reduces renal accumulation.

The metabolites of ISIS 647535 were also identified and their masses confirmed by high resolution mass spectrometric analysis. The cleavage sites and structures of the observed metabolites are presented below. The relative % of the total length of the ASO was calculated according to standard methods, and the results are presented in Table 10a. The major metabolite of ISIS 647535 is a full length ASO without all of the conjugate (ie ISIS 304801) which results from cleavage at cleavage site A below. In addition, additional metabolites generated from other cleavage sites were also observed. These results suggest that the introduction of other cleavable bonds, such as esters, peptides, disulfides, phosphoramidates or acyl hydrazones, between the GalNAc ₃ -1 sugar and ASO, which can be cleaved by enzymes within the cell or which can cleave in the reducing environment of the cytosol, or which are labile in acidic pH within endosomes and lysosomes.

Example 21 Antisense Inhibition of Human ApoC III in ApoC III Transgenic Mice in a Single Administration Study

ISIS 304801, 647535 and 647536, each targeting human ApoC III and described in Table 4, were further evaluated in a single dose study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoC III transgenic mice were maintained on a 12 hour light/dark cycle and allowed free access to Teklad lab food. Prior to the start of the experiment, the animals were acclimatized for at least 7 days in the research laboratory. Prepared ASO in PBS and sterilized by filtration through a 0.2 micron filter. ASO was dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were intraperitoneally injected with a single injection of the dose below, compound ISIS 304801, 647535 or 647536 (described above), or PBS as a control. The experimental group consisted of 3 animals and the control group consisted of 4 animals. Before treatment, as well as after the last dose, blood was taken from each mouse and plasma samples were analyzed. Mice were sacrificed 72 hours after the last injection.

Samples were collected and analyzed to determine levels of ApoC III mRNA and protein in the liver; plasma triglycerides; and cholesterol, including HDL and LDL fractions, which were analyzed as described above (Example 20). The data of these analyzes are presented below in Tables 11-15. Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. ALT and AST levels indicated that the antisense compounds were well tolerated at all doses administered.

These results demonstrate increased potency of antisense compounds containing a GalNAc ₃ -1 conjugate at the 3' end (ISIS 647535 and 647536) compared to an antisense compound not containing a GalNAc ₃ -1 conjugate (ISIS 304801). In addition, ISIS 647536, which contains a GalNAc ₃ -1 conjugate and several phosphodiester bonds, was as effective as ISIS 647535, which contains the same conjugate and all internucleoside bonds in this ASO are thiophosphate.

These results confirm that the GalNAc ₃ -1 conjugate improves the performance of the antisense compound. These results also show equal potency of CalNAc ₃ -1-conjugated antisense compounds in which the antisense oligonucleotides have mixed linkages (ISIS 647536, which has six phosphodiester linkages) and the fully thiophosphate version of the same antisense compound (ISIS 647535).

Thiophosphate bonds provide several properties for antisense compounds. For example, they are resistant to nuclease digestion and bind to proteins resulting in accumulation of the compound in the liver rather than in the kidney/urine. These properties are desirable, especially in the treatment of hepatic indications. However, thiophosphate bonds are also associated with an inflammatory response. Accordingly, reducing the number of thiophosphate bonds in the compound presumably reduces the risk of inflammation, but also reduces the concentration of the compound in the liver, increases the concentration in the kidneys and urine, reduces the stability in the presence of nucleases and reduces the overall effectiveness. The results presented demonstrate that a CalNAc ₃ -1-conjugated antisense compound in which some of the thiophosphate linkages are replaced by phosphodiester linkages is as effective against a liver target as an analog containing only thiophosphate linkages. Such compounds are believed to be less pro-inflammatory (see Example 24, which describes an experiment demonstrating that a decrease in thiophosphates (PS) leads to a decrease in inflammatory action).

Example 22 Effect of Modified CalNAc ₃ -1-Conjugated ASO Targeting SRB-1 in Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 4, were evaluated in a dose-dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below with ISIS 440762, 651900 or PBS as a control. Each experimental group consisted of 4 animals. Mice were sacrificed 48 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), then normalized to a PBS treated control. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to the PBS-treated control, and are labeled "% PBS".

As shown in Table 16, both antisense compounds reduce SRB-1 mRNA levels. In addition, the antisense compound containing the GalNAc ₃ -1 conjugate (ISIS 651900) was significantly more effective than the antisense compound not containing the GalNAc ₃ -1 conjugate (ISIS 440762). These results demonstrate that the performance advantage of GalNAc ₃ -1 conjugates is observed when using antisense oligonucleotides complementary to different targets and having different chemically modified nucleosides, in this case the modified nucleosides contain sterically hindered ethyl sugar moieties (bicyclic sugar moiety).

Example 23 Human Peripheral Blood Mononuclear Cell (hPBMC) Assay Protocol

hPBMC analysis was performed using the BD Vautainer CPT tube method.

Received a whole blood sample from consenting volunteer donors at the US Medical Clinic (Faraday & El Camino Real, Carlsbad) and collected it into 4-15 8 ml BD Vacutainer CPT tubes (VWR, cat. no. BD362753) . The approximate baseline total volume of whole blood in CPT tubes for each donor was recorded on the PBMC Analysis Form.

Prior to centrifugation, the blood sample was immediately remixed by gently inverting the tubes 8-10 times. The CPT tubes were centrifuged at room temperature (18-25° C.) in a horizontal (oversteered) rotor for 30 minutes with a separation factor (RCF) of 1500-1800 with braking (2700 rpm. Beckman Allegra 6R). The cells were removed from the leukocyte interface (between the ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and grouped at 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (without Ca ⁺⁺ , Mg ⁺⁺ ; GIBCO). The tubes were refilled to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330 xg for 15 at room temperature (1215 rpm in a Beckman Allegra 6R) and aspirated as much of the supernatant as possible without disturbing the pellet. The cell pellet was removed by gently swirling the tube and the cells were resuspended in RPMI+10% FBS+penicillin/streptomycin (~1 ml/10 ml of original whole blood volume). 600 µl of sample was pipetted and placed into a sample vial (Beckman Coulter) containing 600 µl of VersaLyse reagent (Beckman Coulter cat #A09777) and gently vortexed for 10-15 seconds. The sample was left to incubate for 10 minutes at room temperature and mixed again before counting. The cell suspension was read on a Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cells (keep dilution factor 1:11 with other parameters). Recorded the number of live cells/ml and viability. The cell suspension was diluted to 1 x 107 live PBMC/ml in RPMI+10% FBS+penicillin/streptomycin.

Cells were plated at 5 x 105 in 50 µl/well of a 96-well tissue culture plate (Falcon Microtest). 50 µl/well 2x concentration of oligomers/control diluted in RPMI+10% FBS+penicillin/streptomycin was added according to the experimental matrix (100 µl/well in total). The plates were placed on a shaker and left to mix for approximately 1 minute. After incubation for 24 hours at 37°C; 5% CO ₂ , the plates were centrifuged at 400 x g for 10 minutes, then the supernatant was removed for analysis of MSD cytokines (ie human IL-6, IL-10, IL-8 and MCP-1).

Example 24 Evaluation of pro-inflammatory effects in the hPBMC assay for GalNAc ₃ -1-conjugated ASO

The antisense oligonucleotides (ASOs) listed in Table 17 were evaluated for proinflammatory effects in the hPBMC assay; using the protocol described in Example 23. ISIS 353512 is an internal standard known to have a high response to IL-6 release in this assay. hPBMC were isolated from fresh samples obtained from volunteer donors and treated with ASO at concentrations of 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM. After 24 hours of treatment, the levels of cytokines were measured.

IL-6 levels were used as the primary indication. EC ₅₀ and E _max were calculated according to standard methods. The results were expressed as the average ratio of E _max /EC ₅₀ for two donors and designated as "E _max /EC ₅₀ ". A lower ratio means a relative decrease in the pro-inflammatory response, and a higher ratio means a relative increase in the pro-inflammatory response.

With respect to the compounds studied, the least pro-inflammatory compound was ASO coupled with PS/PO (ISIS 616468). GalNAc ₃ -1 conjugated ASO, ISIS 647535, was slightly less pro-inflammatory than its unconjugated counterpart, ISIS 304801. These results indicate that the insertion of multiple PO bonds reduces the pro-inflammatory response, and the addition of the GalNAc ₃ -1 conjugate does not make the compound more pro-inflammatory. and may reduce the pro-inflammatory response. Accordingly, an antisense compound containing mixed PS/PO linkages and a GalNAc ₃ -1 conjugate would be expected to elicit weaker pro-inflammatory responses compared to an antisense compound linked via PS alone, with or without a GalNAc ₃ -1 conjugate. These results indicate that CalNAc ₃ -1 conjugated antisense compounds, in particular compounds having lower PS content, are less pro-inflammatory.

Overall, these results suggest that a GalNAc ₃ -1 conjugated compound, in particular a PS-reduced compound, can be administered at a higher dose than an analogous fully PS antisense compound without a GalNAc ₃ -1 conjugate. Since the half-life of these compounds is not expected to vary significantly, this higher dose administration will result in less frequent dosing. In fact, such administration may be even rarer, since CaINAc ₃ -1-conjugated compounds are more effective (see Examples 20-22), and re-dose is necessary only when the concentration of the compound falls below the desired level, while this desired level driven by efficiency.

Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"k" means 6'-(S)-CH ₃ bicyclic nucleoside (eg, cEt); "s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO); and "o'" means -O-P(=O)(OH)-. The superscript "m" stands for 5-methylcytosines. "A _do' -GalNAc ₃ -1 _a "means a conjugate having the GalNAc ₃ -1 structure shown in Example 9 attached to the 3' end of said antisense oligonucleotide.

Example 25 Effect of Modified CalNAc ₃ -1-Conjugated ASO Targeting Human ApoC III in Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells of transgenic mice at a density of 25,000 cells per well were treated with concentrations of 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μM of the modified oligonucleotides. After treatment for about 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR, and hApoC III mRNA levels were adjusted according to total RNA measured by RIBOGREEN.

IC ₅₀ was calculated by standard methods and the results are shown in Table 19. Comparable efficacy was shown to be observed in cells treated with ISIS 647535 compared to the control, ISIS 304801.

In this experiment in vitro did not see such a big advantage in the efficiency of conjugation with GalNAc ₃ -1, as observed in vivo. Subsequent free uptake experiments in primary hepatocytes in vitro showed no increased efficacy of oligonucleotides containing various GalNAc conjugates compared to oligonucleotides not containing GalNAc conjugates (see Examples 60, 82 and 92).

Example 26 Effect of PO/PS Linkages on ASO Activity for ApoC III

Human ApoC III transgenic mice were injected intraperitoneally with 25 mg/kg of ISIS 304801 or ISIS 616468 (both described above) or PBS as control, once a week for two weeks. The experimental group consisted of 3 animals and the control group consisted of 4 animals. Before treatment, as well as after the last dose, blood was taken from each mouse and plasma samples were analyzed. Mice were sacrificed 72 hours after the last injection.

Samples were collected and analyzed to determine liver levels of ApoC III protein as described above (Example 20). The data from these analyzes are presented below in Table 20.

These results demonstrate the reduced efficacy of PO/PS antisense compounds (ISIS 616468) in wings compared to PS-only compounds (ISIS 304801).

Example 27 Compound 56

Compound 56 is commercially available from Glen Research or can be prepared according to the published procedures described by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28 Preparation of Compound 60

Compound 4 was prepared according to the methods described in Example 2. Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.

Compound 57 is illustrative and should not be considered limiting as other mono-protected substituted or unsubstituted alkyl diols, including but not limited to those described herein, may be used to produce phosphoramidites having the desired composition.

Example 29 Preparation of Compound 63

Compounds 61 and 62 were prepared according to the same methods as described by Tober et al., Eur. J. Org. Chem., 2013, 3, 566-577; and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 was prepared according to the same methods as described in the scientific and patent literature by Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., in PCT International Publication WO 2004063208.

Example 30 Preparation of Compound 63b

Compound 63a was prepared according to the same methods as described by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31 Preparation of Compound 63d

Compound 63c was prepared according to the same methods as described by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32 Preparation of Compound 67

Compound 64 was prepared according to the methods presented in Example 2. Compound 65 was prepared according to the same methods as described by Or et al. in PCT International Application WO 2009003009. The protecting groups used for Compound 65 are illustrative and should not be be considered a limitation as other protecting groups may be used, including but not limited to those described herein.

Example 33 Preparation of Compound 70

Compound 64 was prepared according to the methods described in Example 2. Compound 68 is commercially available. The protecting group used for Compound 68 is illustrative and should not be considered limiting as other protecting groups may be used, including but not limited to those described herein.

Example 34 Preparation of Compound 75a

Compound 75 was prepared according to the published procedures described by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35 Preparation of Compound 79

Compound 76 was prepared according to the published procedures described by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36 Preparation of Compound 79a

Compound 77 was obtained according to the methods presented in Example 35.

Example 37 General Method for Preparation of Conjugated Oligomeric Compound 82 Containing a Phosphodiester-Linked GaINAc ₃ -2 Conjugate at the 5' End, on a Solid Support (Method I)

where GalNAc ₃ -2 has the structure:

The GalNAc _{3 cluster portion of the GalNAc 3 -2 conjugating group (GalNAc 3 -2 a )} can be combined with any cleavable moiety to form various conjugating groups. While GalNAc ₃ -2 _a has the formula:

VIMAD-linked oligomeric compound 79b was prepared according to standard methods in an automatic DNA/RNA synthesizer (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite Compounds 56 and 60 were prepared as described in the methods in Examples 27 and 28, respectively. The phosphoramidites depicted are illustrative and should not be considered limiting, as other phosphoramidite building blocks, including but not limited to those presented herein, may be used to prepare an oligomeric compound containing a 5' phosphodiester-linked conjugating group. -end. The order and amount of phosphoramidites added to the solid support can be adjusted to produce the oligomeric compounds described herein which have any desired sequence and composition.

Example 33 Alternative Method for the Preparation of Oligomeric Compound 32 Containing a Phosphodiester-Linked CalNAc ₃ -2 Conjugate at the 5' End (Method II)

VIMAD-linked oligomeric compound 76b was prepared according to standard methods in an automatic DNA/RNA synthesizer (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). GalNAc ₃ -2-clustered phosphoramidite, Compound 79, was prepared according to the methods presented in Example 35. An alternative method allows one-step installation of the phosphodiester-bound GalNAc ₃ -2 conjugate onto the oligomeric compound in the last step of the synthesis. The depicted phosphoramidites are illustrative and should not be considered limiting as other phosphoramidite building blocks, including but not limited to those described herein, may be used to prepare oligomeric compounds containing a phosphodiester conjugate at the 5' end. The order and amount of phosphoramidites added to the solid support can be adjusted to produce the oligomeric compounds described herein which have any desired sequence and composition.

Example 39 General Method for Preparation of Oligomeric Compound 83h Containing a CalNAc ₃ -3 Conjugate at the 5' End (CalNAc ₃ -1 Modified for 5' End Attachment) on a Solid Support

Compound 18 was prepared according to the methods described in Example 4. Compounds 83a and 83b are commercially available. Oligomeric Compound 83e containing phosphodiester-linked hexylamine was prepared by standard methods for the synthesis of oligonucleotides. Treatment of the protected oligomeric compound with aqueous ammonia gave a 5'-GalNAc ₃ -3 conjugated oligomeric compound (83h).

where GalNAc ₃ -3 has the structure:

The GalNAc _{3 cluster portion of the GalNAc 3 -3 conjugating group (GalNAc 3 -3 a )} can be combined with any cleavable moiety to form various conjugating groups. In this case, GalNAc ₃ -3 _a has the formula:

Example 40 General method for preparing oligomeric compound 39 containing a phosphodiester-linked GalNAc ₃ -4 conjugate at the 3' end on a solid support

where GalNAc ₃ -4 has the structure:

Where SM is a split fragment. In some embodiments, the cleavable fragment is:

The GalNAc _{3 cluster portion of the GalNAc 3 -4 conjugating group (GalNAc 3 -4 a )} can be combined with any cleavable moiety to form different conjugating groups. While GalNAc ₃ -4 _a has the formula:

Protected Connection 30 on a functionalized Unylinker solid support is commercially available. Compound 84 was obtained according to the same methods as described in the literature (see Shchepinov et at., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et at., Nucleic Acids Research, 1999, 27, 3035- 3041; and Hornet et at., Nucleic Acids Research, 1997, 25, 4842-4849).

