MODIFIED OLIGOMERIC COMPOUNDS AND USES THEREOF

Field

The present disclosure provides stereo-non-standard 4’-thio nucleosides and oligomeric compounds comprising a modified oligonucleotide having at least one such stereo-non-standard 4’-thio nucleoside.

Background

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 certain instances, antisense compounds result in altered transcription or translation of a target. Such modulation of expression can be achieved by, for example, target RNA degradation or occupancy-based inhibition. Examples of modulation of RNA target function by degradation include, but are not limited to RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound; RISC mediated degradation of the target RNA upon hybridization of an RNA-like antisense compound; and modulation of splicing of pre-mRNA.

Antisense technology is an effective means for modulating the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, therapeutic index, or affinity for a target nucleic acid.

Summary

In certain embodiments, the present disclosure provides stereo-non-standard 4’-thio nucleosides. In certain embodiments, provided herein are oligomeric compounds comprising modified oligonucleotides having one or more such stereo-non-standard 4’-thio nucleosides. In certain embodiments, modified oligonucleotides having one or more stereo-non-standard 4’-thio nucleosides show improved properties compared to similar modified oligonucleotides without one or more stereo-non-standard 4’-thio nucleosides.

In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula I:

wherein one of J ₁ and h is H and the other of J ₁ and h is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula II: wherein one of J ₃ and J ₄ is H and the other of J ₃ and J ₄ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety. In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula III:

wherein one of J ₅ and J ₆ , is H and the other of J ₅ and J ₆ , is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula IV: wherein one of J ₇ and Jx is H and the other of J ₇ and Jx is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula V:

wherein one of J ₉ and J ₁₀ is H and the other of J ₉ and J ₁₀ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula VF wherein one of Jn and J ₁₂ is H and the other of Jn and J ₁₂ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides oligomeric compounds comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard 4’-thio nucleoside having Formula VII:

wherein one of J ₁₃ and J ₁₄ is H and the other of J ₁₃ and J ₁₄ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-non-standard 4’-thio nucleoside having Formula VIII: wherein one of J ₁ or J ₂ is H and the other of J ₁ or J ₂ is selected from OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ;

Ti is H or a hydroxyl protecting group;

T2 is H, a hydroxyl protecting group, or a reactive phosphorus group; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-non-standard 4’-thio nucleoside having Formula IX:

wherein one of J ₃ or J ₄ is H and the other of J ₃ or J ₄ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ;

T ₃ is H or a hydroxyl protecting group;

T4 is H, a hydroxyl protecting group, or a reactive phosphorus group; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-nonstandard 4’-thio nucleoside having Formula X:

X. wherein one of J ₅ or J ₆ , is H and the other of J ₅ or J ₆ , is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ;

T ₅ is H or a hydroxyl protecting group; H, is H. a hydroxyl protecting group, or a reactive phosphorus group; and wherein

Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-non-standard 4’-thio nucleoside having Formula XI:

wherein one of J ₇ or Jx is H and the other of J ₇ or Jx is selected from OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ , T ₇ is H or a hydroxyl protecting group;

Ts is H, a hydroxyl protecting group, or a reactive phosphorus group; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-non-standard 4’-thio nucleoside having Formula XII: wherein one of J ₉ or J ₁₀ is H and the other of J ₉ or J ₁₀ is selected from OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ;

T ₉ is H or a hydroxyl protecting group;

T ₁₀ is H, a hydroxyl protecting group, or a reactive phosphorus group; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-non-standard 4’-thio nucleoside having Formula XIII:

wherein one of Jn or J ₁₂ is H and the other of Jn or J ₁₂ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; Tn is H or a hydroxyl protecting group;

T12 is H, a hydroxyl protecting group, or a reactive phosphorus group; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the present disclosure provides a compound comprising a stereo-non-standard 4’-thio nucleoside having Formula XIV: wherein one of J ₁₃ or J ₁₄ is H and the other of J ₁₃ or J ₁₄ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ;

Ti3 is H or a hydroxyl protecting group;

Ti ₄ is H, a hydroxyl protecting group, or a reactive phosphorus group; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, the modified oligonucleotides having at least one stereo-non-standard 4’-thio nucleoside have an increased maximum tolerated dose when administered to an animal compared to an otherwise identical oligomeric compound, except that the otherwise identical oligomeric compound lacks the at least one stereo-non-standard 4’-thio nucleoside.

In certain embodiments, the modified oligonucleotides having at least one stereo-non-standard 4’- thio nucleoside have an increased therapeutic index, increased nuclease resistance, and/or a longer duration of action compared to an otherwise identical oligomeric compound lacking the at least one stereo -non-standard 4’-thio nucleoside.

Detailed Description

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting.

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

It is understood that the sequence set forth in each SEQ ID NO contained herein is independent of any modification to a sugar moiety, an intemucleoside linkage, or a nucleobase. As such, compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an intemucleoside linkage, or a nucleobase. Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’-OH(H) sugar moiety and a thymine base could be described as a DNA having a modified sugar (2 ’-OH in place of one 2’-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of an uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified nucleobases, such as “AT ^m CGAUCG,” wherein ^m C indicates a cytosine base comprising a methyl group at the 5-position.

As used herein, “2’-MOE” means a 2’-OCH ₂ CH ₂ OCH ₃ group in place of the 2 ’-OH group of a ribosyl sugar moiety. A “2’-MOE sugar moiety” is a sugar moiety with a 2’-OCH ₂ CH ₂ OCH ₃ group in place of the 2’-OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2’-MOE sugar moiety is in the β-D configuration. “MOE” means O-methoxyethyl.

As used herein, “2’-MOE nucleoside” means a nucleoside comprising a 2’-MOE sugar moiety.

As used herein, “2’-OMe” means a 2’-OCH ₃ group in place of the 2’-OH group of a ribosyl sugar moiety. A “2’-OMe sugar moiety” is a sugar moiety with a 2’-OCH ₃ group in place of the 2’-OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2’-OMe sugar moiety is in the β-D configuration. “OMe” means O-methyl.

As used herein, “2’-OMe nucleoside” means a nucleoside comprising a 2’-OMe sugar moiety.

As used herein, “2 ’-substituted nucleoside” means a nucleoside comprising a 2 ’-substituted sugar moiety. As used herein, “2 ’-substituted” in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.

As used herein, a “4’-thiofuranosyl sugar moiety” is a cyclic 5-membered ring having 4 carbons and one sulfur. 4’-thiofuranosyl sugar moieties are the same as furanosyl sugar moieties, except that the oxygen of the furanosyl is instead a sulfur.

As used herein, "administration" or "administering" refers to routes of introducing a compound or composition provided herein to a subject. Examples of routes of administration that can be used include, but are not limited to, administration by inhalation, subcutaneous injection, intrathecal injection, and oral administration.

As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

As used herein, “antisense compound” means a compound comprising an antisense oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.

As used herein, “antisense oligonucleotide” means an oligonucleotide having a nucleobase sequence that is at least partially complementary to a target nucleic acid.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety. As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety, and the bicyclic sugar moiety is a modified bicyclic furanosyl sugar moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

As used herein, “cEt” means a 4’ to T bridge in place of the 2OH-group of a ribosyl sugar moiety, wherein the bridge has the formula of 4'-CH(CH ₃ )-O-2', and wherein the methyl group of the bridge is in the S configuration. A “cEt sugar moiety” is a bicyclic sugar moiety with a 4’ to 2’ bridge in place of the 2ΌH- group of a ribosyl sugar moiety, wherein the bridge has the formula of 4'-CH(CH ₃ ) ^~ '-O-2', and wherein the methyl group of the bridge is in the S configuration. “cEt” means constrained ethyl.

