COMPOUNDS FOR MODULATING BETA GLOBIN EXPRESSION

Sequence Listing

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0371WOSEQ_ST25.txt, created on November 9, 2020, which is 42 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

Field

Provided are oligomeric compounds, methods, and pharmaceutical compositions for modulating the amount of b-globin in a cell or subject. In certain instances, oligomeric compounds, methods, and pharmaceutical compositions modulate splicing of HBB RNA in a cell or subject. In certain instances, oligomeric compounds, methods, and pharmaceutical compositions increase the amount of wild type HBB RNA in a cell or subject. In certain instances, oligomeric compounds, methods, and pharmaceutical compositions increase the amount of hemoglobin in a cell or subject. Such compounds and pharmaceutical compositions are useful to ameliorate at least one symptom or hallmark of b-thalassemia. Such symptoms and hallmarks include a low hemoglobin level, erythrocyte deformities, fatigue, weakness, pale skin or jaundice, abdominal swelling, and decreased longevity.

Background

The human gene HBB encodes human b-globin. Human b-globin is also referred to as HBB, b-globin chain, beta globin, hemoglobin beta, and hemoglobin subunit beta. Human hemoglobin is a tetramer of two a- globin chains and two b-globin chains, and is required in red blood cells to transport oxygen and other gases throughout the body. Hemoglobin also plays a role in iron metabolism. A hemoglobin deficiency resulting from a lack of adequate amounts of b-globin leads to insufficient oxygen transport and iron homeostasis.

A mutation in HBB known as IVS-2-745 (OG) creates an aberrant 5' splice site at nucleotide position 745 of intron 2 of HBB pre-mRNA, and activates a common cryptic 3' splice site at nucleotide position 579 within the same intron. The splicing machinery recognizes these splice sites; as a result, a 165- nucleobase fragment of the intronic sequence between the newly activated splice sites is incorrectly retained in a mutant HBB RNA. The retained 165-micleobase fragment carries an in-frame stop codon that prevents proper translation of the mutant HBB RNA and production of a functional b-globin protein. As a consequence of the production of the mutant HBB RNA, there is a reduction in the production of b-globin. The resulting deficiency in b-globin leads to b-thalassemia. The level of imbalance between a-globin and b- globin chains correlates with the severity of b-thalassemia. Notably, homozygosity of the IVS-2-745 mutation leads to severe transfusion-dependent thalassemia major. Individuals with severe transfusion-dependent thalassemia major typically do not survive beyond five years of age. Summary of the Invention

Provided are compounds, methods, and pharmaceutical compositions for modulating the splicing of HBB RNA in a cell or subject, and in certain embodiments, increasing b-globin in a cell or subject. In certain embodiments, compounds useful for modulating the splicing of HBB RNA are oligomeric compounds or modified oligonucleotides. In certain embodiments, oligomeric compounds comprise a modified oligonucleotide. While not limited to a particular mechanism, it is thought that the modified oligonucleotides described herein obstruct the aberrant 5’ splice site caused by the IVS-2-745 mutation in human HBB, thereby preventing recognition from the spliceosome at the aberrant splice site, and consequently restoring production of wildtype HBB mRNA. In certain embodiments, oligomeric compounds or modified oligonucleotides provide for splicing of HBB pre-mRNA that results in wildtype HBB mRNA. In certain embodiments, oligomeric compounds or modified oligonucleotides described herein increase an amount of wildtype HBB mRNA in a cell or subject.

Also provided are methods useful for ameliorating at least one symptom or hallmark of b- thalassemia. In certain embodiments, symptoms and hallmarks include low hemoglobin level, erythrocyte deformities, fatigue, weakness, pale skin or jaundice, abdominal swelling, decreased longevity, and dark urine. In certain embodiments, amelioration of these symptoms results in improved energy levels and increased longevity. In certain embodiments, methods provided herein result in rebalancing the stoichiometry of a-globin and b-globin chains, reducing toxic a-chain aggregates, and correcting erythrocyte deformities.