Phosphoramidite building blocks, Compounds 60 and 79a, were prepared according to the methods presented in Examples 28 and 36. The depicted phosphoramidites are illustrative and are not intended to be limiting as other phosphoramidite building blocks may be used to prepare an oligomeric compound having a phosphodiester-linked conjugate at 3 '-end, which has the desired sequence and composition. The order and amount of phosphoramidites added to the solid support can be adjusted to produce the oligomeric compounds described herein which have any desired sequence and composition.

Example 41 General Method for the Preparation of ASOs Containing a Phosphodiester-Linked GalNAc ₃ -2 Conjugate (See Example 37, Bx is adenine) at the 5' Position, by Solid Phase Method (Preparation of ISIS 661134)

Unless otherwise noted, all reagents and solutions used for the synthesis of oligomeric compounds were purchased from commercial suppliers. Standard phosphoramidite building blocks and a solid support were used to introduce nucleoside residues, which include, for example, T, A, G and ^m C residues. Phosphoramidite compounds 56 and 60 were used to synthesize a phosphodiester-linked GalNAc ₃ -2 conjugate at the 5' end. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-B-2'-deoxyribonucleoside and 2'-MOE.

Synthesis of antisense oligonucleotides (ASOs) was performed on an ABI 394 synthesizer (1-2 µmol scale) or an AKTA Oligopilot synthesizer from GE Healthcare Bioscience (40-200 µmol scale) by the VIMAD solid support phosphoramidite coupling method (110 µmol/g, Guzaev et at., 2003) packed into a column. For the binding step, the phosphoramidites were added in a 4-fold excess compared to the initial load on the solid support, and the binding of the phosphoramidite was carried out for 10 minutes. All other steps were performed according to protocols provided by the manufacturer. To remove dimethoxytrityl (DMT) groups from the 5'-hydroxyl groups of the nucleotide, a 6% solution of dichloroacetic acid in toluene was used. In the coupling step, 4,5-dicyanoimidazole (0.7 M) in anhydrous CH ₃ CN was used as an activator. Thiophosphate bonds were introduced by sulfonation with a 0.1 M solution of xanthan hydride in a 1:1 mixture of pyridine/CH ₃ CK for 3 minutes of contact time. A 20% solution of tert-butyl hydroperoxide in CH ₃ CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside bonds for 12 minutes of contact time.

After assembling the desired sequence, the cyanoethyl phosphate protecting groups were removed with 20% diethylamine in toluene (v/v) for 45 minutes of contact time. Associated with a solid support ASO suspended in an aqueous solution of ammonia (28-30 wt.%) and heated at 55°C for 6 hours.

The unbound ASO was then filtered off and the ammonia was evaporated by boiling. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH ₃ CN, B = 1.5 M NaBr in A, 0-40% B in 60 min, flow rate 14 ml min-1, λ=260 nm). The residue was desalted by HPLC on a reversed phase column to give the desired ASOs in an isolated yield of 15-30% relative to the initial load on a solid support. ASO was characterized by ion pair HPLC combined with MS analysis on an Agilent 1100 MSD system.

Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-B-2'-deoxyribonucleoside;"k" means 6'-(S)-CH ₃ bicyclic nucleoside (eg, cEt); "s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO); and "o'" means -O-P(=O)(OH)-. The superscript "m" stands for 5-methylcytosines. The structure of GalNAc ₃ -2 _a is shown in Example 37.

Example 42 General Method for the Preparation of ASOs Containing a GalNAc ₃ -3 Conjugate at the 5' Position by Solid Phase Methods (Preparation of ISIS 661166)

The synthesis of ISIS 661166 was carried out according to the same methods as presented in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MY gapmer in which the 5' position contains a GalNAc ₃ -3 conjugate. This ASO was characterized by ion pair HPLC combined with MS analysis on an Agilent 1100 MSD system.

Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO); and "o'" means -O-P(=O)(OH)-. Superscript "t" means 5-methylcytosines. The structure of "5'-GalNAc ₃ -3a" is shown in Example 39.

Example 43 Dose-dependent study of phosphodiester-bound GalNAc ₃ -2 (see Examples 37 and 41, Bx is adenine) at the 5' end targeted to SRB-1 in vivo

ISIS 661134 (see Example 41), containing a phosphodiester-linked GalNAc ₃ -2 conjugate at the 5' end, was tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 and 651900 (GalNAc ₃ -1 conjugate at the 3' end, see Example 9) are included in the study for comparison and are described previously in Table 4.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below with ISIS 440762, 651900, 661134 or PBS as control. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), then normalized to a PBS treated control. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to the PBS-treated control, and are labeled "% PBS". ED ₅₀ was measured by the same methods as described previously, and they are presented below.

As shown in Table 22, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. Indeed, antisense oligonucleotides containing a phosphodiester-linked GalNAc ₃ -2 conjugate at the 5' end (ISIS 661134) or a GalNAc ₃ -1 conjugate linked at the 3' end (ISIS 651900) show a significant improvement in potency compared to unconjugated antisense oligonucleotide (ISIS 440762). In addition, ISIS 661134, which contains a phosphodiester-linked GalNAc ₃ -2 conjugate at the 5' end, was as effective as ISIS 651900, which contains a GalNAc ₃ -1 conjugate at the 3' end.

The structures of 3' GalNAc ₃ -1 and 5' GalNAc ₃ -2 are described previously in Examples 9 and 37.

Pharmacokinetic analysis (PK)

The PK for ASO from the high dose group (7 mg/kg) was investigated and evaluated in the same manner as described in Example 20. Liver samples were minced and extracted according to standard protocols. Full length metabolites 661134 (5' GalNAc ₃ -2) and ISIS 651900 (3' GalNAc ₃ -1) were identified and their masses confirmed by high resolution mass spectrometric analysis. The results indicated that the major metabolite found for ASO containing a phosphodiester-linked GalNAc ₃ -2 conjugate at the 5' end (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites were observed in detectable amounts. In contrast, for ASO containing a GalNAc ₃ -1 conjugate at the 3' end (ISIS 651900), additional metabolites were observed, similar to those described previously in Table 10a. These results suggest that the presence of a phosphodiester-linked GalNAc ₃ -1 or GalNAc ₃ -2 conjugate may improve the PK profile of ASO without compromising their performance.

Example 44 Effect of PO/PS Linkages on Antisense Inhibition of ASOs Containing a GalNAc ₃ -1 Conjugate (see Example 9) at the 3' End Targeting SRB-1

ISIS 655861 and 655862 containing a GalNAc ₃ -1 conjugate at the 3' end, each targeting SRB-1, were tested in a single dose study for their ability to inhibit SRB-1 in mice. The original unconjugated compound, ISIS 353382, was included in the study for comparison.

These ASOs are 5-10-5 MOE gapmers in which the gap region contains ten 2'-deoxyribonucleosides and each wing region contains five 2'-MOE modified nucleosides. These ASOs were prepared by the same methods as shown previously in Example 19 and are described below in Table 23.

Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO); and "o'" means -O-P(=O)(OH)-. Superscript "t" means 5-methylcytosines. The structure of "GalNAc ₃ -1" is presented in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below with compound ISIS 353382, 655861, 655862 or PBS as a control. Each experimental group consisted of 4 animals. Before treatment, as well as after the last dose, blood was taken from each mouse and plasma samples were analyzed. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), then normalized to a PBS treated control. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to the PBS-treated control, and are labeled "% PBS". ED ₅₀ was measured by the same methods as described previously, and they are presented below.

As shown in Table 24, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner compared to a PBS-treated control. Indeed, antisense oligonucleotides containing a GalNAc ₃ -1 conjugate at the 3' end (ISIS 655861 and 655862) show a significant increase in potency compared to an unconjugated antisense oligonucleotide (ISIS 353382). In addition, ISIS 655862 with mixed PS/PO bonds shows an increase in efficiency compared to a connection containing only PS (ISIS 655861).

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Organ weight was also assessed. The results show that no increase in transaminase levels (Table 25) or organ weight (data not shown) was observed in ASO-treated mice compared to PBS controls. In addition, ASO with mixed PS/PO linkages (ISIS 655862) shows similar transaminase levels compared to the PS-only compound (ISIS 655861).

Example 45 Preparation of PFP ester Compound 110a

Compound 4 (9.5 g, 28.8 mmol) was treated separately with compound 103a or 103b (38 mmol) and TMSOTf (0.5 eq.), and molecular sieves in dichloromethane (200 ml), and stirred for 16 hours at room temperature. The organic layer was then filtered through celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting oil was purified by silicate left chromatography (2%-10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compounds 104a and 104b were treated under the same conditions as compounds 100a-d (Example 47) to give compounds 105a and 105b in >90% yield. The LCMS and proton NMR data were consistent with the indicated structure.

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

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

Compounds 107a and 107b were treated under the same conditions as compounds 97a-d (Example 47) to give compounds 108a and 108b in 40-60% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compounds 108a (60%) and 108b (40%) were treated under the same conditions as compounds 100a-d (Example 47) to give compounds 109a and 109b in >80% yield. The LCMS and proton NMR data were consistent with the indicated structure.

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

Example 46 General method for conjugation with PFP esters (oligonucleotide 111); obtaining ISIS 666881 (GalNAc ₃ -10)

The 5'-hexylamino-modified oligonucleotide was synthesized and purified using standard methods for solid phase oligonucleotide production. The 5'-Hexylamino-modified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μl) and 3 equivalents of the selected PFP-esterified GalNAc ₃ cassette dissolved in DMSO (50 μl) were added. If the PFP ester precipitated when the ASO solution was added, then DMSO was added until all the PFP ether went into solution. The reaction was complete after about 16 hours of stirring at room temperature. The resulting solution was diluted with water to 12 ml and then centrifuged at 3000 rpm. in a centrifugal filter with a mass cut-off of 3000 Da. This procedure was repeated twice to remove low molecular weight impurities. The solution was then lyophilized to dryness and redissolved in concentrated aqueous ammonia and stirred at room temperature for 2.5 hours, then concentrated in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC, and lyophilized to give a GalNAc ₃ conjugated oligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc ₃ -10. The GalNAc _{3 cluster portion of the GalNAc 3 -10 conjugating group (GalNAc 3 -10 a )} can be combined with any cleavable moiety to form different conjugating groups. In some embodiments, the cleavable moiety is -P(=O)(OH)-Ad -P(=O)(OH)- as shown below in the oligonucleotide (ISIS 666881) synthesized from GalNAc ₃ -10. The structure of GalNAc ₃ -10 (GalNAc ₃ -10 _a -CM-) is shown below:

ISIS 666881 was prepared using this general method. A 5'-hexylamine modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid phase oligonucleotide preparation methods. ISIS 660254 (40 mg, 5.2 µmol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 µl) and 3 equivalents of PBR ether (Compound 110a) dissolved in DMSO (50 µl) was added. The PBR ester precipitated upon addition of the ASO solution, so additional DMSO (600 µl) was required to completely dissolve the PEP ester.

The reaction was complete after 16 hours of stirring at room temperature. The solution was diluted with water to a total volume of 12 ml and then centrifuged at 3000 rpm. in a centrifugal filter with a mass cut-off of 3000 Da. This procedure was repeated twice to remove low molecular weight impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous ammonia with stirring at room temperature for 2.5 hours, then concentrated in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in 90% w/w yield (42 mg, 4.7 µmol).

GalNAc ₃ -10-conjugated oligonucleotide

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

Example 47 Preparation of Oligonucleotide 102 Containing GalNAc ₃ -8

Triacid compound 90 (4 g, 14.43 mmol) was dissolved in DMF (120 ml) and N,N-diisopropylethylamine (12.35 ml, 72 mmol). Pentafluorophenyl trifluoroacetate (8.9 ml, 52 mmol) was added dropwise under argon atmosphere and the reaction mixture was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added along with N,N-diisopropylethylamine (12.35 ml, 72 mmol) and the reaction mixture was allowed to stir at room temperature for 16 hours. The DMF was then evaporated to >75% under reduced pressure and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and evaporated under reduced pressure to an oily residue. The resulting oily substance was purified by silica gel chromatography (2%~10% methanol/dichloromethane) to give compounds 92a and 92b in an approximate yield of 80%. The LCMS and proton NMR data were consistent with the indicated structure.

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

Compounds 7 or 64 (9.6 mmol) were treated with HBTU (3.7 g, 9.6 mmol) and N,N-diisopropylethylamine (5 ml) in DMFA (20 ml) for 15 minutes. Either compound 93a or 93b (3 mmol) was added to the mixture and left to stir at room temperature for 16 hours. The DMF was then evaporated to >75% under reduced pressure and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and evaporated under reduced pressure to an oily residue. The resulting oily substance was purified by silica gel chromatography (5%->20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compounds 96a-d (0.75 mmol) were individually hydrogenated on Raney nickel for 3 hours in ethanol (75 ml). The catalyst was then removed by filtration through Celite and the ethanol removed under reduced pressure to give compounds 97a-d in 80-90% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compound 23 (0.32 g, 0.53 mmol) was treated with HBTU (0.2 g, 0.53 mmol) N,N-diisopropylethylamine (0.19 ml, 1.14 mmol) in DMF (30 ml) for 15 minutes. Compounds 97a-d (0.38 mmol) were added separately to the mixture and left to stir at room temperature for 16 hours. The DMF was then evaporated to >75% under reduced pressure and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and evaporated under reduced pressure to an oily residue. The resulting oily substance was purified by silica gel chromatography (2%-->20% methanol/dichloromethane) to give compounds 98a-d in 30-40% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compound 99 (0.17 g, 0.76 mmol) was treated with HBTU (0.29 g, 0.76 mmol) and N,N-diisopropylethylamine (0.35 ml, 2.0 mmol) in DMF (50 ml) in within 15 minutes. Compounds 97a-d (0.51 mmol) were added separately to the mixture and left to stir at room temperature for 16 hours. The DMF was then evaporated to >75% under reduced pressure and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and evaporated under reduced pressure to an oily residue. The resulting oily substance was purified by silica gel chromatography (5%-->20% methanol/dichloromethane) to give compounds 100a-d in 40-60% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compounds 100a-d (0.16 mmol) were individually hydrogenated in 10% Pd(OH) ₂ /C for 3 hours in methanol/ethyl acetate (1:1, 50 ml). The catalyst was then removed by filtration through Celite, and the organic solvents were removed under reduced pressure to give compounds 101a-d in 80-90% yield. The LCMS and proton NMR data were consistent with the indicated structure.

Compounds 101a-d (0.15 mmol) were separately dissolved in DMF (15 ml) and pyridine (0.016 ml, 0.2 mmol). Pentafluorophenyl trifluoroacetate (0.034 ml, 0.2 mmol) was added dropwise under argon atmosphere and the reaction mixture was allowed to stir at room temperature for 30 minutes. The DMF was then evaporated to >75% under reduced pressure and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and evaporated under reduced pressure to an oily residue. The resulting oily substance was purified by silica gel chromatography (2%-->5% methanol/dichloromethane) to give compounds 102a-d in an approximate yield of 80%. The LCMS and proton NMR data were consistent with the indicated structure.

Oligomeric Compound 102 containing a GalNAc ₃ -8 conjugation group was prepared according to the general methods presented in Example 46. The GalNAc _{3 cluster portion of the GalNAc 3 -8 conjugation group (GalNAc 3 -8 a )} can be combined with any cleavable moiety to produce various conjugating moieties. groups. In a preferred embodiment, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-.

The structure of GalNAc ₃ -8 (GalNAc ₃ -8 _a -CM-) is shown below:

Example 48 Preparation of Oligonucleotide 119 Containing GalNAc ₃ -7

Compound 112 was synthesized according to the method described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

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

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 ml, 16.2 mmol) were dissolved in anhydrous DMF (17 ml) and the reaction mixture was stirred. mixture at room temperature for 5 minutes. To this mixture was added a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 ml). The reaction mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure to give an oily substance. The residue was dissolved in CH ₂ Cl ₂ (100 ml) and washed with saturated aqueous NaHCO ₃ (100 ml) and brine (100 ml). The organic phase was separated, dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20% MeOH in dichloromethane to give Compound 114 (1.45 g, 30%). The structure was confirmed by LCMS analysis and ¹ H NMR.