As used herein, “cEt nucleoside” means a nucleoside comprising a cEt sugar moiety.

As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases are nucleobase pairs that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine ( ^m C) and guanine (G). Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that such oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

As used herein, “conjugate group” means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups may comprise a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

As used herein, “conjugate linker” means a bond or a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

As used herein, “conjugate moiety” means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

As used herein, “cytotoxic” or “cytotoxicity” in the context of an effect of an oligomeric compound or a parent oligomeric compound on cultured cells means an at least 2-fold increase in caspase activation following administration of 10 mM or less of the oligomeric compound or parent oligomeric compound to the cultured cells relative to cells cultured under the same conditions but that are not administered the oligomeric compound or parent oligomeric compound. In certain embodiments, cytotoxicity is measured using a standard in vitro cytotoxicity assay.

As used herein, “deoxy region” means a region of 5-12 contiguous nucleotides, wherein at least 70% of the nucleosides are stereo-standard DNA nucleosides. In certain embodiments, each nucleoside is selected from a stereo-standard DNA nucleoside, a stereo-non-standard nucleoside comprising a furanosyl sugar moiety, a stereo-non-standard 4’-thio nucleoside of Formula I-VII, a bicyclic nucleoside, and a substituted stereostandard nucleoside. In certain embodiments, a deoxy region supports RNase H activity. In certain embodiments, a deoxy region is the gap of a gapmer.

As used herein, “double-stranded antisense compound” means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an antisense oligonucleotide.

As used herein, “expression” includes all the functions by which a gene’s coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation. As used herein, “modulation of expression” means any change in amount or activity of a product of transcription or translation of a gene. Such a change may be an increase or a reduction of any amount relative to the expression level prior to the modulation.

As used herein, “gapmer” means an oligonucleotide having a central region comprising a plurality of nucleosides that support RNase H cleavage positioned between a 5 ’-region and a 3 ’-region. In certain embodiments, the nucleosides of the 5 ’-region and 3 ’-region each comprise a 2 ’-substituted furanosyl, a 2’- substituted 4’-thiofuranosyl sugar moiety, or a bicyclic sugar moiety, and the 3’- and 5 ’-most nucleosides of the central region each comprise a sugar moiety independently selected from a 2’-deoxyfuranosyl sugar moiety, a 2’-deoxy-4’-thiofuranosyl sugar moiety, or a sugar surrogate. The positions of the central region refer to the order of the nucleosides of the central region and are counted starting from the 5 ’-end of the central region. Thus, the 5 ’-most nucleoside of the central region is at position 1 of the central region. The “central region” may be referred to as a “gap”, and the “5’-region” and “3’-region” may be referred to as “wings”. Gaps of gapmers are deoxy regions.

As used herein, "hybridization" means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, "inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity relative to the expression or activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.

As used herein, the terms “intemucleoside linkage” means a group of atoms or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified intemucleoside linkage” means any intemucleoside linkage other than a naturally occurring, phosphodiester intemucleoside linkage. “Phosphorothioate linkage” means a modified intemucleoside linkage in which one of the nonbridging oxygen atoms of a phosphodiester is replaced with a sulfur atom. Modified intemucleoside linkages may or may not contain a phosphoms atom. A “neutral intemucleoside linkage” is a modified intemucleoside linkage that does not have a negatively charged phosphate in a buffered aqueous solution at pH=7.0. As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).

As used herein, “maximum tolerated dose” means the highest dose of a compound that does not cause unacceptable side effects. In certain embodiments, the maximum tolerated dose is the highest dose of a modified oligonucleotide that does not cause an ALT elevation of three times the upper limit of normal as measured by a standard assay.

As used herein, “mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.

As used herein, “modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism.

As used herein, “motif’ means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or intemucleoside linkages, in an oligonucleotide.

As used herein, “naturally occurring” means found in nature.

As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a modified nucleobase is a group of atoms capable of pairing with at least one unmodified nucleobase. A universal base is a nucleobase that can pair with any one of the five unmodified nucleobases. 5- methylcytosine ( ^m C) is one example of a modified nucleobase.

As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar moiety or intemucleoside linkage modification.

As used herein, “nucleoside” means a moiety comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.

As used herein, "oligomeric compound" means a compound consisting of (1) an oligonucleotide (a single-stranded oligomeric compound) or two oligonucleotides hybridized to one another (a double-stranded oligomeric compound); and (2) optionally one or more additional features, such as a conjugate group or terminal group which may be bound to the oligonucleotide of a single-stranded oligomeric compound or to one or both oligonucleotides of a double -stranded oligomeric compound.

As used herein, "oligonucleotide" means a strand of linked nucleosides connected via intemucleoside linkages, wherein each nucleoside and intemucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 12-30 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or intemucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or intemucleoside modifications. As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, liquids, powders, or suspensions that can be aerosolized or otherwise dispersed for inhalation by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.

As used herein “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the compound and do not impart undesired toxicological effects thereto.

As used herein “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an antisense compound and an aqueous solution.

As used herein, the term “single -stranded” in reference to an antisense compound means such a compound consists of one oligomeric compound that is not paired with a second oligomeric compound to form a duplex. “Self-complementary” in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligomeric compound, wherein the oligonucleotide of the oligomeric compound is self-complementary, is a single -stranded compound. A single-stranded antisense or oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex, in which case the compound would no longer be single-stranded.

As used herein, “stereo-standard nucleoside” means a nucleoside comprising a furanosyl sugar moiety having the configuration of naturally occurring DNA and RNA as shown below. As used herein, “stereostandard 4’-thio nucleoside” means a nucleoside comprising a non-bicyclic 4’-thiofuranosyl sugar moiety having the configuration of naturally occurring DNA and RNA as shown below. A “stereo-standard DNA nucleoside” is a nucleoside comprising a P-D-2’-deoxyribosyl sugar moiety. A “stereo-standard RNA nucleoside” is a nucleoside comprising a β-D-ribosyl sugar moiety. A “substituted stereo-standard nucleoside” is a stereo-standard nucleoside other than a stereo-standard DNA or stereo-standard RNA nucleoside. In certain embodiments, R ₁ is a 2’-substiuent and R ₂ -R ₅ are each H. In certain embodiments, the 2 ’-substituent is selected from OMe, F, OCH ₂ CH ₂ OCH ₃ , O-alkyl, SMe, or NMA. In certain embodiments, R ₁ -R ₄ are H and R ₅ is a 5’- substituent selected from methyl, allyl, or ethyl. In certain embodiments, R ₂ and R ₃ are linked to form a bicyclic nucleoside. In certain embodiments, the heterocyclic base moiety Bx is selected from uracil, thymine, cytosine, 5-methyl cytosine, adenine or guanine. In certain embodiments, the heterocyclic base moiety Bx is other than uracil, thymine, cytosine, 5-methyl cytosine, adenine or guanine.