Detailed Description of the Invention

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. 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. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

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

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

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

DEFINITIONS

As used herein, “2’-deoxyribonucleoside” means a nucleoside comprising a 2’-H(H) deoxyribosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2’- deoxyribonucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil). In certain embodiments, a 2’-deoxyribonucleoside may comprise hypoxanthine. In certain embodiments, a 2’- deoxyribonucleoside is in the b-D configuration, and is referred to as a nucleoside comprising a b -D-2’- deoxyribose 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, “5-methyl cytosine” means a cytosine modified with a methyl group attached to the 5-position. A 5-methyl cytosine is a modified nucleobase.

As used herein, “about” means plus or minus 7% of the provided value.

As used herein, “administering” means providing a pharmaceutical agent to a subject.

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 an oligomeric compound capable of achieving at least one antisense activity.

As used herein, “ameliorate” in reference to a treatment means improvement in at least one symptom relative to the same symptom in the absence of the treatment. In certain embodiments, amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the severity or frequency of a symptom. In certain embodiments, the symptom is a lack of subcutaneous fat, weight loss, hair loss, hypertension, metabolic syndrome, progressive cardiovascular disease resembling atherosclerosis, congestive heart failure, or premature death. In certain embodiments, amelioration of these symptoms results in a reduction of weight loss and increased survival.

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. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

As used herein, “cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell or a subject.

As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of the 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.

As used herein, complementary nucleobases” means nucleobases 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 (mC) 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 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 attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

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

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

As used herein, "contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or intemucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

As used herein, “chirally enriched population” means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. Chirally enriched populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are oligomeric compounds disclosed herein. In certain embodiments, the oligomeric compounds are antisense compounds. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are oligomeric compounds comprising modified oligonucleotides.

As used herein, “gapmer” means a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.” Unless otherwise indicated, “gapmer” refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gap of a gapmer are unmodified 2’-deoxyribosyl. Thus, the term “MOE gapmer” indicates a gapmer having a sugar motif of 2’-MOE nucleosides in both wings and a gap of 2’-deoxynucleosides. Unless otherwise indicated, a MOE gapmer may comprise one or more modified intemucleoside linkages and/or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications.

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, "increasing the amount or activity" refers to more transcriptional expression or activity relative to the transcriptional expression or activity in an untreated or control sample.

As used herein, “decreasing the amount or activity” refers to less transcriptional expression or activity relative to the transcriptional expression or activity in an untreated or control sample.

As used herein, “intemucleoside linkage” is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified intemucleoside linkage” means any intemucleoside linkage other than a phosphodiester intemucleoside linkage. “Phosphorothioate linkage” is a modified intemucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom.

As used herein, the term “intemucleoside linkage” is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified intemucleoside linkage” means any intemucleoside linkage other than a phosphodiester intemucleoside linkage. “Phosphorothioate intemucleoside linkage” is a modified intemucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom.

As used herein, “linker-nucleoside” means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide. As used herein, the term “linker region” in reference to a conjugate moiety refers that part of a conjugate linker that is not a cleavable moiety.

As used herein, “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

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 oligonucleotide are aligned.

As used herein, “MOE” means methoxyethyl. ”2’-MOE,” 2 -b-0-MOE, or “2’-MOE modified sugar” means a 2’-0CH ₂ CH ₂ 0CH ₃ group in place of the 2’-OH group of a ribosyl sugar moiety.

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

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, "nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G).

As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. A “5-methyl cytosine” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases.

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

As used herein, “nucleoside” means a compound 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. Modified nucleosides include abasic nucleosides, which lack a nucleobase. “Linked nucleosides” are nucleosides that are connected in a contiguous sequence (i.e., no additional nucleosides are presented between those that are linked).

As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A “singled-stranded oligomeric compound” is an unpaired oligomeric compound. The term “oligomeric duplex” means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed 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 8-50 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 a subject. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water, distilled water for injection, sterile saline, or sterile buffer solution.

As used herein “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent 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 oligomeric compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

As used herein, “phosphorus moiety” means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.

As used herein “prodrug” means a therapeutic agent in a form outside the body that is converted to a different form within a subject or cells thereof. Typically, conversion of a prodrug within the subject is facilitated by the action of an enzyme (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and/or by physiologic conditions.