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

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 ml, 1.5 mmol) were dissolved in anhydrous DMF (5 ml) and the reaction mixture was stirred. mixture at room temperature for 5 minutes. To this mixture was added a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF, and the reaction mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure and the residue was dissolved in CH ₂ Cl ₂ . The organic layer was washed with saturated aqueous NaHCO ₃ and brine, and dried over anhydrous Na ₂ SO ₄ and filtered. The organic layer was concentrated to dryness and the resulting residue was purified by silica gel column chromatography and eluted with 3 to 15% MeOH in dichloromethane to give Compound 116 (0.84 g, 61%). The structure was confirmed by LCMS analysis and ¹ H NMR.

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

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 ml). To this solution were added N,N-diisopropylethylamine (70 μl, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 μl, 0.42 mmol). The reaction mixture was stirred at room temperature for 12 hours and poured into saturated aqueous NaHCO ₃ . The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na ₂ SO ₄ . The dichloromethane solution was concentrated to dryness and purified by silica gel column chromatography and eluted with 5 to 10% MeOH in dichloromethane to give compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and ¹ H, as well as ¹ H and ¹⁹ F NMR.

Oligomeric Compound 119 containing a GalNAc ₃ -7 conjugating group was prepared according to the general methods presented in Example 46. The GalNAc _{3 cluster portion of the GalNAc 3 -7 conjugating group (GalNAc 3 -7 a )} can be combined with any cleavable moiety to produce various conjugating moieties. groups. In some embodiments, the cleavable moiety is -P(=O)(OH)-A _d -P(=O)(OH)-.

The structure of GalNAc ₃ -7 (GalNAc ₃ -7a-CM-) is shown below:

Example 49 Preparation of oligonucleotide 132 containing GalNAc ₃ -5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 ml). Triethylamine (11.2 ml, 80.35 mmol) was added and stirred for 5 minutes. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 ml) was added. Added additional amount of triethylamine (4.5 ml, 32.28 mmol) and stirred the reaction mixture for 18 hours in an argon atmosphere. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf=0.47). The solvent was removed under reduced pressure. The residue was dissolved in EtOAc (300 ml) and washed with 1 M NaHSO ₄ (3×150 ml), saturated aqueous NaHCO ₃ (3×150 ml) and brine (2×100 ml). The organic layer was dried with Na ₂ SO ₄ . The drying agent was removed by filtration and the organic layer was concentrated on a rotary evaporator. The crude mixture was purified by silica gel column chromatography and eluted with 35-50% EtOAc in hexanes to give compound 122 (15.50 g, 78.13%). The structure was confirmed by LCMS analysis and ¹ H NMR. Mass m/z 589.3 [M+H] ⁺ .

To a cooled solution of compound 122 (7.75 g, 13.16 mmol) dissolved in methanol (15 ml) was added a solution of LiOH (92.15 mmol) in water (20 ml) and THF (10 ml). The reaction mixture was stirred at room temperature for 45 minutes and monitored by TLC (EtOAc:hexane; 1:1). The reaction mixture was concentrated to half volume under reduced pressure. The remaining solution was cooled in an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 ml) and washed with brine (100 ml). On standing overnight, an emulsion formed and cleared.

The organic spy was separated, dried (Na ₂ SO ₄ ), filtered and evaporated to give Compound 123 (8.42 g). The excess weight is likely due to residual salt. The LCMS agreed with the structure. The product was used without any further purification. Mol. mass, calculated: 574.36% mol. mass found: 575.3 [M+H] ⁺ .

Compound 126 was synthesized according to the method described in the literature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in DMF (40 ml) and the resulting reaction mixture cooled in an ice bath. To this mixture was added N,N-diisopropylethylamine (4.42 ml, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by the coupling agent Bop (1.17 g, 2.66 mmol) in argon atmosphere. The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was complete after 1 hour as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 ml) and washed with 1 M NaHSO ₄ (3×100 ml), saturated aqueous NaHCO ₃ (3×100 ml) and brine (2×100 ml). The organic phase was separated, dried (Na ₂ SO ₄ ), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes in EtOAc to 100% EtOAc to give Compound 127 (9.4 g) as a white foam. LCMS and ¹ H NMR were consistent with the structure. Mass m/z 778.4 [M+H] ⁺ .

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

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

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

Compound 130 (75.8 mg, 0.046 mmol), 0.37 M solution of pyridine in DMF (200 µl) was added to a 10 ml volumetric round bottom flask and a stirrer was installed. To this solution, a 0.7 M solution of pentafluorophenyl trifluoroacetate in DMF (100 μl) was added dropwise with stirring. The reaction was complete after 1 hour as determined by LCMS. The solvent was removed under reduced pressure and the residue was dissolved in CHCl ₃ (~10 ml). The organic layer was treated with NaHSO ₄ (1 M, 10 ml), saturated aqueous NaHCO ₃ (10 ml) and brine (10 ml), each three times. The organic phase was separated and dried over Na ₂ SO ₄ , filtered and concentrated to give Compound 131 (77.7 mg). The LCMS agreed with the structure. Applied without further purification. Mass m/z 921.3 [M+2H] ⁺ .

Oligomeric Compound 132 containing a GalNAc ₃ -5 conjugation group was prepared according to the general methods presented in Example 46. The GalNAc _{3 cluster portion of the GalNAc 3 -5 conjugation group (GalNAc 3 -5 a )} can be combined with any cleavable moiety to produce various conjugating moieties. groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-.

The structure of GalNAc ₃ -5 (GalNAc ₃ -5 _a -CM-) is shown below:

Example 50 Preparation of Oligonucleotide 144 Containing GalNAc ₄ -11

Synthesis of Compound 134 VIMAD aminomethyl resin (2.5 g, 450 μmol/g) was added to a Merrifield flask, which was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was allowed to swell in acetone and silt (4 ml). Compound 133 was pre-activated in a 100 ml round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 ml) and DIEA (3.0 mmol, 0.5 ml ). This solution was allowed to stir for 5 minutes and then added to the Merrifield flask with shaking. The suspension was left to shake for 3 hours. The reaction mixture was decanted and the resin was washed with acetonitrile, DMF and DCM. The filling of the new resin was quantified by measuring the absorption of the DMT cation at 500 nm (extinction coefficient = 76,000) in DCM, which was 238 µmol/g. The resin was capped by suspending three times in acetic anhydride solution for ten minutes.

Compound 141 bound to a solid support was synthesized by repeated repetition of Fmoc solid phase peptide synthesis methods. A small amount of solid support was taken and suspended in an aqueous ammonia solution (28-30 wt %) for 6 hours. The cleaved compound was analyzed by LC-MS and observed that the mass is consistent with the structure. Mass m/z 1063.8 [M+2H] ⁺ .

Compound 142 bound to a solid support was synthesized by solid phase peptide synthesis methods.

Compound 143 bound to a solid support was synthesized using standard solid phase synthesis on a DNA synthesizer.

Compound 143 bound to the solid support was suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 16 hours. The solution was cooled and the solid support was filtered off. The filtrate was concentrated and the residue was dissolved in water and purified by HPLC on a strong anion exchange column. Fractions containing full length compound 144 were pooled and desalted. The resulting GalNAc ₄ -11-conjugated oligomeric compound was analyzed by LCMS and observed that the mass was consistent with the structure.

The GalNAc _{4 cluster portion of the GalNAc 4 -11 conjugating group (GalNAc 4 -11 a )} can be combined with any cleavable moiety to form different conjugating groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-.

The structure of GalNAc ₄ -11 (GalNAc ₄ -11 _a -CM) is shown below:

Example 51 Preparation of Oligonucleotide 155 Containing GalNAc ₃ -6

Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH ₂ Cl ₂ (200 ml). Added activated molecular sieves (4 Å, 2 g, powdered) and left the reaction mixture to stir for 30 minutes under nitrogen atmosphere. Added TMS-OTf (4.1 ml, 22.77 mmol) and left the reaction mixture to stir at room temperature overnight. After completion of the reaction, the mixture was quenched by pouring into saturated aqueous NaHCO ₃ (500 ml) with crushed ice (~150 g). The organic layer was separated, washed with brine, dried over MgSO ₄ , filtered and concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH ₂ Cl ₂ to give Compound 112 (16.53 g, 63%). LCMS and ¹ H NMR data were consistent with the putative compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 mL). The reaction mixture was purified by passing a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon, 400 mg) was added and hydrogen gas was bubbled through the solution for 30 minutes. After completion (TLC, 10% MeOH in CH ₂ Cl ₂ , and LCMS), the catalyst was removed by filtration through a Celite pad. The filtrate was concentrated on a rotary evaporator and dried quickly under high vacuum to give Compound 105a (3.28 g). LCMS and ¹ H NMR data were consistent with the desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMFA (100 ml). N,N-diisopropylethylamine (DIEA, 3.9 ml, 22 mmol) was added followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ~15 minutes under nitrogen. To this mixture was added a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF and stirred for 2 hours under nitrogen. The reaction mixture was diluted with EtOAc and washed with saturated aqueous NaHCO ₃ and brine. The organic phase was separated, dried (MgSO ₄ ), filtered and concentrated to an orange syrup. The crude material was purified by column chromatography with 2-5% MeOH in CH ₂ Cl ₂ to give Compound 148 (3.44 g, 73%). LCMS and ¹ H NMR data were consistent with the expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml). The reaction mixture was purified by passing a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon (350 mg) was added. Hydrogen gas was bubbled through the solution for 30 minutes. After completion (TLC, 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated on a rotary evaporator and dried rapidly under high vacuum to give Compound 149 (2.6 g) LCMS data agreed with the desired product The residue was dissolved in dry DMF (10 ml) and used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this was added DIEA (450 μl, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.52 mmol). The reaction mixture was allowed to stir for 15 minutes at room temperature under nitrogen. A solution of compound 149 (2.6 g) in anhydrous DMF (10 ml) was added. The pH of the reaction mixture was adjusted to pH=9-10 by adding DIEA (if necessary). The reaction mixture was allowed to stir at room temperature under nitrogen for 2 hours. After completion of the reaction, the mixture was diluted with EtOAc (100 ml) and washed with saturated aqueous NaHCO ₃ then brine. The organic phase was separated, dried over MgSO ₄ , filtered and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH ₂ Cl ₂ to give Compound 150 (0.62 g, 20%). LCMS and ¹ H NMR data were consistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). The reaction mixture was purified by passing a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon (60 mg) was added. Hydrogen gas was bubbled through the solution for 30 minutes. After completion (TLC, 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (Teflon filter with syringe adapter, 0 45 µm) The filtrate was concentrated on a rotary evaporator and dried rapidly under high vacuum to give Compound 151 (0.57 g).

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 ml) and N,N-diisopropylethylamine (75 μl, 1 mmol) and PFP-TFA (90 μl, 0.76 mmol) were added. On contact, the reaction mixture turned purple and gradually changed color to orange over the next 30 minutes. The progress of the reaction was monitored by TLC and LCMS. After completion (formation of the PFP ester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction mixture was adjusted to pH=9-10 by adding N,N-diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for ~30 minutes. After completion of the reaction, most of the solvent was removed under reduced pressure. The residue was diluted with CH ₂ Cl ₂ and washed with saturated aqueous NaHCO ₃ then brine. The organic phase was separated, dried over MgSO ₄ , filtered and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10% MeOH in CH ₂ Cl ₂ ) to give Compound 152 (0.35 g, 55%). LCMS and ¹ H NMR data were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The reaction mixture was purified by passing a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon (35 mg) was added. Hydrogen gas was bubbled through the solution for 30 minutes. After completion (TLC, 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (Teflon filter with syringe adapter, 0 .45 µm) The filtrate was concentrated on a rotary evaporator and dried rapidly under high vacuum to give Compound 153 (0.33 g, quant.) LCMS data was consistent with the desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 ml) with stirring under nitrogen atmosphere. To this was added N,N-diisopropylethylamine (65 μl, 0.37 mmol) and PFP-TFA (35 μl, 0.28 mmol). The reaction mixture was stirred under nitrogen for ~30 minutes. On contact, the reaction mixture turned purple and gradually changed color to orange. The pH of the reaction mixture was maintained at pH=9-10 by adding more N,N-diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, most of the solvent was removed under reduced pressure. The residue was diluted with CH ₂ Cl ₂ (50 ml) and washed with saturated aqueous NaHCO ₃ then brine. The organic layer was separated, dried over MgSO ₄ , filtered and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10% MeOH in CH ₂ Cl ₂ to give Compound 154 (0.29 g, 79%). LCMS and ¹ H NMR data were consistent with the desired product.

Oligomeric Compound 155 containing a GalNAc ₃ -6 conjugation group was prepared according to the general methods presented in Example 46. The GalNAc _{3 cluster portion of the GalNAc 3 -6 conjugation group (GalNAc 3 -6 a )} can be combined with any cleavable moiety to produce various conjugating moieties. groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-.

The structure of GalNAc ₃ -6 (GalNAc ₃ -6 _a -CM-) is shown below:

Example 52 Preparation of Oligonucleotide 160 Containing GalNAc ₃ -9

Compound 156 was synthesized according to the method described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156 (18.60 g, 29.28 mmol) was dissolved in methanol (200 ml). Palladium on carbon (6.15 g, loading 10 wt % (dry basis), powdered carbon matrix, wet) was added. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction mixture was filtered through a pad of celite and the pad of celite was thoroughly washed with methanol. The combined filtrate was washed and concentrated to dryness. The residue was purified by silica gel column chromatography and eluted with 5-10% methanol in dichloromethane to give Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M-N] ^- .

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 ml). HBTU (3.65 g, 9.61 mmol) and N,N-diisopropylethylamine (13.73 ml, 78.81 mmol) were added and the reaction mixture was stirred at room temperature for 5 minutes. To this was added a solution of compound 47 (2.96 g, 7.04 mmol). The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was poured into saturated aqueous NaHCO ₃ . The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na ₂ SO ₄ ), filtered and evaporated. The resulting residue was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to give Compound 158 (8.25 g, 73.3%). The structure was confirmed by MS and ¹ H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P ₂ O ₅ under reduced pressure. The dried compound was dissolved in anhydrous DMF (50 ml). To this was added 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 ml, 3.81 mmol), and cyanoethyl-N,N,N,N-tetraisopropylphosphorodiamidite (3, 65 ml, 11.50 mmol). The reaction mixture was stirred under argon for 4 hours. The reaction mixture was diluted with ethyl acetate (200 ml). The reaction mixture was washed with saturated NaHCO ₃ solution and brine. The organic phase was separated, dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90% ethyl acetate in hexane to give Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and ³¹ P NMR analysis.

Oligomeric compound 160 containing a GalNAc ₃ -9 conjugating group was prepared using standard methods for the synthesis of oligonucleotides. Three units of compound 159 were bound to a solid support, then to nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous ammonia provided compound 160. The GalNAc _{3 cluster portion of the GalNAc 3 -9 conjugation group (GalNAc 3 -9 a )} can be combined with any cleavable moiety to form various conjugation groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GalNAc ₃ -9 (GalNAc ₃ -9 _a -CM) is shown below:

Example 53 Alternative Preparation of Compound 18 (GalNAc ₃ -1a and GalNAc ₃ -3a)

Lactone 161 was reacted with diaminopropane (3-5 eq.) or mono-Boc-protective diaminopropane (1 eq.) to give alcohol 162a or 162b. Using unprotected propandnam for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected with CbzCl to give 162b as a white solid after purification by column chromatography. Alcohol 162b was then reacted with compound 4 in the presence of TMSOTf to give 163a, which was converted to 163b by removing the Cbz group by catalytic hydrogenation. Pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid compound 113 (see Example 48) with PEP-TFA (3.5 eq.) and pyridine (3.5 eq.) in DMF (0.1 to 0.5 M) . Triester 164 reacted directly with amine 163b (3-4 eq.) and DIPEA (3-4 eq.) to give Compound 18. Example 4.

Example 54 Alternative Preparation of Compound 18 (GalNAc ₃ and GalNAc ₃ -3a)

The tri-PFP ester 164 was prepared from the acid 113 by the method described above in Example 53, this ester was reacted with a mono-Boc-protected diamine to give 165 in almost quantitative yield. All groups were removed with hydrochloric acid or trifluoroacetic acid to give a triamine which was reacted with activated acid PFP 166 in the presence of a suitable base such as DIPEA to give Compound 18.

The PFP-protected acid Gal-NAc 166 was prepared from the corresponding acid by treatment with PFP-TFA (1-1.2 eq.) and pyridine (1-1.2 eq.) in DMF. The precursor acid was in turn prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq.) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by reaction with 1,6-hexanediol (or 1,5-hexanediol or other diol for other n values) (2-4 eq) and TMSOTf using the conditions previously described in Example 47.