Stereo-standard nucleoside Stereo-standard DNA nucleoside

Stereo-standard RNA nucleoside

Stereo-standard 4'-thio nucleoside

As used herein, “stereo-non-standard nucleoside” means a nucleoside comprising afuranosyl sugar moiety having a configuration other than that of a stereo-standard sugar moiety. A “stereo-non-standard 4’- thio nucleoside” means a nucleoside comprising a 4’-thiofuranosyl sugar moiety having a configuration other than that of a stereo-standard 4’-thio sugar moiety. In certain embodiments, a “stereo-non-standard 4’-thio nucleoside” is represented by Formulas I-VII below. In certain embodiments, J ₁ -J ₁₄ are independently selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH _3. A “2 ’-substituted stereo-non-standard 4’- thio nucleoside” has one of formulas I-VII below, wherein either J ₁ , J ₃ , J ₅ , J ₇ , J ₉ , J ₁₁ , and J ₁₃ is other than H and/or or J ₂ , J ₄ , J ₆ , Is. J ₁₀ , J ₁₂ , and J ₁₄ is other than H or OH. In certain embodiments, the heterocyclic base moiety Bx is selected from uracil, thymine, cytosine, 5-methyl cytosine, adenine or guanine. In certain embodiments, the heterocyclic base moiety Bx is other than uracil, thymine, cytosine, 5 -methyl cytosine, adenine or guanine.

As used herein, “stereo-standard sugar moiety” means the sugar moiety of a stereo-standard nucleoside. As used herein, “stereo-standard 4’thio sugar moiety” means the sugar moiety of a stereo-standard 4’-thio nucleoside.

As used herein, “stereo-non-standard sugar moiety” means the sugar moiety of a stereo-non-standard nucleoside. As used herein, “stereo-non-standard 4’-thio sugar moiety” means the sugar moiety of a stereo- non-standard 4’-thio nucleoside.

As used herein, “substituted nucleoside” means a nucleoside comprising a substitutent other than the substitutent corresponding to natural RNA or DNA. As used herein, “substituted sugar moiety” means a furanosyl or 4’-thiofuranosyl sugar moiety comprising a substituent other than the substituent corresponding to natural RNA or DNA. As used herein, “2’ -substituted sugar moiety” means a furanosyl or 4’-thiofuranosyl sugar moiety comprising a substituent at the 2’ position other than (1) both H or (2) one H and the other OH.

As used herein, “substituted stereo-non-standard nucleoside” means a stereo-non-standard nucleoside comprising a substituent other than the substituent corresponding to natural RNA or DNA. As used herein, “substituted stereo-non-standard 4’-thio nucleoside” means a stereo-non-standard 4’-thio nucleoside comprising a substituent other than the substituent corresponding to natural RNA or DNA. Substituted stereo- non-standard 4’-thio nucleosides include but are not limited to nucleosides of Formula I-VII wherein the J groups are other than: (1) both H or (2) one H and the other OH.

As used herein, “subject” means a human or non-human animal selected for treatment or therapy.

As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a β-D-ribosyl moiety, as found in naturally occurring RNA, or a β-D-2’-deoxyribosyl sugar moiety as found in naturally occurring DNA. As used herein, “modified sugar moiety” or “modified sugar” means a sugar surrogate or a furanosyl sugar moiety other than a β-D-ribosyl or a β-D-2’-deoxyribosyl. In certain embodiments, a sugar surrogate is a 4’-thiofuranosyl sugar moiety. Furanosyl and 4’-thiofuranosyl sugar moieties may be modified or substituted at a certain position(s) of the sugar moiety, or unsubstituted, and they may or may not be stereo-non-standard sugar moieties. Modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars. As used herein, "sugar surrogate" means a modified sugar moiety that does not comprise a furanosylor tetrahydrofuranyl ring and that can link a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.

As used herein, “target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” means a nucleic acid that an oligomeric compound, such as an antisense compound, is designed to affect. In certain embodiments, an oligomeric compound comprises an oligonucleotide having a nucleobase sequence that is complementary to more than one RNA, only one of which is the target RNA of the oligomeric compound. In certain embodiments, the target RNA is an RNA present in the species to which an oligomeric compound is administered.

As used herein, “therapeutic index” means a comparison of the amount of a compound that causes a therapeutic effect to the amount that causes toxicity. Compounds having a high therapeutic index have strong efficacy and low toxicity. In certain embodiments, increasing the therapeutic index of a compound increases the amount of the compound that can be safely administered.

As used herein, “treat” refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.

Certain Compounds

In certain embodiments, compounds described herein are oligomeric compounds comprising or consisting of oligonucleotides consisting of linked nucleosides and having at least one stereo-non-standard nucleoside. Oligonucleotides may be unmodified oligonucleotides or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to an unmodified oligonucleotide (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety, a stereo-non-standard nucleoside, and/or a modified nucleobase) and/or at least one modified intemucleoside linkage).

I. Modifications A. Modified Nucleosides Modified nucleosides comprise a stereo-non-standard nucleoside, or a modified sugar moiety, or a modified nucleobase, or any combination thereof.

1. Certain Modified Sugar Moie ties

In certain embodiments, modified sugar moieties are stereo-non-standard sugar moieties. In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties. In certain embodiments, sugar moieties are substituted furanosyl stereo-standard sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic furanosyl sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties. a. Stereo-Non-Standard Sugar Moieties In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula I: wherein one of J ₁ and h is H and the other of J ₁ and h is selected from H, OH, F, OCH ₃ , OCH- ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ , alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula II:

wherein one of J ₃ and J ₄ is H and the other of J ₃ and J ₄ is selected from H, OH, F, OCH ₃ , OCH- 2CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula III: wherein one of J ₅ and F, is H and the other of J ₅ and J ₆ is selected from H, OH, F, OCH ₃ , OCH-

₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula IV:

wherein one of J ₇ and Jx is H and the other of J ₇ and Jx is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula V : wherein one of J ₉ and J ₁₀ is H and the other of J ₉ and J ₁₀ is selected from H, OH, F, OCH ₃ ,

OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula VF

wherein one of J ₁₁ and J ₁₂ is H and the other of Jn and J ₁₂ is selected from H, OH, F, OCH ₃ , OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety.

In certain embodiments, modified sugar moieties are stereo-non-standard 4’-thiofuranosyl sugar moieties shown in Formula VII: wherein one of J ₁₃ and J ₁₄ is H and the other of J ₁₃ and J ₁₄ is selected from H, OH, F, OCH ₃ ,

OCH ₂ CH ₂ OCH ₃ , O-C ₁ -C ₆ alkoxy, and SCH ₃ ; and wherein Bx is a is a heterocyclic base moiety. b. Substituted Stereo-Standard Sugar Moieties In certain embodiments, modified sugar moieties are substituted stereo-standard furanosyl or 4’- thiofuranosyl sugar moieties comprising one or more acyclic substituent, including but not limited to substituents at the 2 ’ , 3 ’ , 4 ’ , and/or 5 ’ positions . In certain embodiments, the furanosyl sugar moiety is a ribosyl sugar moiety. In certain embodiments one or more acyclic substituent of substituted stereo-standard sugar moieties is branched. Examples of 2 ’-substituent groups suitable for substituted stereo-standard sugar moieties include but are not limited to: 2’-F, 2'-OCH ₃ (“2’-OMe” or “2 ’-O-methyl”), and 2'-O(CH ₂ ) ₂ OCH ₃ (“2’-MOE”). In certain embodiments, 2 ’-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF ₃ , OCF ₃ , O- C ₁ -C ₁₀ alkoxy, O- C ₁ -C ₁₀ substituted alkoxy, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ substituted alkyl, S-alkyl, N(R _m )-alkyl, O-alkenyl, S-alkenyl, N(R _m )-alkenyl, O-alkynyl, S-alkynyl, N(R _m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH ₂ ) ₂ SCH3, 0(CH ₂ ) ₂ 0N(R _m )(Rn) or OCH ₂ C(=O)-N(R _m )(R _n ), where each R _m and R _n is, independently, H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl, and the 2 ’-substituent groups described in Cook et al., U.S. 6,531,584; Cook et al., U.S. 5,859,221; and Cook et al., U.S. 6,005,087. Certain embodiments of these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N0 ₂ ), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 3’- substituent groups include 3’-methyl (see Frier, et al., The ups and downs of nucleic acid duplex stability: structure -stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res., 25, 4429-4443, 1997.) Examples of 4’ -substituent groups suitable for substituted stereo-standard sugar moieties include but are not limited to alkoxy ( e.g ., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5 ’-substituent groups suitable for substituted stereo-standard sugar moieties include but are not limited to: 5 ’-methyl (Ror S), 5 ’-allyl, 5 ’-ethyl, 5'-vinyl, and 5 ’-methoxy. In certain embodiments, non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5 '-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836. 2’,4’-difluoro modified sugar moieties have been described in Martinez-Montero, et al., Rigid 2',4'-difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase. J. Org. Chem., 2014, 79:5627-5635. Modified sugar moieties comprising a 2’ -modification (OMe or F) and a 4 ’-modification (OMe or F) have also been described in Malek-Adamian, et al., J. Org. Chem , 2018, 83: 9839-9849.