As used herein, “self-complementary” in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.

As used herein, “stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center having a random stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (5) configuration of the stereorandom chiral center may be but is not necessarily the same as the number of molecules having the (R) configuration of the stereorandom chiral center. The stereochemical configuration of a chiral center is considered random when it is the results of a synthetic method that is not designed to control the stereochemical configuration. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate intemucleoside linkage.

As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2’-OH(H) ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2’-H(H) deoxyribosyl moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moieties have one hydrogen at each of the G, 3’, and 4’ positions, an oxygen at the 3’ position, and two hydrogens at the 5’ position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.

As used herein, “subject” means a human or non-human animal. In certain embodiments, the subject is a human.

As used herein, "sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety 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 target nucleic acids.

As used herein, “symptom or hallmark” means any physical feature or test result that indicates the existence or extent of a disease or disorder. In certain embodiments, a symptom is apparent to a subject or to a medical professional examining or testing said subject. In certain embodiments, a hallmark is apparent upon invasive diagnostic testing, including, but not limited to, post-mortem tests.

As used herein, “target nucleic acid” and “target RNA” mean a nucleic acid that an antisense compound is designed to affect. An antisense compound hybridizes to the target nucleic acid, but may comprise one or more mismatches thereto.

As used herein, “target region” means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

As used herein, "terminal group" means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.

As used herein, “therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to a subject. For example, a therapeutically effective amount improves a symptom of a disease.

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

Embodiment 1. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to an equal length portion of an HBB nucleic acid, and wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar and a modified intemucleoside linkage. Embodiment 2. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOS: 12-163. Embodiment 3. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleobases, wherein the portion is complementary to an equal length portion of nucleobases 1650-2055 of SEQ ID NO: 1 or SEQ ID NO: 2.

Embodiment 4. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleobases, wherein the portion is complementary to: an equal length portion of nucleobases 1650-1693 of SEQ ID NO: 1 or SEQ ID NO: 2; an equal length portion of nucleobases 1739-1858 of SEQ ID NO: 1 or SEQ ID NO: 2; an equal length portion of nucleobases 1864-1938 of SEQ ID NO: 1 or SEQ ID NO: 2; or an equal length portion of nucleobases 1953-2055 of SEQ ID NO: 1 or SEQ ID NO: 2.

Embodiment 5. The oligomeric compound of 4, wherein the nucleobase sequence is selected from: SEQ ID NOS: 14-19; SEQ ID NOS: 20-73; SEQ ID NOS: 79-89, and 92-104; and SEQ ID NOS: 121-163. Embodiment 6. The oligomeric compound of any of embodiments 1-5, wherein the modified oligonucleotide has a nucleobase sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a nucleobase sequence selected from SEQ ID NOS: 1-4, when measured across the entire nucleobase sequence of the modified oligonucleotide.

Embodiment 7. The oligomeric compound of any of embodiments 1-6, wherein the modified oligonucleotide comprises at least one modified nucleoside.

Embodiment 8. The oligomeric compound of embodiment 7, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a modified sugar moiety.

Embodiment 9. The oligomeric compound of embodiment 8, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety.

Embodiment 10. The oligomeric compound of embodiment 9, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar moiety having a 2’ -4’ bridge, wherein the 2 ’-4’ bridge is selected from -O-CH2-; and -O-CE^CEE)-.

Embodiment 11. The oligomeric compound of any of embodiments 7-10, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety. Embodiment 12. The oligomeric compound of embodiment 11, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a non-bicyclic modified sugar moiety comprising a 2’-MOE modified sugar or 2’-OMe modified sugar.

Embodiment 13. The oligomeric compound of any of embodiments 7-12, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a sugar surrogate. Embodiment 14. The oligomeric compound of embodiment 13, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a sugar surrogate selected from morpholino and PNA. Embodiment 15. The oligomeric compound of any of embodiments 7-12, wherein each nucleoside of the modified oligonucleotide comprises the modified sugar moiety.

Embodiment 16. The oligomeric compound of any of embodiments 1-15, wherein the modified oligonucleotide comprises at least one modified intemucleoside linkage.