Example 55 Dose-dependent study of oligonucleotides containing either a 3' or 5' conjugating group (comparison of GalNAc ₃ -1, 3, 8 and 9) targeted to SRB-1 in vivo

The oligonucleotides listed below were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc ₃ conjugating groups was attached to either the 3' or 5' end of the respective oligonucleotide with a phosphodiester-linked 2'-deoxyadenosine nucleoside (cleavable fragment).

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9. The structure of GalNAc ₃ -9 is shown earlier in Example 52. The structure of GalNAc ₃ -3 is shown earlier in Example 39. The structure of GalNAc ₃ -8 is shown earlier in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below of ISIS 353382, 655861, 664078, 661161, 665001 or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 27, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. Indeed, antisense oligonucleotides containing phosphodiester-linked GalNAc ₃ -1 and GalNAc ₃ -9 conjugates at the 3' end (ISIS 655861 and ISIS 664078), as well as GalNAc ₃ -3 and GalNAc ₃ -8 conjugates linked at the 5'- end (ISIS 661161 and ISIS 665001) show a significant improvement in performance over the unconjugated antisense oligonucleotide (ISIS 353382). In addition, ISIS 664078 containing a GalNAc ₃ -9 conjugate at the 3' end was essentially as effective as ISIS 655861 which contains a GalNAc ₃ -1 conjugate at the 3' end. The 5'-conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001, containing GalNAc ₃ -3 or GalNAc ₃ -9, respectively, have increased potency compared to 3'-conjugated antisense compounds (ISIS 655861 and ISIS 664078).

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group. The values of ALT, AST, total bilirubin and BUN are presented in the table below.

Example 56 Dose-dependent study of oligonucleotides containing either a 3' or 5' conjugating group (comparison of GalNAc ₃ -1, 2, 3, 5, 6, 7 and 10) targeted to SRB-1 in vivo

The oligonucleotides listed below were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc ₃ conjugating groups was attached to the 5' end of the respective oligonucleotide with a phosphodiester-linked 2'-deoxyadenosine nucleoside (cleavable fragment), with the exception of ISIS 655861, in which a GalNAc ₃ conjugating group was attached to the 3' end.

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9. The structure of GalNAc ₃ -2 _a is shown earlier in Example 37. The structure of GalNAc ₃ -3 _a is shown earlier in Example 39. The structure of GalNAc ₃ -5 a is shown earlier in Example 49. The structure of GalNAc _{3-6 a} shown earlier in Example 51. Structure of GalNAc _{3-7 a} shown earlier in Example 48. Structure _of GalNAc 3-10 a shown earlier in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below of ISIS 353382, 655861, 664507, 661161, 666224, 666961, 666981, 666881, or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 30, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. Indeed, conjugated antisense oligonucleotides show a significant increase in potency compared to unconjugated antisense oligonucleotide (ISIS 353382). The 5'-conjugated antisense oligonucleotides show a slight increase in potency compared to the 3'-conjugated antisense oligonucleotide.

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group. ALT, AST, total bilirubin, and BUN values are shown in Table 31 below.

Example 57 In Vivo Duration Study of Oligonucleotides Containing a 3'-Conjugating Group Targeting ApoC III

Mice were injected with the dose indicated below once and the levels of ApoC-III and plasma triglycerides (plasma TG) were monitored for 42 days. The study was performed using in each group 3 transgenic mice that express human AROS-III.

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -0-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9.

As can be seen in the table above, the duration of action is increased with the addition of 3'-conjugating groups, as compared to the unconjugated oligonucleotide. An additional increase in duration of action was observed for mixed PO/PS-conjugated oligonucleotide 647536, compared to PS-only conjugated oligonucleotide 647535.

Example 58 Dose-Dependent Study of Oligonucleotides Containing a 3'-Conjugating Group (Comparison of GaINAc3-1 and GaINAc4-11) Targeting SRB-1 in Vivo

The oligonucleotides listed below were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 was included as an unconjugated standard. Each of the conjugating groups was attached to the 3' end of the respective oligonucleotide with a cleavable fragment of a phosphodiester-linked 2'-deoxyadenosine nucleoside.

The structure of GaINAc ₃ -1 _a is shown earlier in Example 9. The structure of GaINAc ₃ -11 _a is shown earlier in Example 50.

Treatment

Six-week-old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below, compound ISIS 440762, 651900, 663748, or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 34, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. Antisense oligonucleotides containing phosphodiester-linked GaINAc ₃ -1 and GaINAc ₄ -11 conjugates at the 3' end (ISIS 651900 and ISIS 663748) show a significant increase in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). These two conjugated oligonucleotides, GaINAc ₃ -1 and GaINAc ₄ -11, were equally effective.

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "k" means 6,-(S)-CH ₃ bicyclic nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group. ALT, AST, total bilirubin, and BUN values are shown in Table 35 below.

Example 59 Action of GaINAc ₃ -1-Conjugated ASOs Targeting FXI in Vivo

The oligonucleotides listed below were tested in a multiple escalating dose study on antisense inhibition of FXI in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugating groups was attached to the 3' end of the respective oligonucleotide with a cleavable fragment of a phosphodiester-linked 2'-deoxyadenosine nucleoside.

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GaINAc ₃ -1 _a is shown earlier in Example 9.

Treatment

Six-week-old male Balb/c mice (Jackson Laboratory, Bar Harbor, Maine) were injected subcutaneously twice weekly for 3 weeks at the dose below with compound ISIS 404071, 656172, 656173 or PBS as control. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic FXI mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. Plasma FXI protein levels were also measured by enzyme-linked immunosorbent assay. FXI mRNA levels were determined relative to total RNA (using RIBOGREEN®), then normalized to a PBS-treated control. The results shown below are presented as the average percentage of FXI mRNA levels for each experimental group. The data was normalized to a PBS-treated control and labeled "% PBS". ED50S was measured by the same methods as described previously and they are presented below.

As shown in Table 37, treatment with anti-smeel oligonucleotides reduces FXI mRNA levels in a dose-dependent manner. Oligonucleotides containing a 3'-GaINAc ₃ -1 conjugating group show a significant increase in potency compared to an unconjugated antisense oligonucleotide (ISIS 404071). Between these two conjugated oligonucleotides, further efficiency gains were achieved by replacing some of the PS bonds with POs (ISIS 656173).

As shown in Table 37a, treatment with antisense oligonucleotides reduces FXI protein levels in a dose-dependent manner. Oligonucleotides containing a 3'-GaINAc ₃ -1 conjugating group show a significant increase in potency compared to an unconjugated antisense oligonucleotide (ISIS 404071). Between these two conjugated oligonucleotides, further efficiency gains were achieved by replacing some of the PS bonds with POs (ISIS 656173).

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin, total albumin, CRE, and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group. The values of ALT, AST, total bilirubin and BUN are presented in the table below.

Example 60 In vitro activity of conjugated ASOs targeting SRB-1

The oligonucleotides listed below were tested in a multiple escalating dose study on antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated standard. Each of the conjugating groups was attached to the 3' or 5' end of the respective oligonucleotide with a cleavable fragment of a phosphodiester-linked 2'-deoxyadenosine nucleoside.

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (P8); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The GaINAc ₃ -1 _a structure is shown earlier in Example 9. The GaINAc ₃ -3a structure is shown earlier in Example 39. The GaINAc ₃ -8a structure is shown earlier in Example 47. The GaINAc ₃ -9a structure is shown earlier in Example 52. The GaINAc ₃ -6a structure shown earlier in Example 51. The structure of GaINAc ₃ -2a is shown earlier in Example 37. The structure of GaINAc ₃ -10a is shown earlier in Example 46. The structure of GaINAc ₃ -5a is shown earlier in Example 49. The structure of GaINAc ₃ -7a is shown earlier in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67, or 20 nM modified oligonucleotide. After treatment for about 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR, and SRB-1 III mRNA levels were adjusted according to total RNA measured by RIBOGREEN®.

IC50 was calculated according to standard methods and the results are presented in Table 40. The results show that under free uptake conditions, in which no reagents or electropulse techniques were used to artificially accelerate the entry of oligonucleotides into cells, oligonucleotides containing the GaINAc conjugate were significantly more effective in hepatocytes than the original oligonucleotide (ISIS 353382), which does not contain the GaINAc conjugate.

Example 61 Preparation of Oligomeric Compound 175 Containing GaINAc ₃ -12

Compound 169 is commercially available. Compound 172 was prepared by adding 6-enzyl(perfluorophenyl)glutarate to compound 171. Benzyl(perfluorophenyl)glutarate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric Compound 175 containing the GaINAc ₃ -12 conjugation group was prepared from compound 174 by the general methods presented in Example 46. The GaINAc _{3 cluster portion of the GaINAc 3 -12 conjugation group (GaTNAc 3 -12a )} can be combined with any cleavable moiety to obtain different conjugating groups. In some embodiments, the cleavable moiety is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -12 (GaINAc ₃ -12 _a -CM-) is shown below:

Compound 176 was prepared according to the general method presented in Example 2. Oligomeric compound 180 containing the GaINAc ₃ -13 conjugation group was obtained from compound 177 according to the general methods presented in Example 49. The GaINAc ₃ cluster portion of the GaINAc ₃ -13 conjugation group (GaINAc ₃ -13 _a ) can be combined with any cleavable moiety to obtain various conjugating groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc _3-13 (GaINAc ₃ -13 _a -CM-) is shown below:

Example 63 Preparation of Oligomeric Compound 188 Containing GaINAc ₃ -14

Compounds 181 and 185 are commercially available. Oligomeric Compound 188 containing the GaINAc ₃ -14 conjugation group was prepared from compound 187 by the general methods presented in Example 46. The GaINAc _{3 cluster portion of the GaINAc 3 -14 conjugation group (GaINAc 3 -14 a )} can be combined with any cleavable moiety to obtaining various conjugating groups. In some embodiments, the cleavable moiety is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -14 (GaINAc ₃ -14 _a -CM-) is shown below:

Example 64 Preparation of Oligomeric Compound 197 Containing GaINAc ₃ -15

Compound 189 is commercially available. Compound 195 was obtained according to the general method presented in Example 31. Oligomeric compound 197 containing the conjugating group GaINAc ₃ -15 was obtained from compounds 194 and 195 using standard methods for the synthesis of oligonucleotides. The GaINAc _{3 cluster portion of the GaINAc 3 -15 conjugating group (GaINAc 3 -15 a )} can be combined with any cleavable moiety to form different conjugating groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -15 (GaINAc ₃ -15 _a -CM-) is shown below:

Example 65 Dose-dependent study of oligonucleotides containing a 5'-conjugating group (comparison of GaINAc ₃ -3, 12, 13, 14 and 15) targeted to SRB-1 in vivo

The oligonucleotides listed below were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GaINAc ₃ conjugating groups was attached to the 5' end of the respective oligonucleotide with a cleavable fragment of a phosphodiester-linked 2'-deoxyadenosine nucleoside.

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GaINAc ₃ -3 _a is shown earlier in Example 39. The structure of GaINAc ₃ -12a is shown earlier in Example 61. The structure of GaINAc ₃ -13a is shown earlier in Example 62. The structure of GaINAc ₃ -14a is shown earlier in Example 63. The structure of GaINAc ₃ -15a shown earlier in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Maine) were injected once or twice subcutaneously with the dose below, compound ISIS 353382, 661161, 671144, 670061, 671261, 671262, or saline. Mice dosed twice were given a second dose three days after the first dose. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RTBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 42, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. No significant difference in target knockdown was observed between single-dose and two-dose animals (see ISIS 353382 at 30 and 2 x 15 mg/kg; and ISIS 661161 at 5 and 2 x 2.5 mg/kg ). Antisense oligonucleotides containing phosphodiester-linked conjugates GaINAc ₃ -3, 12, 13, 14 and 15 show a significant increase in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group (data not shown). ALT, AST, total bilirubin and BUN values are presented in Table 43 below.

Example 66 Effect of Various Cleavable Moieties on In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing the 5'-GaINAc ₃ Cluster

The oligonucleotides listed below were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice. Each of the GaINAc ₃ conjugating groups was attached to the 5' end of the respective oligonucleotide with a phosphodiester-linked nucleoside (cleavable fragment (CM)).

Capital letters indicate the nitrogenous base for each nucleoside, and ^m C stands for 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GaINAc ₃ -3 _a is shown earlier in Example 39. The structure of GaINAc ₃ -13a is shown earlier in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below of ISIS 661161, 670699, 670700, 670701, 671165 or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 45, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. All antisense oligonucleotides containing different cleavable fragments show the same efficiency.

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group (data not shown). ALT, AST, total bilirubin, and BUN values are shown in Table 46 below.

Oligomeric compound 199 containing a GaINAc ₃ -16 conjugation group was prepared according to the general methods presented in Examples 7 and 9. The GaINAc _{3 cluster portion of the GaINAc 3 -16 conjugation group (GaINAc 3 -16 a )} can be combined with any cleavable moiety to obtain various conjugating groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -16 (GaINAc ₃ -16 _a -CM-) is shown below:

Oligomeric compound 200 containing a GaINAc ₃ -17 conjugation group was prepared according to the general methods presented in Example 46. The GaINAc _{3 cluster portion of the GaINAc 3 -17 conjugation group (GaINAc 3 -17 a )} can be combined with any cleavable moiety to produce various conjugating moieties. groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -17 (GaINAc ₃ -17 _a -CM-) is shown below:

Example 69 Preparation of Oligomeric Compound 201 Containing GaINAc ₃ -18

Oligomeric compound 201 containing a GaINAc ₃ -18 conjugation group was prepared according to the general methods presented in Example 46. The GaINAc _{3 cluster portion of the GaINAc 3 -18 conjugation group (GaINAc 3 -18 a )} can be combined with any cleavable moiety to produce various conjugating moieties. groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -18 (GaINAc ₃ -18 _a -CM-) is shown below:

Example 70 Preparation of Oligomeric Compound 204 Containing GaINAc ₃ -19

Oligomeric Compound 204 containing the GaINAc ₃ -19 conjugation group was prepared from compound 64 by the general methods presented in Example 52. The GaINAc _{3 cluster portion of the GaINAc 3 -19 conjugation group (GaINAc 3 -19 a )} can be combined with any cleavable moiety to obtaining various conjugating groups. In some embodiments, the cleavable moiety is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -19 (GaINAc ₃ -19 _a -CM-) is shown below:

Example 71 Preparation of Oligomeric Compound 210 Containing GaINAc ₃ -20

Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which was prepared by adding trifluoroacetic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80°C, then cooled to room temperature. Oligomeric Compound 210 containing the GaINAc ₃ -20 conjugation group was prepared from compound 208 by the general methods presented in Example 52. The GaINAc _{3 cluster portion of the GaINAc 3 -20 conjugation group (GaINAc 3 -20 a )} can be combined with any cleavable moiety to obtaining various conjugating groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -20 (GaINAc ₃ -20 _a -CM-) is shown below:

Example 72 Preparation of Oligomeric Compound 215 Containing GaINAc ₃ -21

Compound 211 is commercially available. Oligomeric Compound 215 containing a GaINAc ₃ -21 conjugation group was prepared from compound 213 by the general methods presented in Example 52. The GaINAc _{3 cluster portion of the GaINAc 3 -21 conjugation group (GaINAc 3 -21 a )} can be combined with any cleavable moiety to obtaining various conjugating groups. In some embodiments, the cleavable fragment is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GaINAc ₃ -21 (GaINAc ₃ -21 _a -CM-) is shown below:

Example 73 Preparation of Oligomeric Compound 221 Containing GaINAc ₃ -22

Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide. Oligomeric compound 221 containing a GalNAc ₃ -21 conjugation group was prepared from compound 220 by the general method shown in Example 52. The GalNAc _{3 cluster portion of the GalNAc 3 -22 conjugation group (GalNAc 3 -22 a )} can be combined with any cleavable moiety to obtaining various conjugating groups. In some embodiments, the cleavable moiety is -P(=O)(OH)-A _d -P(=O)(OH)-. The structure of GalNAc ₃ -22 (GalNAc ₃ -22 _a -CM-) is shown below:

Example 74 Effect of Various Cleavable Moieties on In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing 5'-GalNAc ₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study i antisense inhibition of SRB-1 in mice. Each of the GalNAc ₃ conjugating groups was attached to the 5' end of the corresponding oligonucleotide.