In certain embodiments, a 2 ’-substituted stereo-standard nucleoside comprises a sugar moiety comprising a non-bridging 2 ’-substituent group selected from: F, NH ₂ , N3, OCF _3, OCH ₃ , SCH ₃ , O(CH ₂ ) ₃ NH ₂ , CH ₂ CH=CH ₂ , OCH ₂ CH=CH ₂ , OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ 0N(Rm)(Rn),

O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ , and N-substituted acetamide (OCH ₂ C(=O)-N(R _m )(Rn)), where each R _m and R _n is, independently, H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

In certain embodiments, a 2 ’-substituted stereo-standard nucleoside comprises a sugar moiety comprising a non-bridging 2 ’-substituent group selected from: F, OCF _3, OCH 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 OCH ₂ C(=O)-N(H)CH ₃ (“NMA”).

In certain embodiments, a 2 ’-substituted stereo-standard nucleoside comprises a sugar moiety comprising a 2 ’-substituent group selected from: F, OCH ₂ , and OCH ₂ CH ₂ OCH ₃ .

In certain embodiments, nucleosides comprise a 4’-thiofuranosyl sugar moiety, In certain embodiments, nucleosides comprise a 2’-deoxy-4’-thiofuranosyl sugar moiety, to form 4’-thio DNA (see Takahashi, et al., Nucleic Acids Research 2009, 37: 1353-1362). This modification can be combined with other modifications detailed herein. In certain such embodiments, the sugar moiety is further modified at the 2’ position. In certain embodiments the sugar moiety comprises a 2’-fluoro. A thymidine with this sugar moiety has been described in Watts, et al., J. Org. Chem. 2006, 71(3): 921-925 (4’-S-fluoro5-methylarauridine or FAMU). c. Bicyclic Nucleosides

Certain nucleosides comprise modifed sugar moieties that comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of sugar moieties comprising such 4’ to 2’ bridging sugar substituents include but are not limited to bicyclic sugars comprising: 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -O-2' (“ENA”), 4'-CH ₂ -S-2', 4'-(CH ₂ ) ₂ -O-2' (“ENA”), 4'-CH(CH ₃ )-O-2' (referred to as “constrained ethyl” or “cEf ’ when in the S configuration), 4’-CH ₂ -O-CH ₂ -2’, 4’-CH ₂ -N(R)-2’, 4'-CH(CH ₂ OCH ₃ )-O-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. 7,399,845, Bhat et al., U.S. 7,569,686, Swayze et al., U.S. 7,741,457, and Swayze et al., U.S. 8,022,193), 4'-C(CH ₃ )(CH ₃ )-O-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'-CH ₂ -N(OCH ₃ )-2' and analogs thereof (see, e.g., Prakash etal., U.S. 8,278,425), 4'-CH ₂ - 0-N(CH ₃ )-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745), 4'-CH ₂ -C(H)(CH ₃ )- 2' (see, e.g., Zhou, et al, J. Org. Chem., 2009, 74, 118-134), 4'-CH ₂ -C(=CH ₂ )-2' and analogs thereof (see e.g., Seth et al., U.S. 8,278,426), 4’-C(R _a R _b )-N(R)-O-2’, 4’-C(R _a R _b )-O-N(R)-2’, 4'-CH ₂ -O-N(R)-2', and 4'-CH ₂ - N(R)-O-2', wherein each R, R _a , and R _b is, independently, H, a protecting group, or C1-C12 alkyl (see, e.g. Imanishi et al., U.S. 7,427,672), 4’-C(=O)-N(CH ₃ ) ₂ -2’, 4’-C(=O)-N(R) ₂ -2’, 4’-C(=S)-N(R) ₂ -2’ and analogs thereof (see, e.g. , Obika et al . , WO2011052436A 1 , Yusuke, WO2017018360A 1 ) .

Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al, J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; 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., 2017, 129, 8362-8379; Elayadi et al,; Christiansen, et al., J. Am. Chem. Soc. 1998, 120, 5458- 5463 / Wengel et a., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al. U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the α-L configuration or in the β-D configuration.

LNA (β-D-configuration) α-L-LNA (α-L-configuration) bridge = 4'-CH ₂ -O-2' bridge = 4'-CH ₂ O-2' α-L-methyleneoxy (4’-CH ₂ -O-2’) or a-L-LNA bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA) are identified in exemplified embodiments herein, they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 ’-substituted and 4’-2’ bridged sugars).

The term “substituted” following a position of the furanosyl ring, such as ”2’ -substituted” or “2 ’-4’- substituted”, indicates that is the only position(s) having a substituent other than those found in unmodified sugar moieties in oligonucleotides. d. Sugar Surrogates

In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4’-sulfur atom and a substitution at the 2'- position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,' 939,677) and/or the 5’ position.

In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), altritol nucleic acid (“ANA”), mannitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA (“F-HNA”, see e.g. Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906; F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran) .

In certain embodiments, sugar surrogates comprise rings having no heteroatoms. For example, nucleosides comprising bicyclo [3.1.0] -hexane have been described (see, e.g., Marquez, et al., J. Med. Chem. 1996, 39:3739-3749). In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506). As used here, the term “morpholino” means a sugar surrogate comprising the following structure:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are refered to herein as “modifed morpholinos.” In certain embodiments, morpholino residues replace a full nucleotide, including the intemucleoside linkage, and have the structures shown below, wherein Bx is a heterocyclic base moiety.

In certain embodiments, sugar surrogates comprise acyclic moieites. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), glycol nucleic acid (“GNA”, see Schlegel, et al., J. Am. Chem. Soc. 2017, 139:8537-8546) and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides. Certain such ring systems are described in Hanessian, et al., J. Org. Chem., 2013, 78: 9051-9063 and include bcDNA andtcDNA. Modifications to bcDNA andtcDNA, such as 6’-fluoro, have also been described (Dogovic and Leumann, J. Org. Chem., 2014, 79: 1271-1279).

2. Modified Nucleohases

In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine,

5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N- methyladenine, 2-propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH ₃ ) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N- benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size- expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2- one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications , Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273- 288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163-166 and 442-443. In certain embodiments, modified nucleosides comprise double-headed nucleosides having two nucleobases. Such compounds are described in detail in Sorinas et al., J. Org. Chem, 2014 79: 8020-8030.

Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manoharan et al., US2003/0158403; Manoharan et al., US2003/0175906;; Dinh et al., U.S. 4,845,205; Spielvogel et al., U.S. 5,130,302; Rogers et al., U.S. 5,134,066; Bischofberger et al., U.S. 5,175,273; Urdea et al., U.S. 5,367,066; Benner et al., U.S. 5,432,272; Matteucci et al., U.S. 5,434,257; Gmeiner et al., U.S. 5,457,187; Cook et al., U.S. 5,459,255; Froehler et al., U.S. 5,484,908; Matteucci et al., U.S. 5,502,177; Hawkins et al., U.S. 5,525,711; Haralambidis et al., U.S. 5,552,540; Cook et al., U.S. 5,587,469; Froehler et al., U.S. 5,594,121; Switzer et al., U.S. 5,596,091; Cook et al., U.S. 5,614,617; Froehler et al., U.S. 5,645,985; Cook et al., U.S. 5,681,941; Cook et al., U.S. 5,811,534; Cook et al., U.S. 5,750,692; Cook et al., U.S. 5,948,903; Cook et al., U.S. 5,587,470; Cook et al., U.S. 5,457,191; Matteucci et ak, U.S. 5,763,588; Froehler et al., U.S. 5,830,653; Cook et ak, U.S. 5,808,027; Cook et ak, 6,166,199; and Matteucci et ak, U.S. 6,005,096.

In certain embodiments, compounds comprise or consist of a modified oligonucleotide complementary to an target nucleic acid comprising one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5- methylcytosine. B. Modified Internucleoside Linkages

In certain embodiments, compounds described herein having one or more modified intemucleoside linkages are selected over compounds having only phosphodiester intemucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

In certain embodiments, compounds comprise or consist of a modified oligonucleotide complementary to a target nucleic acid comprising one or more modified intemucleoside linkages. In certain embodiments, the modified intemucleoside linkages are phosphorothioate linkages. In certain embodiments, each intemucleoside linkage of an antisense compound is a phosphorothioate intemucleoside linkage.

In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any intemucleoside linkage. The two main classes of intemucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing intemucleoside linkages include unmodified phosphodiester intemucleoside linkages, modified phosphotriesters such as THP phosphotriester and isopropyl phosphotriester, phosphonates such as methylphosphonate, isopropyl phosphonate, isobutyl phosphonate, and phosphonoacetate, phosphoramidates, phosphorothioate, and phosphorodithioate (“HS- P=S”). Representative non-phosphorus containing intemucleoside linkages include but are not limited to methylenemethylimino (-CH ₂ -N(CH ₃ )-O-CH ₂ -), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (- O-S1H2-O-); formacetal, thioacetamido (TANA), alt-thioformacetal, glycine amide, and N,N'- dimethylhydrazine (-CH ₂ -N(CH ₃ )-N(CH ₃ )-). Modified intemucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are well known to those skilled in the art.

Representative intemucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising intemucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom intemucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate intemucleoside linkages wherein all of the phosphorothioate intemucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. All phosphorothioate linkages described herein are stereorandom unless otherwise specified. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate intemucleoside linkages in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et ak, JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017/015555. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (rip) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (/Zp) configuration. In certain embodiments, modified oligonucleotides comprising (/Zp) and/or (.S'p) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:

Unless otherwise indicated, chiral intemucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.

Neutral intemucleoside linkages include, without limitation, phosphotriesters, phosphonates, 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'), formacetal (3'-O- CH ₂ -O-5'), methoxypropyl, and thioformacetal (3'-S-CH ₂ -O-5'). Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH ₂ component parts.

In certain embodiments, nucleic acids can be linked 2’ to 5’ rather than the standard 3’ to 5’ linkage. Such a linkage is illustrated below.

In certain embodiments, nucleosides can be linked by vicinal 2’, 3’-phosphodiester bonds. In certain such embodiments, the nucleosides are threofuranosyl nucleosides (TNA; see Bala, et al., J Org. Chem. 2017, 82:5910-5916). A TNA linkage is shown below.

Additional modified linkages include a,β-D-CNA type linkages and related conformationally- constrained linkages, shown below. Synthesis of such molecules has been described previously (see Dupouy, et ak, Angew. Chem. Int. Ed. Engl., 2014, 45: 3623-3627; Borsting, et al. Tetahedron, 2004, 60:10955- 10966; Ostergaard, et al., ACS Chem. Biol. 2014, 9: 1975-1979; Dupouy, et al., Eur. J. Org. Chem.., 2008, 1285-1294; Martinez, et al., PLoS One, 2011, 6:e25510; Dupouy, et al., Eur. J. Org. Chem., 2007, 5256- 5264; Boissonnet, et ak, New J. Chem., 2011, 35: 1528-1533.)

II. Certain Motifs In certain embodiments, oligomeric compounds described herein comprise or consist of oligonucleotides. Modified oligonucleotides can be described by their motif, e.g. a pattern of unmodified and/or modified sugar moieties, nucleobases, and/or intemucleoside linkages. In certain embodiments, modified oligonucleotides comprise one or more stereo-non-standard 4’-thio nucleosides. In certain embodiments, modified oligonucleotides comprise one or more stereo-standard nucleosides. In certain embodiments, modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified intemucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns or motifs of sugar moieties, nucleobases, and intemucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or intemucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).

A. Certain Sugar Motifs

In certain embodiments, oligomeric compounds described herein comprise or consist of oligonucleotides. In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include without limitation any of the sugar modifications discussed herein.

In certain embodiments, a modified oligonucleotide comprises or consists of a gapmer. The sugar motif of a gapmer defines the regions of the gapmer: 5 ’-region, central region (gap), and 3 ’-region. The central region is linked directly to the 5 ’-region and to the 3 ’-region with no nucleosides intervening. The central region is a deoxy region. The nucleoside at the first position (position 1) from the 5 ’-end of the central region and the nucleoside at the last position of the central region are adjacent to the 5’-region and 3’- region, respectively, and each comprise a sugar moiety independently selected from a 2’-deoxyfuranosyl sugar moiety, a 2’-deoxy-4’-thiofuranosyl sugar moiety, or a sugar surrogate. In certain embodiments, the nucleoside at position 1 of the central region and the nucleoside at the last position of the central region are DNA nucleosides, selected from stereo-standard DNA nucleosides or stereo-non-standard DNA nucleosides having any of Formulas I-VII, wherein each J is H. In certain embodiments, the nucleoside at the first and last positions of the central region adjacent to the 5’ and 3’ regions are stereo-standard DNA nucleosides. Unlike the nucleosides at the first and last positions of the central region, the nucleosides at the other positions within the central region may comprise a 2’ -substituted stereo-standard sugar moiety or a substituted stereo-non-standard sugar moiety or a bicyclic sugar moiety. In certain embodiments, each nucleoside within the central region supports RNase H cleavage. In certain embodiments, a plurality of nucleosides within the central region support RNase H cleavage.