Embodiment 17. The oligomeric compound of embodiment 16, wherein each intemucleoside linkage of the modified oligonucleotide is a modified intemucleoside linkage.

Embodiment 18. The oligomeric compound of embodiment 16 or 17, wherein at least one intemucleoside linkage is a phosphorothioate intemucleoside linkage.

Embodiment 19. The oligomeric compound of embodiment 16 or 18, wherein the modified oligonucleotide comprises at least one phosphodiester intemucleoside linkage.

Embodiment 20. The oligomeric compound of any of embodiments 16, 18 or 19, wherein each intemucleoside linkage is either a phosphodiester intemucleoside linkage or a phosphorothioate intemucleoside linkage.

Embodiment 21. The oligomeric compound of any of embodiments 1-20, wherein the modified oligonucleotide comprises at least one modified nucleobase.

Embodiment 22. The oligomeric compound of embodiment 21, wherein the modified nucleobase is a 5- methyl cytosine.

Embodiment 23. The oligomeric compound of any of embodiments 1-22, wherein the modified oligonucleotide consists of 12-30, 12-22, 12-20, 14-20, 15-25, 16-20, 18-22 or 18-20 linked nucleosides. Embodiment 24. The oligomeric compound of any of embodiments 1-23, wherein the modified oligonucleotide consists of 18 linked nucleosides.

Embodiment 25. The oligomeric compound of any of embodiments 1-24 consisting of the modified oligonucleotide.

Embodiment 26. The oligomeric compound of any of embodiments 1-24 comprising a conjugate group comprising a conjugate moiety and a conjugate linker.

Embodiment 27. The oligomeric compound of embodiment 26, wherein the conjugate group comprises a GalNAc cluster comprising 1-3 GalNAc ligands.

Embodiment 28. The oligomeric compound of embodiment 26 or 27, wherein the conjugate linker consists of a single bond.

Embodiment 29. The oligomeric compound of embodiment 26 or 27, wherein the conjugate linker is cleavable.

Embodiment 30. The oligomeric compound of embodiment 29, wherein the conjugate linker comprises 1-3 linker-nucleosides . Embodiment 31. The oligomeric compound of any of embodiments 26-30, wherein the conjugate group is attached to the modified oligonucleotide at the 5 ’-end of the modified oligonucleotide.

Embodiment 32. The oligomeric compound of any of embodiments 26-30, wherein the conjugate group is attached to the modified oligonucleotide at the 3 ’-end of the modified oligonucleotide. Embodiment 33. The oligomeric compound of any of embodiments 1-32 comprising a terminal group.

Embodiment 34. The oligomeric compound of any of embodiments 1-33 wherein the oligomeric compound is a singled-stranded oligomeric compound.

Embodiment 35. The oligomeric compound of any of embodiments 1-29 or 31-33, wherein the oligomeric compound does not comprise linker-nucleosides. Embodiment 36. An oligomeric duplex comprising an oligomeric compound of any of embodiments 1-33 or 35.

Embodiment 37. An antisense compound comprising or consisting of an oligomeric compound of any of embodiments 1-35 or an oligomeric duplex of embodiment 36.

Embodiment 38. A pharmaceutical composition comprising an oligomeric compound of any of embodiments 1-35 or an oligomeric duplex of embodiment 36 and a pharmaceutically acceptable carrier or diluent.

Embodiment 39. A modified oligonucleotide according to the following formula:

(SEQ ID NO: 58), or a salt thereof.

Embodiment 40. A modified oligonucleotide according to the following structure:

(SEQ ID NO: 59), or a salt thereof. Embodiment 41. A modified oligonucleotide according to the following structure:

(SEQ ID NO: 60) or a salt thereof.

Embodiment 42. The modified oligonucleotide of any one of embodiments 39-41, which is a sodium salt of the formula.

Embodiment 43. A modified oligonucleotide according to the following formula:

(SEQ ID NO: 58).

Embodiment 44. A modified oligonucleotide according to the following formula:

Embodiment 45. A modified oligonucleotide according to the following formula:

Embodiment 46. A chirally enriched population of the modified oligonucleotide of any of embodiments 39- 45, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having a particular stereochemical configuration.