In all tables, capital letters indicate the nitrogenous base for each) nucleoside, and ^m C means 5-methylcytosine. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"s" means thiophosphate internucleoside bond (PS); "o" means a phosphodiester internucleoside bond (PO); and "o'" means -O-P(=O)(OH)-. Conjugate groups are shown in bold.

The structure of GalNAc ₃ -3 _a is shown earlier in Example 39. The structure of GalNAc ₃ -17a is shown earlier in Example 68 and the structure of GalNAc ₃ -18a is shown earlier in Example 69.

Treatment

Six to eight week old C57dl6 mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below) of the oligonucleotide listed in Table 47 or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 48, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner. Antisense oligonucleotides containing a GalNAc conjugate show equal potency and are significantly more effective than the original oligonucleotide lacking a GalNAc conjugate.

Serum levels of liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured relative to saline-injected mice using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group (data not shown). ALT, AST, total bilirubin, and BUN values are shown in Table 49 below.

Example 75 Pharmacokinetic Analysis of Oligonucleotides Containing a 5'-Conjugating Group

The FA of ASO shown in Tables 41, 44 and 47 above was evaluated using liver samples obtained after performing the treatments described in Examples 65, 66 and 74. Liver samples were minced and extracted according to standard protocols and analyzed using IP- HPLC-MS along with internal standard. The total level (μg/g) of all metabolites in tissue was measured by integrating the corresponding UV peaks, and tissue levels of full-length ASO containing no conjugate ("base", in this case Isis #353382) were measured using appropriate ion-extraction chromatograms ( EIC).

The results presented in Table 50 above demonstrate that higher levels were observed in the liver of oligonucleotides containing the GalNAc3 conjugation group than the original oligonucleotide not containing the GalNAc ₃ conjugation group (ISIS 353382) 72 hours after the administration of the oligonucleotide, especially considering the administration of various doses for oligonucleotides with and without a GalNAc ₃ conjugate group. In addition, after 72 hours, 40-98% of each oligonucleotide containing a GalNAc ₃ conjugating group was metabolized to the parent compound, indicating that the GalNAc ₃ conjugating groups were cleaved in these oligonucleotides.

Example 76 Preparation of Oligomeric Compound 230 Containing GalNAc ₃ -23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500 ml) for 16 hours. The reaction mixture was then evaporated to an oil, dissolved in EtOAc and washed with water, saturated NaHCO ₃ solution, brine and dried over Na ₂ SO ₄ . Ethyl acetate was concentrated to dryness and purified by column chromatography, eluting with EtOAc in hexanes (1:1) then 10% methanol in CH ₂ Cl ₂ to give compound 223 as a colorless oil. The LCMS and NMR data were consistent with the indicated structure. 10 g (32.86 mmol) of 1-tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO (100 mP) at room temperature for 17 hours. The reaction mixture was then poured into water and extracted with EtOAc. The organic layer was washed three times with water and dried over Na ₂ SO ₄ . The organic layer was concentrated to dryness to give 5.3 g of compound 224 (92%). The LCMS and NMR data were consistent with the indicated structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4A molecular sieves (5 g) and TMSOTf (1.65 mp, 9.11 mmol) in dichloromethane (100 ml) under an inert atmosphere. After 14 hours the reaction mixture was filtered to remove sieves and the organic layer was washed with saturated NaHCO ₃ , water, brine and dried over Na ₂ SO ₄ . The organic layer was concentrated to dryness and purified by column chromatography, eluting with a gradient of 2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMR data were consistent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in EtOAc/methanol (4:1, 250 mL) over a Pearlman catalyst. After 8 hours the catalyst was removed by filtration and the solvents were removed to dryness to give compound 226. LCMS and NMR data were consistent with the structure.

To obtain compound 227, a solution of nitromethanetripropionic acid (4.17 g, 15.04 mmol) and Hunig's base (10.3 ml, 60.17 mmol) in DMF (100 ml) was treated dropwise with pentafluorotrifluoro acetate (9.05 ml, 52 .65 mmol). After 30 minutes the reaction mixture was poured into ice water and extracted with EtOAc. The organic layer was washed with water, brine and dried over Na ₂ SO ₄ . The organic layer was concentrated to dryness and then recrystallized from heptane to give compound 227 as a white solid. The LCMS and NMR data were consistent with the indicated structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 ml) for 2 hours. The reaction mixture was then evaporated to dryness and purified by column chromatography eluting with a gradient of 2 to 10% methanol in dichloromethane to give compound 228. LCMS and NMR data were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney nickel (ca. 2 g, wet) in ethanol (100 ml) under a hydrogen atmosphere. After 12 hours the catalyst was removed by filtration and the organic layer was evaporated to a solid which was used directly in the next step. The LCMS and NMR data were consistent with the indicated structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 µl, 1 .6 mmol) in DMF (5 ml). After 16 hours, DMF was removed under reduced pressure at 65°C to an oily substance, and this oily substance was dissolved in dichloromethane. The organic layer was washed with saturated NaHCO ₃ , brine and dried over Na ₂ SO ₄ . After evaporation of the organic layer, the compound was purified by column chromatography and eluted with a gradient of 2 to 20% methanol in dichloromethane to give the bound product. The LCMS and NMR data were consistent with the indicated structure. The benzyl ether was deprotected on a Pearlman catalyst under a hydrogen atmosphere for 1 hour. The catalyst was then removed by filtration and the solvents were removed to dryness to give the acid. The LCMS and NMR data were consistent with the indicated structure. This acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 ml). Pyridine (53.61 µl, 0.66 mmol) was added and the reaction mixture was purged with argon. Pentafluorotrifluoroacetate (46.39 µl, 0.4 mmol) was slowly added to the reaction mixture. The color of the reaction mixture changed from pale yellow to burgundy, and a light haze appeared, which escaped with a stream of argon. The reaction mixture was allowed to stir at room temperature for one hour (completion of the reaction was confirmed by LCMS). The solvent was removed under reduced pressure (rotary evaporator) at 70°C. The residue was diluted with DCM and washed with 1N. NaHSO ₄ , brine, brine, and again brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give 225 mg of compound 229 as a brittle yellow foam. The LCMS and NMR data were consistent with the indicated structure.

Oligomeric Compound 230 containing a GalNAc ₃ -23 conjugation group was prepared from compound 229 by the general methods presented in Example 46. The GalNAc _{3 cluster portion of the GalNAc 3 -23 conjugation group (GalNAc 3 -23 a )} can be combined with any cleavable moiety to obtaining various conjugating groups. The structure of GalNAc ₃ -23 (GalNAc ₃ -23 _a -CM) is shown below:

Example 77 In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing a GalNAc ₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, GalNAc ₃ -3 _a is shown in Example 39, GalNAc ₃ -9a is shown in Example 52, GalNAc ₃ -10a is shown in Example 46, GalNAc ₃ -19 _a is shown in Example 70, GalNAc ₃ -20 _a is shown in Example 71 and GalNAc ₃ -23 _a is shown in Example 76.

Treatment

Six to eight week old C57dl6 mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below, the oligonucleotide listed in Table 51, or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Table 52, treatment with anti-smeel oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner.

Hepatic transaminase, alanine aminotransferase (ALT), and serum aspartate aminotransferase (AST) levels were also measured using standard protocols. Total bilirubin and blood urea nitrogen (BUN) were also assessed. The change in body weight was checked, which was not significantly different from the saline group (data not shown). ALT, AST, total bilirubin and BUN values are presented below in

Example 78 In Vivo Antisense Inhibition by Angiotensinogen Targeting Oligonucleotides Containing a GalNAc ₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent antisense angiotensinogen (AGT) inhibition study in normotensive Sprague-Dawley mice.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9.

Treatment

Six-week-old male Sprague-Dawley rats were injected subcutaneously once a week with the dose below, for a total of three doses of the oligonucleotide listed in Table 54, or PBS. Each experimental group consisted of 4 animals. Rats were sacrificed 72 hours after the last dose. Liver AGT mRNA levels were measured using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, OR) according to standard protocols. Plasma AGT protein levels were measured using a total angiotensinogen enzyme-linked immunosorbent assay (Cat. No. JP27412, IBL International, Toronto, Ontario) with a plasma dilution of 1:20,000. The results shown below are presented as the mean percentage of liver AGT mRNA levels or plasma AGT protein levels for each experimental group, normalized to PBS control.

As shown in Table 55, treatment with antisense oligonucleotides reduced hepatic AGT mRNA levels and plasma protein levels in a dose-dependent manner, and the oligonucleotide containing the GalNAc conjugate was significantly more effective than the original oligonucleotide not containing the GalNAc conjugate.

Hepatic transaminase, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) plasma levels were also measured, as well as body weight at the time of sacrifice using standard protocols. The results are presented below in Table 56.

Example 79 Duration of In Vivo Action of Oligonucleotides Targeting APOC-III Containing a GalNAc ₃ Conjugate

The oligonucleotides listed in Table 57 below were tested in a single dose duration study in mice.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, GalNAc ₃ -3 _a is shown in Example 39, GalNAc ₃ -7 _a is shown in Example 48, GalNAc ₃ -10 _a is shown in Example 46, and GalNAc ₃ -13 _a is shown in Example 62.

Treatment

Six to eight week old transgenic mice expressing human APOC-III were given a single subcutaneous injection of the oligonucleotide listed in Table 57 or PBS. Each experimental group consisted of 3 animals. Blood samples were taken pre-dose to determine baseline, and 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks post-dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results shown below are presented as the mean percentage of triglyceride and APOC-III plasma levels for each experimental group, normalized and baseline, and they demonstrate that oligonucleotides containing the GalNAc conjugation group have a longer duration of action than the original oligonucleotide without the conjugation group (ISIS 304801) even though the dose of the original compound was three times higher than the dose of the oligonucleotides containing the GalNAc conjugation group.

Example 80 In vivo antisense inhibition by oligonucleotides targeting alpha-1 antitrypsin (A1AT) containing a GalNAc ₃ conjugate

The oligonucleotides listed in Table 59 below were tested in a dose dependent A1AT inhibition study in mice.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, GalNAc ₃ -3 _a is shown in Example 39, GalNAc ₃ -7 _a is shown in Example 48, GalNAc ₃ -10 _a is shown in Example 46, and GalNAc ₃ -13 _a is shown in Example 62.

Treatment

Six week old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) were injected subcutaneously once a week with the dose below, for a total of three doses of the oligonucleotide listed in Table 59 or PBS. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection. Liver A1AT mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. Plasma A1AT protein levels were determined using an enzyme-linked immunosorbent assay for mouse alpha 1 antitrypsin (Cat. No. 41-A1AMS-E01, Alpco, Salem, NH). The results shown below are presented as the mean percentage of liver A1AT mRNA and plasma protein levels for each experimental group, normalized to the PBS control.

As shown in Table 60, antisense oligonucleotide treatment reduces hepatic A1AT mRNA levels and plasma A1AT protein levels in a dose-dependent manner. Oligonucleotides containing the GalNAc conjugate were significantly more effective than the parent compound (ISIS 476366).

At the time of sacrifice, liver transaminase and plasma BUN levels were measured according to standard protocols. Body weights and organ weights were also measured. The results are shown in Table 61 below. Body weight is presented as % of baseline. Organ weights are presented as % of body weight relative to the PBS control group.

Example 81 In vivo duration of action of A1AT-targeted oligonucleotides containing the GalNAc ₃ cassette

The oligonucleotides listed in Table 59 were tested in a single dose duration study in mice.

Treatment

Six week old male C57BL/6 mice were given a single subcutaneous injection of the oligonucleotide listed in Table 59 or PBS. Each experimental group consisted of 4 animals. Blood samples were taken the day before the dose to determine the initial value, as well as 5, 12, 19 and 25 days after | dose administration. Plasma A1AT protein levels were measured by enzyme-linked immunosorbent assay (see Example 80). The results shown below are presented as the average percentage of A1AT protein levels in plasma for each experimental group, normalized to baseline levels. The results demonstrate that the oligonucleotides containing the GalNAc conjugate were more effective and lasted longer than the parent compound without the GalNAc conjugate (ISIS 476366). In addition, oligonucleotides containing 5'-GalNAc conjugate (ISIS 678381, 678382, 678383 and 678384) were, in general, even more effective with even longer duration of action than oligonucleotide containing 3'-GalNAc conjugate (ISIS 656326).

Example 82 In Vitro Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing a GalNAc ₃ Conjugate

Primary mouse liver hepatocytes were plated in 96-well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 63 were added at 2, 10, 50 or 250 nM in William E medium and the cells were incubated overnight at 37° C. in 5% CO ₂ . Cells were lysed 16 hours after adding the oligonucleotide and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. IC ₅₀ values were determined using the Prism 4 program (GraphPad). The results demonstrate that oligonucleotides containing a variety of different GalNAc conjugation groups and a variety of different cleavable moieties are significantly more efficient in an in vitro free uptake experiment than the original oligonucleotides lacking a GalNAc conjugation group (ISIS 353382 and 666841).

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, GalNAc ₃ -3 _a is shown in Example 39, GalNAc ₃ -5 _a is shown in Example 49, GalNAc ₃ -6 _a is shown in Example 51, GalNAc ₃ -7 _a is shown in Example 48, GalNAc ₃ -8 _a is shown in Example 47, GalNAc ₃ -9 _a is shown in Example 52, GalNAc ₃ -10 _a is shown in Example 46, GalNAc ₃ -12 _a is shown in Example 61, GalNAc ₃ -13 _a is shown in Example 62, GalNAc ₃ -14 _a shown in Example 63, GalNAc ₃ -15 _a shown in Example 64, GalNAc ₃ -17 _a shown in Example 68, GalNAc ₃ -18 _a shown in Example 69, GalNAc ₃ -19 _a shown in Example 62 70, GalNAc ₃ -20 _a is shown in Example 71 and GalNAc ₃ -23 _a is shown in Example 76.

Example 83 In Vivo Antisense Inhibition by Factor XI Targeting Oligonucleotides Containing the GalNAc ₃ Cluster

The oligonucleotides listed in Table 64 below were tested in a dose dependent factor XI inhibition study in mice.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, GalNAc ₃ -3 _a is shown in Example 39, GalNAc ₃ -7 _a is shown in Example 48, GalNAc ₃ -10 _a is shown in Example 46, and GalNAc ₃ -13 _a is shown in Example 62.

Treatment

Six to eight week old mice were injected subcutaneously once a week with the dose below, for a total of three doses of the oligonucleotide listed below, or PBS. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last dose. Liver factor XI mRNA levels were measured by real-time PCR and normalized to cyclophilin according to standard protocols. Liver transaminases, BUN and bilirubin were also measured. The results shown below are presented as the average percentage for each experimental group, normalized to the PBS control.

As shown in Table 65, treatment with antisense oligonucleotides reduces hepatic factor XI mRNA levels in a dose-dependent manner. The results demonstrate that the oligonucleotides containing the GalNAc conjugate were more effective than the parent compound without the GalNAc conjugate (ISIS 404071). In addition, oligonucleotides containing 5'-GalNAc conjugate (ISIS 663086, 678347, 678348 and 678349) were even more effective than oligonucleotide containing 3'-GalNAc conjugate (ISIS 656173).

Example 84 Duration of In Vivo Action of Oligonucleotides Targeting Factor XI Containing GalNAc ₃ Conjugate

The oligonucleotides listed in Table 64 were tested in a single dose duration study in mice.

Treatment

Six to eight weeks old mice were given a single subcutaneous injection of the oligonucleotide listed in Table 64 or PBS. Each experimental group consisted of 4 animals. Blood samples were taken from the tail vein the day before dosing to determine the initial value, as well as 3, 10 and 17 days after dosing. Plasma factor XI protein levels were determined by enzyme-linked immunosorbent assay using immobilized and biotinylated factor XI detection antibodies from R&D Systems, Minneapolis, Minnesota (p/n AF2460 and p/n BAF2460, respectively) and OptEIA reagent kit B (p/n. No. 550534, BD Biosciences, San Jose, CA). The results shown below are presented as the mean percentage of factor XI plasma protein levels for each treatment group, normalized to baseline levels. The results demonstrate that the oligonucleotides containing the GalNAc conjugate were more effective and lasted longer than the parent compound without the GalNAc conjugate (ISIS 404071). In addition, oligonucleotides containing 5'-GalNAc conjugate (ISIS 663086, 678347, 678348 and 678349) were even more effective with even longer duration of action than oligonucleotide containing 3'-GalNAc conjugate (ISIS 656173).