In certain embodiments, the central region comprises at least one stereo-non-standard 4’-thio nucleoside selected from Formula I-VII. In certain embodiments, the central region comprises at least two, at least three, at least four, at least five, or at least six stereo-non-standard 4’-thio nucleosides selected from Formula I-VII. In certain embodiments, the central region comprises exactly one stereo-non-standard 4’-thio nucleoside. In certain embodiments, the central region comprises exactly two stereo-non-standard 4’-thio nucleosides. In certain embodiments, the central region comprises exactly three stereo-non-standard 4’-thio nucleosides. In certain embodiments, the central region comprises exactly four stereo-non-standard 4’-thio nucleosides. In certain embodiments, the central region comprises exactly five stereo-non-standard 4’-thio nucleosides. In certain embodiments, the central region comprises exactly 6, 7, 8, 9, or 10 stereo-nonstandard 4’-thio nucleosides. In certain embodiments, the remainder of the nucleosides of the central region are stereo-standard DNA nucleosides. In certain embodiments, exactly one nucleoside of the central region is a 2 ’-substituted stereo-non-standard 4’-thio nucleoside, and the remainder of the nucleosides of the central region are stereo-standard DNA nucleosides. In certain embodiments, exactly one nucleoside of the central region is a 2’-OMe stereo-non-standard 4’-thio nucleoside, and the remainder of the nucleosides of the central region are stereo-standard DNA nucleosides. In certain embodiments, one or more nucleosides of the central region is a stereo-non-standard nucleoside, the nucleoside at position 2 of the central region is a stereo-standard 2’-OMe nucleoside, and the remainder of the nucleosides of the central region are stereostandard DNA nucleosides. In certain embodiments, each nucleoside of the central region is a stereo-nonstandard 4’-thio nucleoside.

In certain embodiments, the nucleoside at the first position of the central region is a stereo-nonstandard DNA nucleoside. In certain embodiments, the nucleoside at the last position of the central region is a stereo-non-standard DNA nucleoside.

In certain embodiments, the nucleoside at the second position of the central region is a stereo-nonstandard 4’-thio nucleoside. In certain embodiments, the nucleoside at the third position of the central region is a stereo-non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the fourth position of the central region is a stereo-non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the fifth position of the central region is a stereo-non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the sixth position of the central region is a stereo-non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the seventh position of the central region is a stereo-non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the eighth position of the central region is a stereo- non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the ninth position of the central region is a stereo-non-standard 4’-thio nucleoside. In certain embodiments, the nucleoside at the tenth position of the central region is a stereo-non-standard 4’-thio nucleoside. In any of such embodiments, the stereo-non-standard 4’-thio nucleoside may be a substituted stereo-non-standard 4’-thio nucleoside.

The 3 ’-most nucleoside of the 5 ’-region and the 5 ’-most nucleoside of the 3 ’-region are substituted stereo-standard nucleosides or bicyclic nucleosides. In certain embodiments, each nucleoside of the 5 ’-region and the 3 ’-region is either a stereo-standard nucleoside or a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5 ’-region and the 3 ’-region is either a substituted stereo-standard nucleoside or a bicyclic nucleoside. In certain embodiments, the bicyclic sugar moiety in the 5’ and 3 ’-regions is a 4 ’-2 ’-bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety in the 5’ and 3’ regions is a cEt. In certain embodiments, the stereo-standard sugar moiety is a 2’-MOE-P-D-ribofuranosyl sugar moiety.

Herein, the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [# of nucleosides in the 5 ’-region] - [# of nucleosides in the central region] - [# of nucleosides in the 3 ’-region]. Thus, a 3-10-3 gapmer consists of 3 linked nucleosides in each of the 3’ and 5’ regions and 10 linked nucleosides in the central region. Where such nomenclature is followed by a specific modification, that modification is the modification of each sugar moiety of each 5’ and 3 ’-region and the central region nucleosides comprise stereo-standard DNA sugar moieties. Thus, a 5-10-5 MOE gapmer consists of 5 linked nucleosides each comprising 2’-MOE-stereo-standard sugar moieties in the 5 ’-region, 10 linked nucleosides each comprising a stereo-standard DNA sugar moiety in the central region, and 5 linked nucleosides each comprising 2’-MOE-stereo-standard sugar moieties in the 3’-region. A 5-10-5 MOE gapmer having a substituted stereo-non-standard 4’-thio nucleoside at position 2 of the gap has a gap of 10 nucleosides wherein the 2 ^nd nucleoside of the gap is a substituted stereo-non-standard 4’-thio nucleoside rather than the stereo-standard DNA nucleoside. Such oligonucleotide may also be described as a 5-1-1-8-5 MOE/substituted stereo-non-standard/MOE gapmer. A 3-10-3 cEt gapmer consists of 3 linked nucleosides each comprising a cEt in the 5 ’-region, 10 linked nucleosides each comprising a stereo-standard DNA sugar moiety in the central region, and 3 linked nucleosides each comprising a cEt in the 3’-region. A 3-10-3 cEt gapmer having a substituted stereo-non-standard 4’-thio nucleoside at position 2 of the gap has a gap of 10 nucleoside wherein the 2 ^nd nucleoside of the gap is a substituted stereo-non-standard 4’-thio nucleoside rather than the stereo-standard DNA nucleoside. Such oligonucleotide may also be described as a 3-1-1-8-3 cEt/substituted stereo-non-standard/cEt gapmer.

The sugar motif of a gapmer may also be denoted by a notation where different letters indicate various nucleosides. For example: kkk-dx*d(8)-kkk, wherein each “k” represents a cEt nucleoside, each “d” represents a stereo standard DNA and x* represents a substituted stereo-non-standard 4’-thio nucleoside. Certain MOE gapmers may be denoted by the following notations eeeee-dx*(8)-eeeee or e(5)-dx*(8)-e(5), wherein each “e” represents a 2’-MOE-stereo standard nucleosides, each “d” represents a stereo standard DNA, and each x* represents a substituted stereo-non-standard 4’-thio nucleoside. Sugar motifs are independent of the nucleobase sequence, the intemucleoside linkage motif, and any nucleobase modifications.

B. Certain Nucleobase Motifs

In certain embodiments, oligomeric compounds described herein comprise or consist of oligonucleotides. In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5 -methylcytosine s .

In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3 ’-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 3 ’-end of the oligonucleotide. In certain embodiments, the block is at the 5 ’-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5 ’-end of the oligonucleotide.

In certain embodiments, one nucleoside comprising a modified nucleobase is in the central region of a modified oligonucleotide. In certain such embodiments, the sugar moiety of said nucleoside is a 2 ^' -b-ϋ- deoxyribosyl moiety. In certain such embodiments, the modified nucleobase is selected from: 5-methyl cytosine, 2-thiopyrimidine, 2-thiothymine, 6-methyladenine, inosine, pseudouracil, or 5-propynepyrimidine.

C. Certain Internucleoside Linkage Motifs

In certain embodiments, oligomeric compounds described herein comprise or consist of oligonucleotides. In certain embodiments, oligonucleotides comprise modified and/or unmodified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each intemucleoside linkage is a phosphodiester intemucleoside linkage (P=O). In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is a phosphorothioate intemucleoside linkage (P=S). In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage and phosphodiester intemucleoside linkage. In certain embodiments, each phosphorothioate intemucleoside linkage is independently selected from a stereorandom phosphorothioate, a (.Vp) phosphorothioate, and a (rip) phosphorothioate. In certain embodiments, the intemucleoside linkages within the central region of a modified oligonucleotide are all modified. In certain such embodiments, some or all of the intemucleoside linkages in the 5 ’-region and 3 ’-region are unmodified phosphate linkages. In certain embodiments, the terminal intemucleoside linkages are modified. In certain embodiments, the intemucleoside linkage motif comprises at least one phosphodiester intemucleoside linkage in at least one of the 5 ’-region and the 3’- region, wherein the at least one phosphodiester linkage is not a terminal intemucleoside linkage, and the remaining intemucleoside linkages are phosphorothioate intemucleoside linkages. In certain such embodiments, all of the phosphorothioate linkages are stereorandom. In certain embodiments, all of the phosphorothioate linkages in the 5 ’-region and 3 ’-region are (rip) phosphorothioates, and the central region comprises at least one rip, rip, rip motif. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such intemucleoside linkage motifs.