Embodiment 47. The chirally enriched population of embodiment 46, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having the (Sp) configuration.

Embodiment 48. The chirally enriched population of embodiment 46 or 47, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having the (7/p) configuration. Embodiment 49. The chirally enriched population of embodiment 46, wherein the population is enriched for modified oligonucleotides having a particular, independently selected stereochemical configuration at each phosphorothioate intemucleoside linkage

Embodiment 50. The chirally enriched population of embodiment 49, wherein the population is enriched for modified oligonucleotides having the (.S'p) configuration at each phosphorothioate intemucleoside linkage. Embodiment 51. The chirally enriched population of embodiment 49, wherein the population is enriched for modified oligonucleotides having the (7y) configuration at each phosphorothioate intemucleoside linkage. Embodiment 52. The chirally enriched population of embodiment 46 or embodiment 49, wherein the population is enriched for modified oligonucleotides having at least 3 contiguous phosphorothioate intemucleoside linkages in the .S'p- S'p -/y configuration, in the 5’ to 3’ direction.

Embodiment 53. A population of modified oligonucleotides of any of embodiments 39-45, wherein all of the phosphorothioate intemucleoside linkages of the modified oligonucleotide are stereorandom.

Embodiment 54. A pharmaceutical composition comprising the modified oligonucleotide of any of embodiments 39-45 and a pharmaceutically acceptable diluent or carrier.

Embodiment 55. The pharmaceutical composition of embodiment 54, wherein the pharmaceutically acceptable diluent is phosphate buffered saline (PBS).

Embodiment 56. The pharmaceutical composition of embodiment 55, wherein the pharmaceutical composition consists essentially of the modified oligonucleotide and PBS.

Embodiment 57. A method comprising contacting a cell with the oligomeric compound of any one of embodiments 1-35, the oligomeric duplex of embodiment 36, or the modified oligonucleotide of any one of embodiments 39-45.

Embodiment 58. The method of embodiment 57, wherein the cell is a human cell comprising an HBB gene, and wherein the HBB gene comprises a guanine at nucleotide position 745 of intron 2.

Embodiment 59. The method of embodiment 58, wherein an amount of mutant HBB RNA is reduced. Embodiment 60. The method of embodiment 58 or 59, wherein an amount of wildtype HBB mRNA is increased.

Embodiment 61. An oligomeric compound comprising a modified oligonucleotide according to the following formula: mCes Tes Tes Tes Aes Ges Aes Aes Tes Ges Ges Tes Ges mCes Aes Aes Aes Ge (SEQ ID NO: 58); wherein, A = an adenine, mC = a 5-methylcytosine, G = a guanine, T = a thymine, e = a 2’-0- methoxyethylribose modified sugar, and s = a phosphorothioate intemucleoside linkage.

Embodiment 62. An oligomeric compound comprising a modified oligonucleotide according to the following formula: Tes Tes mCes Tes Tes Tes Aes Ges Aes Aes Tes Ges Ges Tes Ges mCes Aes Ae (SEQ ID NO: 59); wherein, A = an adenine, mC = a 5-methylcytosine, G = a guanine, T = a thymine, e = a 2’-0- methoxyethylribose modified sugar, and s = a phosphorothioate intemucleoside linkage. Embodiment 63. An oligomeric compound comprising a modified oligonucleotide according to the following formula: Tes Aes Tes Tes mCes Tes Tes Tes Aes Ges Aes Aes Tes Ges Ges Tes Ges mCe (SEQ ID NO: 60); wherein, A = an adenine, mC = a 5-methylcytosine, G = a guanine, T = a thymine, e = a 2’-0- methoxyethylribose modified sugar, and s = a phosphorothioate intemucleoside linkage.

Certain Oligonucleotides

In certain embodiments, provided herein are oligomeric compounds comprising oligonucleotides, which consist of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA. That is, modified oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified intemucleoside linkage.

Certain Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modifed sugar moiety and a modified nucleobase.

Certain Sugar Moieties

In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic 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.