Example 85 In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing a GalNAc ₃ Conjugate

The oligonucleotides listed in Table 63 were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were injected subcutaneously once a week with the dose below, for a total of three doses of the oligonucleotide listed in Table 63 or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 48 hours after the last injection to determine hepatic SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. The results shown below are presented as the average percentage of liver SRB-1 mRNA levels for each experimental group, normalized to a saline treated control.

As shown in Tables 67 and 68, treatment with antisense oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner.

The levels of liver transaminase, total bilirubin, BUN, and body weight were also measured according to standard protocols. The mean values for each experimental group are presented below in Table 69.

Example 86 In Vivo Antisense Inhibition by TTR Targeting Oligonucleotides Containing the GalNAc ₃ Cluster

The oligonucleotides listed in Table 70 below were tested in a dose-dependent human transthyretin (TTR) antisense inhibition study in transgenic mice expressing the human TTR gene.

Treatment

Eight-week-old TTR transgenic mice once a week for three weeks, for a total of three doses, were injected subcutaneously with the oligonucleotide and the doses listed in the tables below, or with PBS. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection. During the experiment, at various time points, blood samples were taken from the tail vein and plasma levels of TTR protein, ALT and AST were measured, which are presented in Tables 72-74. Plasma levels of ALT, AST, and human TTR were measured after sacrifice, as well as body weights, organ weights, and human TTR mRNA levels in the liver. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc., Eugene, OR) were used according to standard protocols to determine liver levels of human TTR mRNA. The results shown in Tables 71-74 are the mean values for each experimental group. mRNA levels are averages relative to the average for the PBS group. Plasma protein levels are mean values relative to the mean of the PBS group at baseline. Body weights are the average percentage change in weight from baseline to sacrifice for each individual experimental group. The reported organ weights are normalized to the body weight of the animal, and then the mean normalized organ weight for each experimental group is presented relative to the mean normalized organ weight for the PBS group.

In Tables 71-74, "BL" refers to baseline measurements taken just before the first dose. As shown in Tables 71 and 72, treatment with antisense oligonucleotides reduces TTR expression levels in a dose-dependent manner. Oligonucleotides containing the GalNAc conjugate were more effective than the parent compound without the GalNAc conjugate (ISIS 420915). In addition, oligonucleotides containing a GalNAc conjugate and mixed PS/PO internucleoside bonds were even more effective than an oligonucleotide containing a GalNAc conjugate and only PS bonds.

The legend for Table 72 is shown in Example 74. The structure of GalNAc ₃ -1 is shown in Example 9. The structure of GalNAc ₃ -3 _a is shown in Example 39. The structure of GalNAc ₃ -7 _a is shown in Example 48. The structure of GalNAc ₃ -10 _a is shown in Example 46. The structure of GalNAc ₃ -13 _a is shown in Example 62. The structure of GalNAc ₃ -19 _a is shown in Example 70.

Example 87 Duration of In Vivo Action of Single Doses of TTR Targeting Oligonucleotides Containing the GalNAc ₃ Cluster

ISIS Nos. 420915 and 660261 (see Table 70) were tested in a single dose duration study in mice. ISIS Nos. 420915, 682883 and 682885 (see Table 70) were also tested in a single dose duration study in mice.

Treatment

Eight week old male transgenic mice that express human TTR were given a single subcutaneous injection of 100 mg/kg ISIS #420915 or 13.5 mg/kg ISIS #660261. Each experimental group consisted of 4 animals. Blood samples were taken from the tail vein before dosing for baseline measurements and on days 3, 7, 10, 17, 24 and 39 post-dose. Plasma TTR protein levels were measured as described in Example 86. The results shown below are presented as the average percentage of plasma TTR levels for each experimental group, normalized to baseline levels.

Treatment

Eight week old female transgenic mice that express human TTR were given a single subcutaneous injection of 100 mg/kg ISIS #420915, 10.0 mg/kg ISIS #682883, or 10.0 mg/kg ISIS #682885. Each experimental group consisted of 4 animals. Blood samples were taken from the tail vein before dosing for baseline measurements and on days 3, 7, 10, 17, 24 and 39 post-dose. Plasma TTR protein levels were measured as described in Example 86. The results shown below are presented as the average percentage of plasma TTR levels for each experimental group, normalized to baseline levels.

The results in Tables 75 and 76 demonstrate that the oligonucleotides containing the GalNAc conjugate are more effective and have a longer duration of action than the original oligonucleotide without the conjugate (ISIS 420915).

Example 88 In Vivo Splicing Modulation by SMN-Targeting Oligonucleotides Containing a GalNAc3 Conjugate

The oligonucleotides listed in Table 77 were tested for splicing modulation of human survival motor neuron (SMN) genes in mice.

The structure of GalNAc ₃ -7 _a is shown previously in Example 48. "X" is a 5'-primary amine obtained from Gene Tools (Philomat, Oregon), and GalNAc ₃ -7 _b is a GalNAc ₃ -7 _a structure containing no part -NH-C ₆ -O bond as shown below:

ISIS Nos. 703421 and 703422 are morpholino oligonucleotides, with each nucleotide of the two oligonucleotides being a morpholino nucleotide.

Treatment

Six-week-old transgenic mice expressing human SMN were given a single subcutaneous injection of oligonucleotide a listed in Table 78 or saline. Each experimental group consisted of 2 males and 2 females. Mice were sacrificed 3 days post-dose to determine liver levels of human SMN mRNA with and without exon 7 using real-time PCR according to standard protocols. Total RNA was measured using the Ribogreen reagent. SMN mRNA levels were normalized to total mRNA and then normalized to the average values in the saline treated group. The resulting average ratios of exon 7-containing SMN mRNA to exon-7-free SMN mRNA are shown in Table 78. The results demonstrate that fully modified oligonucleotides that modulate splicing and contain a GalNAc conjugate are significantly more effective in altering splicing in the liver, than the original oligonucleotides that do not contain the conjugate GalNAc. In addition, this trend holds for the chemistry of multiple modifications, including 2'-MOE and morpholino-modified oligonucleotides.

Example 89 In vivo antisense inhibition by oligonucleotides targeting apolipoprotein A (Apo(a)) containing a GalNAc ₃ conjugate

The oligonucleotides listed in Table 79 below were tested in a dose dependent Apo(a) inhibition study in transgenic mice.

The structure of GalNAc ₃ -7 _a is shown in Example 48.

Treatment

Eight week old female C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) were injected subcutaneously with the dose below once a week for a total of six doses of the oligonucleotide listed in Table 79 or PBS. Each experimental group consisted of 3-4 animals. Blood samples were taken from the tail vein the day before the first dose and weekly after each dose to determine plasma Apo(a) protein levels. Mice were sacrificed two days after the last injection. Hepatic Apo(a) mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantitation reagent (Molecular Probes, Inc., Eugene, Oregon) according to standard protocols. Plasma Apo(a) protein levels were determined by enzyme-linked immunosorbent assay and liver transaminase levels were determined. The results of the analysis of mRNA and protein in plasma in Table 80 are presented as the average percentage for the experimental group relative to the group treated with PBS. Plasma protein levels were further normalized to baseline (BL) for the PBS group. Mean absolute transaminase levels and body weight (% relative to mean baseline) are presented in Table 81.

As shown in Table 80, oligonucleotide treatment reduces liver Apo(a) mRNA levels and plasma protein levels in a dose-dependent manner. In addition, the oligonucleotide containing the GalNAc conjugate was significantly more effective with a longer duration of action than the original oligonucleotide not containing the GalNAc conjugate. As shown in Table 81, the presented oligonucleotides do not affect transaminase levels and body weight, indicating good tolerability of the oligonucleotides.

Example 90 In Vivo Antisense Inhibition by TTR Targeting Oligonucleotides Containing the GalNAc ₃ Cluster

The oligonucleotides listed in Table 82 below were tested in a dose-dependent human transthyretin (TTR) antisense inhibition study in transgenic mice expressing the human TTR gene.

Treatment

TTR transgenic mice once a week for three weeks for a total of three doses were injected subcutaneously with the oligonucleotide and the doses listed in Table 83 or PBS. Each experimental group consisted of 4 animals. Prior to the first dose, a blood sample was taken from the tail vein to determine plasma TTR protein levels at baseline (BL). Mice were sacrificed 72 hours after the last injection. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc., Eugene, OR) were used according to standard protocols to determine liver levels of human TTR mRNA. The results shown in Table 83 are the averages for each experimental group. mRNA levels are averages relative to the average for the PBS group. Plasma protein levels are mean values relative to the mean of the PBS group at baseline. "BL" refers to baseline measurements taken just before the first dose. As shown in Table 83, treatment with antisense oligonucleotides reduces TTR expression levels in a dose-dependent manner. The oligonucleotide containing the GalNAc conjugate was more effective than the parent compound without the GalNAc conjugate (ISIS 420915), and the oligonucleotides containing the phosphodiester or deoxyadenosine cleavage moiety showed a significant increase in efficiency compared to the parent compound not containing the conjugate (see ISIS no. 682883 and 666943 versus 420915, and also see Examples 86 and 87).

The legend for Table 82 is shown in Example 74. The structure of GalNAc ₃ -3 _a is shown in Example 39. The structure of GalNAc ₃ -7 _a is shown in Example 48. The structure of GalNAc ₃ -10 _a is shown in Example 46. The structure of GalNAc ₃ -13 _a is shown in Example 62.

Example 91 In Vivo Antisense Inhibition by Factor VII Targeting Oligonucleotides Containing a GalNAc ₃ Conjugate in Non-Human Primates

The oligonucleotides listed in Table 84 below were tested in a non-terminal dose escalation study for factor VII antisense inhibition in monkeys.

Treatment

Previously unexperimented monkeys were given subcutaneous injections of increasing doses of the oligonucleotides listed in Table 84 or PBS on days 0, 15 and 29. Each experimental group consisted of 4 males and 1 female. Before the introduction of the first dose and at various time points after it took blood samples to determine the levels of factor VII protein in plasma. Factor VII protein levels were determined by ELISA. The results shown in Table 85 are the mean values for each experimental group relative to the mean value for the PBS group at baseline (BL), measured immediately before the first dose. As shown in Table 85, treatment with antisense oligonucleotides reduces factor VII expression levels in a dose-dependent manner, and the oligonucleotide containing the GalNAc conjugate was significantly more effective in monkeys than the oligonucleotide not containing the GalNAc conjugate.

The legend for Table 84 is shown in Example 74. The structure of GalNAc ₃ -10 a is shown in Example 46.

Example 92 Antisense Inhibition in Primary Hepatocytes by Apo-CIII Targeting Oligonucleotides Containing a GalNAc ₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well and the oligonucleotides listed in Table 86 were added, targeting mouse ApoC-III at a concentration of 0.46, 1.37, 4.12 or 12.35, 37.04 , 111.11 or 333.33 nM, or 1.00 µM. After incubation with oligonucleotides for 24 hours, cells were lysed and total RNA was purified using RNeasy (Qiagen). ApoC-III mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) according to standard protocols. IC ₅₀ values were determined using the Prism 4 software (GraphPad). The results demonstrate that regardless of whether the cleavage fragment is a phosphodiester or a phosphodiester-linked deoxyadenosine, the oligonucleotides containing the GalNAc conjugate were significantly more effective than the original oligonucleotide containing no conjugate.

The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, GalNAc ₃ -3 _a is shown in Example 39, GalNAc ₃ -7 _a is shown in Example 48, GalNAc ₃ -10 _a is shown in Example 46, and GalNAc ₃ -13 _a is shown in Example 62, and GalNAc ₃ -19 _a is shown in Example 70.

Example 93 In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing Mixed Wings and 5'-GalNAc ₃ Conjugate

The oligonucleotides listed in Table 87 were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice.

The structure of GalNAc ₃ -3 _a is shown earlier in Example 39, and the structure of GalNAc ₃ -7 _a is shown earlier in Example 48. Subscripts: "e" means 2'-MOE modified nucleoside; "d" means β-D-2'-deoxyribonucleoside;"k" means 6'-(S)-CH ₃ bicyclic nucleoside (cEt); "s" stands for thiophosphate internucleoside linkages (PS); "o" means phosphodiester internucleoside bonds (RO). The superscript "m" stands for 5-methylcytosines.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of the dose below, the oligonucleotide listed in Table 87, or saline. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection. Liver SRB-1 mRNA levels were measured by real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results shown below are presented as the mean percentage of SRB-1 mRNA levels for each experimental group compared to the saline treated control group. As shown in Table 88, treatment with antisense oligonucleotides reduces SRB-1 mRNA levels in a dose-dependent manner, and gapmer oligonucleotides containing a GalNAc conjugate and having wings that are either all-cEt or mixed sugar modifications were significantly more effective than the original oligonucleotide, conjugate-free and fully cEt modified wings.

Body weights, liver transaminase levels, total bilirubin, and BUN were also measured, and the mean values for each experimental group are presented in Table 88. Body weight is shown as the mean percentage of body weight relative to baseline body weight (% BL) measured immediately before dosing the oligonucleotide .

Example 94 In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing 2'-Sugar Modifications and a 5'-GalNAc ₃ Conjugate

The oligonucleotides listed in Table 89 were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice.

The subscript "m" denotes a 2'-O-methyl modified nucleoside. The full legend to the table is provided in Example 74. The structure of GalNAc ₃ -3 _a is shown earlier in Example 39, and the structure of GalNAc ₃ -7a is shown earlier in Example 48.

Treatment

The study was performed according to the protocol described in Example 93. The results are presented below in Table 90 and demonstrate that both 2'-MOE and 2'-OMe-modified oligonucleotides containing the GalNAc conjugate were significantly more effective than the corresponding original oligonucleotides, not containing a conjugate. The results of measurements of body weight, liver transaminase, total bilirubin and BUN demonstrate that these compounds are well tolerated.

Example 95 In Vivo Antisense Inhibition by SRB-1 Targeting Oligonucleotides Containing Bicyclic Nucleosides and 5'-GalNAc ₃ Conjugate

The oligonucleotides listed in Table 91 were tested in a dose-dependent study of antisense inhibition of SRB-1 in mice.

The subscript "g" means a fluoro-HNA nucleoside, the subscript "l" means a closed nucleoside containing a 2'-O-CH ₂ -4' bridge. Other abbreviations are shown in the table legend of Example 74. The structure of GalNAc ₃ -1 _a is shown earlier in Example 9, the structure of GalNAc ₃ -3 _a is shown earlier in Example 39, and the structure of GalNAc ₃ -7a is shown earlier in Example 48.

Treatment

The study was performed according to the protocol described in Example 93. The results are presented in Table 92 below and demonstrate that oligonucleotides containing the GalNAc conjugate and various bicyclic nucleoside modifications were significantly more effective than the original oligonucleotide containing no conjugate and containing bicyclic nucleoside modifications. In addition, the oligonucleotide containing the conjugate of GalNAc and the fluoro-HNA modification was significantly more effective than the parent compound containing no conjugate and containing the fluoro-HNA modification. The results of measurements of body weight, liver transaminase, total bilirubin and BUN demonstrate that these compounds are well tolerated.

Example 96 Plasma Protein Binding by Antisense Oligonucleotides Containing a GalNAc ₃ Conjugating Group

The oligonucleotides listed in Table 57 targeting ApoC-III and the oligonucleotides in Table 93 targeting Apo(a) were tested in an ultrafiltration assay to assess plasma protein binding.

The table legend is shown in Example 74. The structure of GalNAc ₃ -7a is shown earlier in Example 48.