In certain embodiments, oligonucleotides comprise a region having an alternating intemucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified intemucleoside linkages. In certain such embodiments, the intemucleoside linkages are phosphorothioate intemucleoside linkages. In certain embodiments, all of the intemucleoside linkages of the oligonucleotide are phosphorothioate intemucleoside linkages. In certain embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester or phosphate and phosphorothioate. In certain embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester or phosphate and phosphorothioate and at least one intemucleoside linkage is phosphorothioate. In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate intemucleoside linkages. In certain such embodiments, at least one such block is located at the 3’ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3’ end of the oligonucleotide.

In certain embodiments, oligonucleotides comprise one or more methylphosphonate linkages. In certain embodiments, modified oligonucleotides comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosphonate linkages. In certain embodiments, one methylphosphonate linkage is in the central region of an oligonucleotide.

In certain embodiments, it is desirable to arrange the number of phosphorothioate intemucleoside linkages and phosphodiester intemucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate intemucleoside linkages and the number and position of phosphodiester intemucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate intemucleoside linkages may be decreased and the number of phosphodiester intemucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate intemucleoside linkages may be decreased and the number of phosphodiester intemucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments it is desirable to decrease the number of phosphorothioate intemucleoside linkages while retaining nuclease resistance. In certain embodiments it is desirable to increase the number of phosphodiester intemucleoside linkages while retaining nuclease resistance.

III. Certain Modified Oligonucleotides

In certain embodiments, oligomeric compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, the above modifications (sugar, nucleobase, intemucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modifications, motifs, and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each intemucleoside linkage of a modified oligonucleotide may be modified or unmodified and may or may not follow the modification pattern of the sugar moieties. Likewise, such modified oligonucleotides may comprise one or more modified nucleobase independent of the pattern of the sugar modifications. Furthermore, in certain instances, a modified oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a region of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions or segments, A, B, and C, wherein region or segment A consists of 2-6 linked nucleosides having a specified sugar moiety, region or segment B consists of 6-10 linked nucleosides having a specified sugar moiety, and region or segment C consists of 2-6 linked nucleosides having a specified sugar moiety. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of 20 for the overall length of the modified oligonucleotide. Unless otherwise indicated, all modifications are independent of nucleobase sequence except that the modified nucleobase 5- methylcytosine is necessarily a “C” in an oligonucleotide sequence. In certain embodiments, when a DNA nucleoside or DNA-like nucleoside that comprises a T in a DNA sequence is replaced with a RNA-like nucleoside, the nucleobase T is replaced with the nucleobase U. Each of these compounds has an identical target RNA.

In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,

46, 47, 48, 49, and 50; provided that X<Y. For example, in certain embodiments, oligonucleotides consist of

12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18,

13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25,

14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19,

16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29,

17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29,

19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25,

22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30,

26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.

In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.

IV. Certain Conjugated Compounds

In certain embodiments, the oligomeric compounds described herein comprise or consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker that links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3’ and/or 5 ’-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3 ’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3 ’-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5 ’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5 ’-end of oligonucleotides.

Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.

Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et ak, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiochole sterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-O-hexadecyl-rac-glycero-3-H- phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, i, 923 -937), ₌ a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; doi:10.1038/mtna.2014.72 and Nishina et ύ., Molecular The rapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).

1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (.V)-(+)-pranoprofcn, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fmgolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

2. Conjugate linkers

Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain oligomeric compounds, a conjugate linker is a single chemical bond (i.e. conjugate moiety is attached to an oligonucleotide via a conjugate linker through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to oligomeric compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on an oligomeric compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted Ci- Cio alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5- methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid.

For example, an oligomeric compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such a compound is more than 30. Alternatively, an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such a compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides.

In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated oligonucleotide. Thus, certain conjugate may comprise one or more cleavable moieties, typically within the conjugate linker. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate or phosphodiester linkage between an oligonucleotide and a conjugate moiety or conjugate group.

In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is a nucleoside comprising a 2'-deoxyfuranosyl that is attached to either the 3' or 5 '-terminal nucleoside of an oligonucleotide by a phosphodiester intemucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphodiester or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is a nucleoside comprising a 2’-P-D-deoxyribosyl sugar moiety. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.

3. Certain Cell-Targeting Conjugate Moieties In certain embodiments, a conjugate group comprises a cell-targeting conjugate moiety. In certain embodiments, a conjugate group has the general formula: wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2 or greater, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1.

In certain embodiments, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.

In certain embodiments, the cell-targeting moiety comprises a branching group comprising one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system.

In certain embodiments, each tether of a cell-targeting moiety comprises one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amino, oxo, amide, phosphodiester, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, amino, oxo, and amid, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, amino, and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group. In certain embodiments, each tether comprises a chain from about 6 to about 20 atoms in length. In certain embodiments, each tether comprises a chain from about 10 to about 18 atoms in length. In certain embodiments, each tether comprises about 10 atoms in chain length.

In certain embodiments, each ligand of a cell-targeting moiety has an affinity for at least one type of receptor on a target cell. In certain embodiments, each ligand has an affinity for at least one type of receptor on the surface of a mammalian lung cell.

In certain embodiments, each ligand of a cell-targeting moiety is a carbohydrate, carbohydrate derivative, modified carbohydrate, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain such embodiments, the conjugate group comprises a carbohydrate cluster (see, e.g., Maier et ah, “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et ak, “Design and Synthesis of Novel N- Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, 47, 5798-5808, which are incorporated herein by reference in their entirety). In certain such embodiments, each ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, such as sialic acid, a-D-galactosamine, b- 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 A-sulfo-D-glucosaminc. and A-glycoloyl-a- neuraminic acid. For example, thio sugars may be selected from 5-Thio-P-D-glucopyranose, methyl 2,3,4-tri- O-acetyl-l-thio-6-O-trityl-a-D-glucopyranoside, 4-thio-P-D-galactopyranose, and ethyl 3,4,6,7-tetra-O- acetyl-2-deoxy-l,5-dithio-a-D-g/wco-heptopyranoside.

In certain embodiments, oligomeric compounds described herein comprise a conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et ak, J Biol Chem, 1982, 257, 939-945; Pavia et ak, Int J Pep Protein Res, 1983, 22, 539-548; Lee et ak, Biochem, 1984, 23, 4255-4261; Lee et ak, Glycoconjugate J, 1987, 4, 317-328; Toyokuni et ak, Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et ak, JMed Chem, 1995, 38, 1538-1546; Valentijn et ak, Tetrahedron, 1997, 53, 759- 770; Kim et ak, Tetrahedron Lett, 1997, 38, 3487-3490; Lee et ak, Bioconjug Chem, 1997, 8, 762-765; Kato et ak, Glycobiol, 2001, 11, 821-829; Rensen et ak, JBiol Chem, 2001, 276, 37577-37584; Lee et ak, Methods Enzymol, 2003, 362, 38-43; Westerlind et ak, Glycoconj J, 2004, 21, 227-241; Lee et ak, Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et ak, Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et ak, Bioorg Med Chem, 2008, 16, 5216-5231; Lee et ak, Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et ak, Analyt Biochem, 2012, 425, 43-46; Pujol et ak, Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., JMed Chem, 1995, 38, 1846-1852; Sliedregt et al., JMed Chem, 1999, 42, 609-618; Rensen et al., JMed 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., JOrg Chem, 2012, 77, 7564-7571; Biessen et al., FASEBJ, 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., OrgLett, 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 WO1998/013381; WO2011/038356;

WO 1997/046098; W02008/098788; W02004/101619; WO2012/037254; WO2011/120053; W02011/100131; WO2011/163121; WO2012/177947; W02013/033230; W02013/075035; WO2012/083185; W02012/083046; W02009/082607; WO2009/134487; W02010/144740; W02010/148013; WO 1997/020563; W02010/088537; W02002/043771; W02010/129709;

WO2012/068187; WO2009/126933; W02004/024757; WO2010/054406; WO2012/089352;

WO2012/089602; WO2013/166121; WO2013/165816; U.S. Patents 4,751,219; 8,552,163; 6,908,903;

7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;

6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent

Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132.