In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2’, 4’, and/or 5’ positions. In certain embodiments one or more non-bridging substituent of non-bicyclic modified sugar moieties is branched. Examples of 2’- substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2'- OCH3 (“OMe” or “O-methyl”), and 2'-0(CH ₂ ) ₂ 0CH ₃ (“MOE”). In certain embodiments, 2 ’-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-Ci-Cio alkoxy, O- C1-C10 substituted alkoxy, O-Ci-Cio alkyl, O-Ci-Cio 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, OiCEh^SCEE, 0(CH ₂ ) ₂ 0N(R _m )(Rn) or OCH ₂ C(=0)-N(R _m )(Rn), where each R _m and R _n is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, and the 2’- substituent groups described in Cook et ah, U.S. 6,531,584; Cook et ah, 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 (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4 ’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et ah, WO 2015/106128. Examples of 5 ’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5 ’-methyl (R or S), 5'- vinyl, and 5 ’-methoxy. In certain embodiments, non-bicyclic modified sugar moieties 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 ak, WO 2008/101157 and Rajeev et ah,

US2013/0203836.).

In certain embodiments, a 2 ’-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2 ’-substituent group selected from: F, NEE, N3, OCF3 _, OCH3,

0(CH ₂ ) ₃ NH ₂ , CH ₂ CH=CH ₂ , OCH ₂ CH=CH ₂ , OCH ₂ CH ₂ OCH ₃ , 0(CH ₂ ) ₂ SCH ₃ , 0(CH ₂ ) ₂ 0N(R _m )(R _n ), 0(CH ₂ ) ₂ 0(CH ₂ ) ₂ N(CH3) ₂ , and N-substituted acetamide (OCH ₂ C(=0)-N(R _m )(Rn)), where each R _m and R _n is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl.

In certain embodiments, a 2 ’-substituted nucleoside non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2 ’-substituent group selected from: F, OCF3 _, OCH3,

OCH ₂ CH ₂ OCH ₃ , 0(CH ₂ ) ₂ SCH ₃ , 0(CH ₂ ) ₂ 0N(CH ₃ ) ₂ , 0(CH ₂ ) ₂ 0(CH ₂ ) ₂ N(CH3) ₂ , and 0CH ₂ C(=0)-N(H)CH ₃ (“NMA”).

In certain embodiments, a 2 ’-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2 ’-substituent group selected from: F, OCH3, and OCEECEEOCH3.

Certain modifed sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form 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. Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -0-2' (“ENA”), 4'-CH ₂ -S-2', 4'-(CH ₂ ) ₂ -0-2' (“ENA”), 4'-CH(CH ₃ )-0-2' (referred to as “constrained ethyl” or “cEt”), 4’-CH ₂ - O-CEb-2’, 4’-CH ₂ -N(R)-2’, 4'-CH(CH ₂ 0CH ₃ )-0-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et ak, U.S. 7,399,845, Bhat et ak, U.S. 7,569,686, Swayze et ak, U.S. 7,741,457, and Swayze et ak, U.S. 8,022,193), 4'-C(CH ₃ )(CH ₃ )-0-2' and analogs thereof (see, e.g., Seth et ak, U.S. 8,278,283), 4'- CH ₂ -N(OCH ₃ )-2' and analogs thereof (see, e.g., Prakash et ak, U.S. 8,278,425), 4'-CH ₂ -0-N(CH ₃ )-2' (see, e.g., Allerson et ak, U.S. 7,696,345 and Allerson et ak, 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 ak, U.S. 8,278,426), 4’C(-R _a R _b )-N(R)-0-2’, 4’-C(R _a R _¾ )-0-N(R)-2’, 4'-CH ₂ -0-N(R)-2', and 4'-CH ₂ -N(R)-0-2', wherein each R, R _a , and R, is, independently, H, a protecting group, or Ci-Ci ₂ alkyl (see, e.g. Imanishi et ak, U.S. 7,427,672). In certain embodiments, such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently selected from: -|C(R _a )(R _b )| _n -. -|C(R _a )(R _b )| _n -0-. -C(R _a )=C(R _b )-, -C(R _a )=N-, C(=-NR _a )-, - C(=0)-, -C(=S)-, -0-, -Si(R _a ) ₂ -, -S(=0) _x -, and -N(R _a )-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R _a and R _b is, independently, H, a protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C ₅ -C ₇ alicyclic radical, substituted C ₅ -C ₇ alicyclic radical, halogen, OJi, NJ ₁ J ₂ , SJi, N ₃ , COOJi, acyl (C(=0)- H), substituted acyl, CN, sulfonyl (S(=0) ₂ -Ji), or sulfoxyl (S(=0)-Ji); and each Ji and J ₂ is, independently, H, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, acyl (C(=0)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ -C ₁₂ aminoalkyl, or a protecting group.