Ultrafree-MC ultrafiltration elements (nominal molecular weight limit 30,000, low binding regenerated cellulose membrane, Millipore, Bedford, MA) were preconditioned with 300 µl of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, then with 300 µl 300 μg/ml solution of the control oligonucleotide in H ₂ O and centrifuged at 2000 g for 16 minutes. To assess non-specific binding to the filters of each test oligonucleotide from Tables 57 and 93 used in the tests, 300 μl of a 250 ng/ml solution of the oligonucleotide in in at pH 7.4 was placed in preconditioned filters and centrifuged at 2000 g for 16 minutes. Unfiltered and filtered samples were analyzed by enzyme-linked immunosorbent assay to determine the concentrations of oligonucleotides. Three copies were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample was used to determine the percentage of oligonucleotide that passed through the filter without plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from healthy, unmedicated human volunteers, cynomolgus monkeys, and CD-1 mice were purchased from Bioreclamation LLC (Westbury, NY). The test oligonucleotides were added to 1.2 ml plasma aliquots at two concentrations (5 and 150 μg/ml). An aliquot (300 µl) of each labeled plasma sample was placed on a preconditioned filter element and incubated at 37°C for 30 minutes, followed immediately by centrifugation at 2000 g for 16 minutes. Aliquots of filtered and unfiltered labeled plasma samples were analyzed by enzyme-linked immunosorbent assay to determine the concentration of oligonucleotide in each sample. Used three copies of each concentration to determine the average percentage of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample was used to determine the percentage of plasma oligonucleotide not bound to plasma proteins (% unbound). The final unbound oligonucleotide values were corrected for non-specific binding by dividing the % unbound by the % recovery for each oligonucleotide. The final % bound oligonucleotide values were determined by subtracting the final % unbound values from 100. The results are shown in Table 94 for two concentrations of the tested oligonucleotide (5 and 150 μg/ml) in plasma of each species. The results demonstrate that the GalNAc conjugating groups do not significantly affect plasma protein binding. In addition, oligonucleotides containing only PS internucleoside bonds and mixed PO/PS bonds both bind plasma proteins, while oligonucleotides containing only PS bonds bind plasma proteins to a slightly greater extent than oligonucleotides containing mixed PO/PS bonds. .

Example 97 Modified TTR Targeting Oligonucleotides Containing a GalNAc ₃ Conjugation Group

The oligonucleotides shown in Table 95 containing the GalNAc conjugate are designed to affect the TTR.

The legend for Table 95 is shown in Example 74. The structure of GalNAc ₃ -1 is shown in Example 9. The structure of GalNAc ₃ -3 _a is shown in Example 39. The structure of GalNAc ₃ -7 _a is shown in Example 48. The structure of GalNAc ₃ -10 _a is shown in Example 46. The structure of GalNAc ₃ -13 _a is shown in Example 62. The structure of GalNAc ₃ -19 _a is shown in Example 70.

Example 98 Evaluation of the pro-inflammatory effect of oligonucleotides containing a GalNAc conjugate in the hPMBC assay

The oligonucleotides listed in Table 96 were tested for pro-inflammatory activity in the hPMBC assay described in Examples 23 and 24 (see Tables 17, 70, 82 and 95 for a description of the oligonucleotides). ISIS 353512 has a high response and was used as a positive control, and other oligonucleotides are described in Tables 70, 82 and 95. The results presented in Table 96 are from blood obtained from a single volunteer donor. The results demonstrate that oligonucleotides containing mixed PO/PS internucleoside bonds elicit significantly weaker pro-inflammatory responses compared to the same oligonucleotides containing only PS bonds. In addition, in this assay, the GalNAc conjugating group was not significantly affected.

Example 99 Binding Affinities of Oligonucleotides Containing a GalNAc Conjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 97 (see Table 63 for a description of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competing ligand, α1-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 u. neuraminidase-agarose for 16 hours at 37°C, and >90% dezalylation was confirmed by analysis with sialic acid or size exclusion chromatography (SEC). For iodination of AHF, iodine monochloride was used according to the method described by Atsma et al. (See J Lipid Res. January 1991; 32(1):173-81.) In this method, desalylated α1-acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na ¹²⁵ I and 1 M glycine in 0.25 M NaOH. After incubation for 10 minutes at room temperature, ¹²⁵ I-labeled de-AGP was separated from free ¹²⁵ I by doubling the mixture using a spin column with a nominal molecular weight cutoff of 3 kD. The protein was tested for labeling efficiency and purity on an HPLC system equipped with an Agilent SEC-3 (7.8 x 300 mm) column and B-RAM counter. Competitive studies using 1251-labeled de-AGP and ASO containing various GalNAc clusters were performed as follows. Human HepG2 cells (10 ⁶ cells/ml) were plated in 6-well plates in 2 ml of appropriate growth medium. Used medium MEM supplemented with 10% fetal bovine serum (FBS), 2 mm L-glutamine and 10 mm HEPES. Cells were grown for 16-20 hours at 37°C with 5% and 10% CO ₂ , respectively. Before the experiment, the cells were washed with the medium without FBS. Cells were grown for 30 minutes at 37°C with 1 ml of a competitive mixture containing the appropriate nutrient medium with 2% FBS, 10 ^-8 M ¹²⁵ I-labeled de-AGP and ASO containing the GalNAc cluster in concentrations ranging from 10 ^{- 11} to 10 ^-5 M. Non-specific binding was determined in the presence of 10 ^-2 M GalNAc sugar. Cells were washed twice with FBS-free medium to remove unbound ¹²⁵ I-labeled de-AGP and competing GalNAc ASO. Cells were lysed using Qiagen's RLT buffer containing 1% β-mercaptoethanol. The lysates were transferred to round bottom assay tubes after a quick 10 minute freeze/thaw cycle and analyzed on a γ-counter. Non-specific binding was subtracted, and then the ¹²⁵ I protein pulses were divided by the value of the pulses at the lowest concentration of GalNAc-ASO. Inhibition curves were constructed from the monosite competitive binding equation using a non-linear regression algorithm to calculate binding affinity (K _D ).

The results in Table 97 were obtained from experiments performed on five different days. The results for oligonucleotides marked with a superscript "a" are the average of experiments performed on two different days. The results demonstrate that oligonucleotides containing a GalNAc conjugation group at the 5' end bind the human cell asialoglycoprotein receptor HepG2 with an affinity that is 1.5-16 times greater than oligonucleotides containing a GalNAc conjugation group at the 3' end.

Example 100 In vivo Antisense Inhibition by Oligonucleotides Containing a GalNAc Conjugating Group Targeting Apo(a) in Vivo

The oligonucleotides listed in Table 98a below were tested in a single dose duration study in mice.

The structure of GalNAc ₃ -7 _a is shown in Example 48.

Treatment

Each female transgenic mouse expressing human Apo(a), once a week for a total of 6 doses, was injected subcutaneously with the oligonucleotide and the dose listed in Table 98b or PBS. Each experimental group consisted of 3 animals. Blood samples were taken the day before dosing to determine baseline plasma levels of Apo(a) protein, as well as 72 hours, 1 week and 2 weeks after the first dose. Additional blood samples were taken at 3 weeks, 4 weeks, 5 weeks and 6 weeks after the first dose. Plasma Apo(a) protein levels were measured by enzyme-linked immunosorbent assay. The results in Table 98b are presented as the mean percentage of Apo(a) plasma protein levels for each experimental group, normalized to baseline levels (% BL). The results show that oligonucleotides containing a GalNAc conjugation group show an effective reduction in Apo(a) expression. This powerful effect was observed for an oligonucleotide containing only PS internucleoside bonds and for an oligonucleotide containing mixed PO and PS bonds.

Example 101 Antisense Inhibition by Oligonucleotides Containing a GalNAc Cluster Linked Via a Stable Fragment

The oligonucleotides listed in Table 99 were tested for inhibition of murine APOC-III expression in vivo. C57B1/6 mice were given a single subcutaneous injection of the oligonucleotide listed in Table 99 or PBS. Each experimental group consisted of 4 animals. Each mouse treated with ISIS 440670 was dosed at 2, 6, 20, or 60 mg/kg. Each mouse treated with ISIS 680772 or 696847 was injected with 0.6, 2, 6 or 20 mg/kg. The conjugating group of GalNAc in ISIS 696847 is linked through a stable moiety, a thiophosphate bond, instead of an easily cleavable phosphodiester-containing bond. Animals were sacrificed 72 hours after dosing. AROS-III mRNA levels in the liver were measured by real-time PCR. AROS-III mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented in Table 99 as the mean percentage of APOC-III mRNA levels for each experimental group compared to the saline-treated control group. The results demonstrate that oligonucleotides containing a GalNAc conjugation group were significantly more effective than an oligonucleotide without a conjugation group. In addition, an oligonucleotide containing a GalNAc conjugation group linked to the oligonucleotide via a cleavable fragment (ISIS 680772) was even more effective than an oligonucleotide containing a GalNAc conjugating group linked to the oligonucleotide via a stable fragment (ISIS 696847).

The structure of GalNAc ₃ -7a is shown in Example 48.

Example 102 Hepatic Distribution of Antisense Oligonucleotides Containing a GalNAc Conjugate

Liver distribution of ISIS 353382 (see Table 23) which does not contain GalNAc conjugate and ISIS 655861 (see Table 23) which contains GalNAc conjugate was assessed. Male balb/c mice were given a single subcutaneous injection of ISIS 353382 or 655861 at the dose listed in Table 100. Each experimental group consisted of 3 animals, except for the 18 mg/kg dose group for ISIS 655861, which consisted of 2 animals. Animals were sacrificed 48 hours post-dose to determine liver distribution of oligonucleotides. To measure the number of antisense oligonucleotide molecules per cell, a ruthenium(II) tris-bipyridine label (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide sample used to detect antisense oligonucleotides. The results presented in Table 100 are the average oligonucleotide concentrations for each experimental group in units of millions of oligonucleotide molecules per cell. The results demonstrate that at equal doses, the oligonucleotide containing the GalNAc conjugate was contained in higher concentrations in general in the liver and in hepatocytes than the oligonucleotide not containing the GalNAc conjugate. In addition, the oligonucleotide containing the GalNAc conjugate was present at lower concentrations in non-parenchymal liver cells than the oligonucleotide not containing the GalNAc conjugate. And while the concentrations of ISIS 655861 in hepatocytes and non-parenchymal cells of the liver were the same per cell, the content of hepatocytes in the liver is about 80% by volume. Therefore, the main part of the ISIS 655861 oligonucleotide contained in the liver was located in hepatocytes, while the main part of the ISIS 353382 oligonucleotide contained in the liver was located in non-parenchymal cells of the liver.

Example 103 In vivo duration of action of oligonucleotides targeting APOC-III containing a GalNAc ₃ conjugate

The oligonucleotides listed in Table 101 below were tested in a single dose duration study in mice.

The structure of GalNAc ₃ -3 _a is shown in Example 39 and GalNAc ₃ -19a is shown in Example 70.

Treatment

Female transgenic mice expressing human AROS-III were given a single subcutaneous injection of the oligonucleotide listed in Table 101 or PBS. Each experimental group consisted of 3 animals. Blood samples were taken from pre-dose to determine baseline and on days 3, 7, 14, 21, 28, 35 and 42 post-dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 102 are presented as the mean percentage of triglyceride and APOC-III plasma levels for each treatment group, normalized to baseline levels. Comparison of the results in Table 58 of Example 79 with the results shown in Table 102 below demonstrates that oligonucleotides containing a mixture of phosphodiester and thiophosphate internucleoside bonds have an increased duration of action than equivalent oligonucleotides containing only thiophosphate internucleoside bonds.

Example 104 Synthesis of Oligonucleotides Containing 5'-GalNAc ₂ Conjugate

Compound 120 is commercially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol) and HOBt (1.64 g, 4.33 mmol) was dissolved in DMF (10 ml) and N,N-diisopropylethylamine (1.75 ml, 10.1 mmol) was added. After about 5 minutes, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction mixture. After 3 hours, the reaction mixture was poured into 100 ml of 1 M NaHSO4 solution and extracted with 2 × 50 ml of ethyl acetate. The organic layers were combined and washed with 3 x 40 ml saturated NaHCO ₃ solution and 2 x brine, dried with Na 2 SO ₄ , filtered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:hexanes, 1:1:1) to give compound 231. LCMS and NMR data were consistent with the structure. Compound 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 ml) and trifluoroacetic acid (10 ml) was added. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 10 ml). The residue was dried under reduced pressure to give compound 232 as the trifluoroacetate salt. The synthesis of compound 166 is described in Example 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 ml). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 ml) and N,N-diisopropylethylamine (1.55 ml) was added. The reaction mixture was stirred at room temperature for 30 minutes, then poured into water (80 ml) and the aqueous layer was extracted with EtOAc (2×100 ml). The organic phase was separated and washed with saturated aqueous NaHCO ₃ (3 x 80 yl), 1 M NaHSO ₄ (3 x 80 ml) and brine (2 x 80 ml), then dried (Na ₂ SO ₄ ), filtered and concentrated . The residue was purified by silica gel column chromatography to give compound 233. LCMS and NMR data were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 ml) and ethyl acetate (2.2 ml). Palladium on carbon (10 wt.% Pd/C, wet, 0.07 g) was added and the reaction mixture was stirred under a hydrogen atmosphere for 3 hours. The reaction mixture was filtered through a pad of celite and concentrated to give the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, non-clustered acid) was dissolved in DMF (3.2 ml). To this was added N,N-diisopropylethylamine (0.3 ml, 1.73 mmol) and PFP-TFA (0.30 ml, 1.73 mmol). After 30 minutes of stirring at room temperature, the reaction mixture was poured into water (40 ml) and extracted with EtOAc (2×50 ml). Followed the standard product isolation procedure as described above to give compound 234. LCMS and NMR data were consistent with the structure. Oligonucleotide 235 was prepared by the general method described in Example 46. The GalNAc ₂ (GalNAc ₂ -24 _a ) cluster portion of the GalNAc2-24 conjugation group can be combined with any cleavable moiety contained in the oligonucleotide to form various conjugation groups. The structure of GalNAc ₂ -24 (GalNAc ₂ -24 _a -CM) is shown below:

Example 105 Synthesis of Oligonucleotides Containing _a GalNAc 1-25 Conjugate

The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared according to the general method described in Example 46.

Alternatively, oligonucleotide 236 was synthesized according to the scheme shown below, and compound 238 was used to prepare oligonucleotide 236 according to the methods described in Example 10.

The cluster portion of the GalNAc ₁ (GalNAc ₁ -25 _a ) conjugating group GalNAc ₁ -25 can be combined with any cleavable fragment contained in the oligonucleotide to form different conjugating groups. The structure of GalNAc ₁ -25 (GalNAc ₁ -25 _a -CM) is shown below:

Example 106 In vivo antisense inhibition by oligonucleotides targeting SRB-1 containing a 5'-GalNAc ₂ or 5'-GalNAc ₃ conjugate

The oligonucleotides listed in Tables 103 and 104 were tested in dose-dependent antisense inhibition studies of SRB-1 in mice.

Treatment

Six-week-old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) were given a single subcutaneous injection of 2, 7, or 20 mg/kg ISIS No. 440762; or 0.2, 0.6, 2, 6 or 20 mg/kg ISIS No. 686221, 686222 or 708561; or saline solution. Each experimental group consisted of 4 animals. Mice were sacrificed 72 hours after the last injection. Liver SRB-1 mRNA levels were measured by real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. Antisense oligonucleotides reduce SRB-1 mRNA levels in a dose-dependent manner, and ED ₅₀ results are shown in Tables 103 and 104. Although previous studies have shown that trivalent GalNAc-conjugated oligonucleotides are significantly more effective than bivalent GalNAc-conjugated oligonucleotides, which in turn , are significantly more efficient than monovalent GalNAc-conjugated oligonucleotides (see, for example, Khorev et al., Bioorg. & Med. Chem., vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides containing monovalent, divalent and trivalent GalNAc clusters resulted in the reduction of SRB-1 mRNA levels with equal efficiency, as shown in Tables 103 and 104.

The table legend is shown in Example 93. The structure of GalNAc ₃ -13a is shown in Example 62 and the structure of GalNAc ₂ -24a is shown in Example 104.

The table legend is shown in Example 93. The structure of GalNAc ₁ -25a is shown in Example 105.

The concentrations of the oligonucleotides presented in Tables 103 and 104 in the liver were also evaluated using the methods described in Example 75. The results shown in Tables 104a and 104b below are the average values of the total content of antisense oligonucleotides in tissues for each experimental group, measured by UV, in units of µg oligonucleotide a per gram of liver tissue. The results demonstrate that oligonucleotides containing a GalNAc conjugation group accumulate in the liver at significantly higher levels than the same dose of an oligonucleotide lacking a GalNAc conjugation group. In addition, antisense oligonucleotides containing one, two or three GalNAc ligands in their respective conjugate groups accumulate in the liver in equal amounts. This result is unexpected given the data presented above in the literature by Khorev et al., and is consistent with the activity data presented above in Tables 103 and 104.