Compositions and Methods for Formulating Pharmaceutical Compositions

Oligomeric compounds described herein may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

Certain embodiments provide pharmaceutical compositions comprising one or more oligomeric compounds or a salt thereof. In certain embodiments, the oligomeric compounds comprise or consist of a modified oligonucleotide. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more oligomeric compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more oligomeric compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more oligomeric compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one oligomeric compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and phosphate- buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more oligomeric compound and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An oligomeric compound described herein complementary to a target nucleic acid can be utilized in pharmaceutical compositions by combining the oligomeric compound with a suitable pharmaceutically acceptable diluent or carrier and/or additional components such that the pharmaceutical composition is suitable for injection. In certain embodiments, a pharmaceutically acceptable diluent is phosphate buffered saline. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an oligomeric compound complementary to a target nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is phosphate buffered saline. In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide provided herein.

Pharmaceutical compositions comprising oligomeric compounds provided herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. In certain embodiments, the oligomeric compound comprises or consists of a modified oligonucleotide. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

Certain Mechanisms

In certain embodiments, oligomeric compounds described herein comprise or consist of modified oligonucleotides having at least one stereo-non-standard 4’-thio nucleoside. In certain such embodiments, the oligomeric compounds described herein are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, compounds described herein selectively affect one or more target nucleic acid. Such compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in a significant undesired antisense activity. In certain antisense activities, hybridization of a compound described herein to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain compounds described herein result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, compounds described herein are sufficiently “DNA- like” to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in in the RNA:DNA duplex is tolerated.

Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or animal.

Certain oligomeric compounds

In certain embodiments, oligomeric compounds described herein having one or more stereo-nonstandard 4’-thio nucleosides are selected over compounds lacking such stereo-non-standard 4’-thio nucleosides because of one or more desirable properties. In certain embodiments, oligomeric compounds described herein having one or more stereo-non-standard 4’-thio nucleosides have enhanced cellular uptake. In certain embodiments, oligomeric compounds described herein having one or more stereo-non-standard 4’- thio nucleosides have enhanced bioavailability. In certain embodiments, oligomeric compounds described herein having one or more stereo-non-standard 4’-thio nucleosides have enhanced affinity for target nucleic acids. In certain embodiments, oligomeric compounds described herein having one or more stereo-nonstandard 4’-thio nucleosides have increased stability in the presence of nucleases. In certain embodiments, oligomeric compounds described herein having one or more stereo-non-standard 4’-thio nucleosides have increased interactions with certain proteins. In certain embodiments, oligomeric compounds described herein having one or more stereo-non-standard 4’-thio nucleosides have decreased interactions with certain proteins. In certain embodiments, oligomeric compounds described herein having one or more stereo-non-standard 4’- thio nucleosides have increased RNase H activity. In certain embodiments, incorporation of one or more stereo-non-standard 4’-thio nucleosides into a modified oligonucleotide within the central region can significantly reduce toxicity with only a modest loss in potency, if any. In certain embodiments, incorporation of one or more stereo-non-standard 4’-thio nucleosides into a modified oligonucleotide at positions 2, 3 or 4 of the central region can significantly reduce toxicity with only a modest loss in potency, if any. In certain embodiments, incorporation of one or more stereo-non-standard 4’-thio nucleosides into a modified oligonucleotide at position 2 of the central region can significantly reduce toxicity with only a modest loss in potency, if any. In certain such embodiments, the stereo-non-standard 4’-thio nucleoside is a stereo-non-standard 4’-thio nucleoside of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, or Formula VII.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, compounds described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, a pre-mRNA and corresponding mRNA are both target nucleic acids of a single compound. In certain such embodiments, the target region is entirely within an intron of a target pre-mRNA. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

Certain Compounds

Certain compounds described herein ( e.g ., modified oligonucleotides) have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as ( R ) or (5), as a or b such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms. All tautomeric forms of the compounds provided herein are included unless otherwise indicated.

The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the 'H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to: ² H or ³ H in place of ¹ H, ¹³ C or ¹⁴ C in place of ¹² C, ¹⁵ N in place of ¹⁴ N, ¹⁷ O or ¹⁸ O in place of ¹⁶ O, and ³³ S, ³⁴ S, ³⁵ S, or ³⁶ S in place of ³² S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.

EXAMPLES

The following examples are intended to illustrate certain aspects of the invention and are not intended to limit the invention in any way. Example 1: Synthesis of 4’-thio nucleoside amidites for use in solid-phase oligonucleotide synthesis

Described below are synthetic methods to prepare 4’-thio nucleoside amidites that can then be incorporated using standard solid-phase oligonucleotide synthesis.

Shown below is the synthesis of a 4’-thio-α-D-deoxyribosyl nucleoside of Formula VIII, wherein J ₁ andJ ₂ are each H, Ti is a hydroxyl protecting group, and T ₂ is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula I, wherein J ₁ and J ₂ are each H.

Shown below is the synthesis of a 4’-thio-β-D-deoxyxylosyl nucleoside of Formula IX, wherein J ₃ and J ₄ are each H, T ₃ is a hydroxyl protecting group, and T4 is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula II, wherein J ₃ and J ₄ are each H.

Shown below is the synthesis of a 4’-thio-α-L-deoxyxylosyl nucleoside of Formula X, wherein J ₅ and J ₆ are each H, T ₅ is a hydroxyl protecting group, and T ₆ is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula III, wherein J ₅ and F are each H. Shown below is the synthesis of a 4’-thio-β-L-deoxyribosyl nucleoside of Formula XI, wherein J ₇ and Is are each H, T ₇ is a hydroxyl protecting group, and Tx is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula IV, wherein J ₇ and Jx are each H.

Shown below is the synthesis of a 4’-thio-α-L-deoxyribosyl nucleoside of Formula XII, wherein J ₉ and J ₁₀ are each H, T ₉ is a hydroxyl protecting group, and T ₁₀ is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula V, wherein J ₉ and J ₁₀ are each H.

Shown below is the synthesis of a 4’-thio-β-L-deoxyxylosyl nucleoside of Formula XIII, wherein Jn and J ₁₂ are each H, Tn is a hydroxyl protecting group, and T ₁₂ is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula VI, wherein Jn and J ₁₂ are each H.

Shown below is the synthesis of a 4’-thio-α-D-deoxyxylosyl nucleoside of Formula XIV, wherein J ₁₃ and J ₁₄ are each H, T ₁₃ is a hydroxyl protecting group, and T ₁₄ is a reactive phosphorous group. Incorporation of this amidite into an oligonucleotide yields a modified oligonucleotide comprising a stereo-non-standard 4’- thio nucleoside having Formula VII, wherein J ₁₃ and J ₁₄ are each H.