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., 20017, 129, 8362-8379;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 ak, 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 UNA nucleoside (described herein) may be in the a-U configuration or in the b-D configuration.

LNA (b-D-configuration) a-L-LNA («.-/.-configuration) bridge = 4'-CH ₂ -0-2' bridge = 4'-CH ₂ -0-2' a-L-methyleneoxy (4’-CH ₂ -0-2’) or a-L-LNA bicyclic nucleosides have been incorporated into 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 or cEt) are identified in exemplified embodiments herein, they are in the b-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).

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”), anitol nucleic acid (“ANA”), manitol 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), and nucleosides comprising additional modified THP compounds having the formula: wherein, independently, for each of said modified THP nucleoside:

Bx is a nucleobase moiety;

T3 and T4 are each, independently, an intemucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an intemucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group; qi, q ₂ . q3, q4, qs, qeand q7 are each, independently, H, C 1 -G, alkyl, substituted C 1 -G alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and each of Ri and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N3, OC(=X)Ji, OC(=X)NJIJ2, NJ3C(=X)NJIJ2, and CN, wherein X is O, S or NJi, and each Ji, J2, and J3 is, independently, H or C1-C6 alkyl.

In certain embodiments, modified THP nucleosides are provided wherein qi, q2, q3, q4, qs, qe and q ₇ are each H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q ₇ is other than H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q ₇ is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is F and R2 is H, in certain embodiments, Ri is methoxy and R2 is H, and in certain embodiments, Ri is methoxyethoxy and R2 is

H.

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 having 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 referred to herein as “modifed morpholinos.” 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), 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 (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).

Certain Modified Nucleobases

In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.

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 0-6 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 (-CºC-CH3) 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 fhiorinated 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.

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.

Certain Modified Internucleoside Linkages

In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any intemucleoside linkage. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing intemucleoside linkages include but are not limited to phosphodiesters (“P=0”) (also referred to as unmodified or naturally occurring linkages or phosphate linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (“P=S”), and phosphorodithioates (“HS-P=S”). Representative non-phosphorus containing intemucleoside linking groups include but are not limited to methylenemethylimino (-CH ₂ -N(CH ₃ )-0-CH ₂ -), thiodiester, thionocarbamate (-0-C(=0)(NH)-S-); siloxane (-0-SiH ₂ -0-); and N,N'-dimethylhydrazine (-CH -N(CH )- N(0¾)-). Modified intemucleoside linkages, compared to naturally occurring phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. 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. 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, methylphosphonates, MMI (3'-CH ₂ -N(CH ₃ )-0-5'), amide-3 (3'-CH ₂ -C(=0)-N(H)-5'), amide-4 (3'-CH ₂ -N(H)-C(=0)-5'), formacetal (3'-0-CH ₂ -0-5'), methoxypropyl, and thioformacetal (3'-S-CH ₂ -0-5'). 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 CEE component parts.

Certain Motifs

In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. 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 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).

Certain Sugar Motifs

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 but are not limited to any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which is defined by two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer motif (the 5 ’-wing, the gap, and the 3 ’-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3 ’-most nucleoside of the 5 ’-wing and the 5 ’-most nucleoside of the 3 ’-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside. In certain embodiments, at least one nucleoside of each wing of a gapmer is a modified nucleoside. In certain embodiments, at least two nucleosides of each wing of a gapmer are modified nucleosides. In certain embodiments, at least three nucleosides of each wing of a gapmer are modified nucleosides. In certain embodiments, at least four nucleosides of each wing of a gapmer are modified nucleosides.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2’-deoxy nucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In embodiments, the nucleosides on the gap side of each wing/gap junction are unmodified 2’-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In certain embodiments, each nucleoside of the gap is an unmodified 2 ’-deoxy nucleoside. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2 ’-modification.