Example 107 Synthesis _of Oligonucleotides Containing _a GalNAc 1-26 or GalNAc 1-27 Conjugate

Oligonucleotide 239 was synthesized by coupling compound 47 (see Example 15) with acid 64 (see Example 32) using HBTU and DIEA in DMF. The resulting amide-containing compound was thiophosphorylated, then attached to the 5' end of the oligonucleotide according to the methods described in Example 10. The GalNAc ₁ (GalNAc ₁ -26 _a ) cluster portion of the GalNAc ₁ -26 conjugating group can be combined with any cleavable moiety contained in oligonucleotide, with obtaining various conjugating groups. The structure of GalNAc ₁ -26 (GalNAc ₁ -26 _a -CM) is presented below:

To attach the conjugating groups of GalNAc ₁ to the 3'-end of the oligonucleotide, the amide formed by the reaction of compounds 47 and 64 was attached to a solid support according to the methods described in Example 7. Then, the synthesis of the oligonucleotide was performed according to the methods described in Example 9, obtaining oligonucleotide 240 .

The cluster portion of the GalNAc ₁ (GalNAc ₁ -27 _a ) conjugating group GalNAc ₁ -27 can be combined with any cleavable fragment contained in the oligonucleotide to form different conjugating groups. The structure of GalNAc ₁ -27 (GalNAc ₁ -27a-CM) is shown below:

Example 108 In vivo antisense inhibition by oligonucleotides containing a GalNAc conjugating group targeting Apo(a) in vivo

The oligonucleotides listed in Table 105 below were tested in a single dose study in mice.

The structure of GalNAc ₃ -7 _a is shown in Example 48.

Treatment

Male transgenic mice expressing human Apo(a) were given a single subcutaneous injection of oligonucleotide a and the doses listed in Table 106 or PBS. Each experimental group consisted of 4 animals. Blood samples were taken the day before dosing to determine baseline plasma levels of Apo(a) protein and also 1 week after the first dose. Blood samples were taken additionally weekly for about 8 weeks. Plasma Apo(a) protein levels were measured by enzyme-linked immunosorbent assay. The results in Table 106 are presented as the mean percentage of Apo(a) plasma protein levels for each experimental group, normalized to baseline levels (% BL). The results show that antisense oligonucleotides reduce Apo(a) protein expression. In addition, oligonucleotides containing a GalNAc conjugation group show an even more effective reduction in Apo(a) expression than an oligonucleotide without a conjugation group.

Example 109 Synthesis _of Oligonucleotides Containing _a GalNAc 1-28 or GalNAc 1-29 Conjugate

Oligonucleotide 241 was synthesized by the same methods as described in Example 71 to obtain a phosphorus amidite intermediate, followed by the steps described in Example 10 to synthesize the oligonucleotide. The cluster portion of the GalNAc ₁ (GalNAc ₁ -28 _a ) of the GalNAc ₁ -28 conjugating group can be combined with any cleavable moiety contained in the oligonucleotide to form different conjugating groups. The structure of GalNAc ₁ -28 (GalNAc ₁ -28 _a -CM) is shown below:

To attach the GalNAc ₁ conjugating groups to the 3' end of the oligonucleotide, the same methods as described in Example 71 were used to obtain a hydroxyl intermediate, which was then attached to a solid support according to the methods described in Example 7. The synthesis of the oligonucleotide was then performed according to the methods described in Example 9 to give oligonucleotide 242.

The GalNAc ₁ cluster portion (GalNAc ₁ -29 _a ) of the GalNAc ₁ -29 conjugating group can be combined with any cleavable moiety contained in the oligonucleotide to form different conjugating groups. The structure of GalNAc ₁ -29 (GalNAc ₁ -29 _a -CM) is shown below:

Example 110 Synthesis of Oligonucleotides Containing a GalNAc _1-30 Conjugate

Oligonucleotide 246 containing a conjugating group GalNAc _1-30 , where Y is selected from O, S, substituted or unsubstituted C ₁ -C ₁₀ alkyl, amino, substituted amino, azido, alkenyl, or alkynyl, was synthesized as shown above. The GalNAc ₁ cluster portion (GalNAc ₁ -30a) of the GalNAc ₁ -30 conjugating group can be combined with any cleavable moiety to form different conjugating groups. In some embodiments of the invention, Y is part of a cleavable fragment. In some embodiments of the invention, Y is part of a stable fragment, and the cleavable fragment is in the oligonucleotide. The structure of GalNAc ₁ -30 _a is shown below:

Example 111 Synthesis of Oligonucleotides Containing a GalNAc ₂ -31 or GalNAc ₂ -32 Conjugate

Oligonucleotide 250 containing a conjugating group GalNAc ₂ -31, where Y is selected from O, S, substituted or unsubstituted C ₁ -C ₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, was synthesized as shown above. The GalNAc ₂ cluster portion (GalNAc ₂ -31 _a ) of the GalNAc ₂ -31 conjugating group can be combined with any cleavable moiety to form different conjugating groups. In some embodiments of the invention, the Y-containing group, which is located immediately near the 5'end of the oligonucleotide, is part of the cleavable fragment. In some embodiments of the invention, the Y-containing group, which is located immediately near the 5'-end of the oligonucleotide a, is part of a stable fragment, and the cleavable fragment is in the oligonucleotide. The structure of GalNAc ₂ -31 _a is shown below:

The synthesis of oligonucleotide a containing the GalNAc ₂ -32 conjugate is shown below.

Oligonucleotide 252 containing a conjugating group GalNAc ₂ -32, where Y is selected from O, S, substituted or unsubstituted C ₁ -C ₁₀ alkyl, amino, substituted amino, azido, alkenyl, or alkynyl, was synthesized as shown above. The GalNAc ₂ cluster portion (GalNAc ₂ -32 _a ) of the GalNAc ₂ -32 conjugating group can be combined with any cleavable moiety to form different conjugating groups. In some embodiments of the invention, the Y-containing group, which is located immediately near the 5'end of the oligonucleotide, is part of the cleavable fragment. In some embodiments of the invention, the Y-containing group, which is located immediately near the 5'end of the oligonucleotide, is part of a stable fragment, and the cleavable fragment is in the oligonucleotide. The structure of GalNAc ₂ -32 _a is shown below:

Example 112 Modified Oligonucleotides Containing GalNAc ₁ Conjugate

The oligonucleotides in Table 107 targeting SRB-1 were synthesized with a GalNAc ₁ conjugate group to further test the efficacy of oligonucleotides containing conjugation groups that contain the GalNAc ligand.

Example 113 Modified Oligonucleotides Containing a GalNAc Conjugation Group Targeting Hepatitis B Virus (HBV)

The oligonucleotides listed in Table 108 below are intended to target HBV. In some embodiments, the cleavable moiety is a phosphodiester bond.

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<400> 1

aattccacaa cctttcacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60

gctggtggct ccagttcagg agcagtaaac cctgttccga ctactgcctc tcccttatcg 120

tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180

ctaggacccc ttctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240

300

cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcctg tcctccaact 360

tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg 420

ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480

ctaattccag gatcctcaac caccagcacg ggaccatgcc gaacctgcat gactactgct 540

caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc 600

tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660

cgttctctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720

actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780

ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttggggtata catttaaacc 840

ctaacaaaac aaagagatgg ggttactctc tgaattttat gggttatgtc attggaagtt 900

atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 960

1020

ctgccccatt tacacaatgt ggttatcctg cgttaatgcc cttgtatgca tgtattcaat 1080

ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 1140

acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200

ccactggctg gggcttggtc atgggccatc agcgcgtgcg tggaaccttt tcggctcctc 1260

tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320

acattatcgg gactgataac tctgttgtcc tctcccgcaa atatacatcg tatccatggc 1380

1440

cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 1500

gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 1560

cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620

cgtgaacgcc caccgaatgt tgcccaaggt cttacataag aggactcttg gactctctgc 1680

aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 1740

gttgggggag gagattagat taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800

ctgcgcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct 1860

actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat cgacccttat 1920

aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttca 1980

gtacgagatc ttctagatac cgcctcagct ctgtatcggg aagccttaga gtctcctgag 2040

cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg 2100

actctagcta cctgggtggg tgttaatttg gaagatccag catctagaga cctagtagtc 2160

agttatgtca acactaatat gggcctaaag ttcaggcaac tcttgtggtt tcacatttct 2220

2280

2340

actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400

aggtctcaat cgccgcgtcg cagaagatct caatctcggg aacctcaatg ttagtattcc 2460

ttggactcat aaggtgggga actttactgg tctttattct tctactgtac ctgtctttaa 2520

2580

atgtgaacag tttgtaggcc cacttacagt taatgagaaa agaagattgc aattgattat 2640

gcctgctagg ttttatccaa aggttaccaa atatttacca ttggataagg gtattaaacc 2700

ttattatcca gaacatctag ttaatcatta cttccaaact agacactatt tacacactct 2760

atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat tttgtgggtc 2820

accatattct tgggaacaag atctacagca tggggcagaa tctttccacc agcaatcctc 2880

tgggattctt tcccgaccac cagttggatc cagccttcag agcaaacaca gcaaatccag 2940

attgggactt caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag 3000

cattcgggct gggtttcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc 3060

agggcatact acaaactttg ccagcaaatc cgcctcctgc ctccaccaat cgccagacag 3120

gaaggcagcc taccccgctg tctccacctt tgagaaacac tcatcctcag gccatgcagt 3180

gg 3182

<210> 2

<211> 938

<212> DNA

<213> Homo sapiens

<400> 2

gttgactaag tcaataatca gaatcagcag gtttgcagtc agattggcag ggataagcag 60

cctagctcag gagaagtgag tataaaagcc ccaggctggg agcagccatc acagaagtcc 120

actcattctt ggcaggatgg cttctcatcg tctgctcctc ctctgccttg ctggactggt 180

atttgtgtct gaggctggcc ctacggggcac cggtgaatcc aagtgtcctc tgatggtcaa 240

agttctagat gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg tgttcagaaa 300

ggctgctgat gacacctggg agccatttgc ctctgggaaa accagtgagt ctggagagct 360

gcatgggctc acaactgagg aggaatttgt agaagggata tacaaagtgg aaatagacac 420

caaatcttac tggaaggcac ttggcatctc cccattccat gagcatgcag aggtggtatt 480

cacagccaac gactccggcc cccgccgcta caccattgcc gccctgctga gcccctactc 540

ctattccacc acggctgtcg tcaccaatcc caaggaatga gggacttctc ctccagtgga 600

cctgaaggac gagggatggg atttcatgta accaaggta ttccatttt actaaagcag 660

tgttttcacc tcatatgcta tgttagaagt ccaggcagag acaataaaac attcctgtga 720

aaggcacttt tcattccact ttaacttgat tttttaaatt cccttattgt cccttccaaa 780

aaaaagagaa tcaaaatttt acaaagaatc aaaggaattc tagaaagtat ctgggcagaa 840

cgctaggaga gatccaaatt tccattgtct tgcaagcaaa gcacgtatta aatatgatct 900

gcagccatta aaaagacaca ttctgtaaaa aaaaaaaa 938

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 3

gcagaggtga agcgaagtgc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 4

ccaatttatg cctacagcct 20

<210> 5

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 5

ggcatagcag caggatg 17

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 6

aggagttccg cagtatggat 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 7

gtgaagcgaa gtgcacacgg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 8

gtgcagaggt gaagcgaagt 20

<210> 9

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 9

aggtgaagcg aagtgc 16

<210> 10

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 10

tccgcagtat ggatcg 16

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 11

aattatgcc tacagcct 18

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 12

tcttggttac atgaaatccc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 13

cttggttaca tgaaatccca 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 14

ggaatactct tggttacatg 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 15

tggaatactc ttggttacat 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 16

ttttttgtc tctgcctgga 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 17

gaatgtttta ttgtctctgc 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 18

aggaatgttt tattgtctct 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 19

acaggaatgt tttattgtct 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 20

agcttcttgt ccagctttat 20

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 21

agcttcttgt ccagctttat a 21

<210> 22

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 22

tcagtcatga cttc 14

<210> 23

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 23

tcagtcatga cttca 15

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 24

gctgattaga gagaggtccc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 25

tcccatttca ggagacctgg 20

<210> 26

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 26

atcagtcatg acttc 15

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 27

cggtgcaagg cttaggaatt 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 28

gcttcagtca tgacttcctt 20

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 29

gcttcagtca tgacttcctt a 21

<210> 30

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 30

agcttcagtc atgacttcct t 21

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 31

tggtaatcca ctttcagagg 20

<210> 32

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 32

tggtaatcca ctttcagagg a 21

<210> 33

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 33

tgcttcagtc atgacttcct t 21

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 34

cactgatttt tgcccaggat 20

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 35

cactgatttt tgcccaggat a 21

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 36

aagcttcttg tccagcttta t 21

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 37

acccaattca gaaggaagga 20

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 38

acccaattca gaaggaagga a 21

<210> 39

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 39

aacccaattc agaaggaagg a 21

<210> 40

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 40

atggtaatcc actttcagag g 21

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 41

tcttggttac atgaaatccc 20

<210> 42

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 42

tcttggttac atgaaatccc a 21

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 43

attcactttc ataatgctgg 20

<210> 44

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 44

attcactttc ataatgctgg a 21

<210> 45

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 45

atcttggtta catgaaatcc c 21

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 46

atgcatggtg atgcttctga 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 47

cagctttatt agggacagca 20

<210> 48

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 48

cagctttatt agggacagca a 21

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 49

acagctttat tagggacagc a 21

<210> 50

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 50

ttcagtcatg acttcc 16

<210> 51

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature (misc_feature)

<222> (1)..(4)

<223> bases at the indicated positions are RNA

<220>

<221> misc_feature (misc_feature)

<222> (15)..(18)

<223> bases at the indicated positions are RNA

<400> 51

gcuucagtca tgactucc 18

<210> 52

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 52

tgctccgttg gtgcttgttc a 21

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 53

tgctccgttg gtgcttgttc 20

<---

Claims (32)

1. An oligomeric compound, wherein the anionic form of the oligomeric compound is characterized by the following chemical structure:

(SEQ ID NO: 12). 2. Oligomeric compound characterized by the following chemical structure:

(SEQ ID NO: 12), or a salt thereof. 3. A compound according to claim 2 which is a sodium salt or a potassium salt. 4. Oligomeric compound characterized by the following chemical structure:

(SEQ ID NO: 12). 5. An oligomeric compound containing a modified oligonucleotide and a conjugating group, characterized by the following formula: GalNAC ₃ -7 _ao ,Tes ^m C _eo T _eo T _eo G _eo G _ds T _ds T _ds A _ds ^m C _ds A _ds T _ds G _ds A _ds A _ds A _eo T _eo ^m C _es ^m C _es ^m C _e (SEQ ID NO: 12); where A=adenine nitrogenous base, ^m C=5-methylcytosine nitrogenous base, G=guanine nitrogenous base, T=thymine nitrogenous base, e=2'-OCH ₂ CH ₂ OCH ₃ ribosyl sugar moiety, d=β-D-2'-deoxyribosyl sugar moiety, s=thiophosphate internucleoside bond, o=phosphodiester internucleoside bond and GalNAc ₃ -7 _ao '-=

where the cleavable fragment (CM) is 5'-P(OH)(=O)-O-3'. 6. Pharmaceutical composition for the treatment of transthyretin amyloidosis, containing a therapeutically effective amount of a compound according to any one of paragraphs. 1-5 and a pharmaceutically acceptable carrier or diluent. 7. The use of a compound according to any one of paragraphs. 1-5 in the manufacture of a medicament for the treatment of transthyretin amyloidosis. 8. Use of the composition according to claim 6 in the manufacture of a medicament for the treatment of transthyretin amyloidosis. 9. The use according to claim 7 or 8, wherein the transthyretin amyloidosis is senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) or familial amyloid cardiopathy (FAC). 10. A method of treating transthyretin amyloidosis in a subject, comprising administering to the subject a therapeutically effective amount of an oligomeric compound according to any one of paragraphs. 1-5. 11. A method of treating transthyretin amyloidosis in a subject, comprising administering to the subject a therapeutically effective amount of a composition according to claim 6. 12. A method of treating transthyretin amyloidosis according to claim 10 or 11, wherein the transthyretin amyloidosis is senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), or familial amyloid cardiopathy (FAC). 13. The use of an oligomeric compound according to any one of paragraphs. 1-5 for the treatment of transthyretin amyloidosis. 14. Use of a composition according to claim 6 for the treatment of transthyretin amyloidosis. 15. Use according to claim 13 or 14, characterized in that the transthyretin amyloidosis is senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) or familial amyloid cardiopathy (FAC).