Herein, the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [# of nucleosides in the 5’-wing] - [# of nucleosides in the gap] - [# of nucleosides in the 3’- wing]. Thus, a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap. Where such nomenclature is followed by a specific modification, that modification is the modification in each sugar moiety of each wing and the gap nucleosides comprise unmodified deoxynucleoside sugars. Thus, a 5-10-5 MOE gapmer consists of 5 linked MOE modified nucleosides in the 5’-wing, 10 linked deoxynucleosides in the gap, and 5 linked MOE nucleosides in the 3’-wing.

In certain embodiments, modified oligonucleotides are 5-10-5 MOE gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 BNA gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 cEt gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 LNA gapmers.

In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety (uniformly modified sugar motif). In certain embodiments, the uniformly modified sugar motif is 7 to 20 nucleosides in length. In certain embodiments, each nucleoside of the uniformly modified sugar motif is a 2 ’-substituted nucleoside, a sugar surrogate, or a bicyclic nucleoside. In certain embodiments, each nucleoside of the uniformly modified sugar motif comprises either a 2’-0CH ₂ CH ₂ 0CH ₃ group or a 2’-OCH ₃ group. In certain embodiments, modified oligonucleotides having at least one fully modified sugar motif may also have at least 1, at least 2, at least 3, or at least 42’-deoxyribonucleosides.

In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety (fully modified oligonucleotide). In certain embodiments, a fully modified oligonucleotide comprises different 2 ’-modifications. In certain embodiments, each nucleoside of a fully modified oligonucleotide is a 2 ’-substituted nucleoside, a sugar surrogate, or a bicyclic nucleoside. In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises either a 2’-0CH2CH20CH3 group and at least one 2’-OCH ₃ group.

In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises the same 2’- modification (uniformly modified oligonucleotide). In certain embodiments, each nucleoside of a uniformly modified oligonucleotide is a 2 ’-substituted nucleoside, a sugar surrogate, or a bicyclic nucleoside. In certain embodiments, each nucleoside of a uniformly modified oligonucleotide comprises either a 2’- OCH2CH2OCH3 group or a 2’-OCH3 group.

In certain embodiments, modified oligonucleotides comprise at least 12, at last 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleosides comprising a modified sugar moiety. In certain embodiments, each nucleoside of a modified oligonucleotide is a 2 ’-substituted nucleoside, a sugar surrogate, a bicyclic nucleoside, or a p-D-2’-deoxyribonucleoside. In certain embodiments, each nucleoside of a modified oligonucleotide comprises a 2’-0CH2CH20CH3 group, a 2’- H(H) deoxyribosyl sugar moiety, or a cEt modified sugar.

Certain Nucleobase Motifs

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-methyl cytosines.

In certain embodiments, all of the cytosine nucleobases are 5 -methyl cytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases. 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, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2’-deoxyribosyl moiety. In certain embodiments, the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine. In certain embodiments, the modified nucleobase is a hypoxanthine.

Certain Internucleoside Linkage Motifs

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 linking group is a phosphodiester intemucleoside linkage (P=0). In certain embodiments, each intemucleoside linking group 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 (.S'p) phosphorothioate, and a (7/p) phosphorothioate. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the intemucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the intemucleoside linkages in the wings are unmodified phosphodiester intemucleoside linkages. In certain embodiments, the terminal intemucleoside linkages are modified. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer, and the intemucleoside linkage motif comprises at least one phosphodiester intemucleoside linkage in at least one wing, 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 wings are (Sp) phosphorothioates, and the gap comprises at least one Sp, Sp, Rp motif. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such intemucleoside linkage motifs.

Certain Lengths

It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target RNA, albeit to a lesser extent than the oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.

In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. 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.

Certain 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 modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each intemucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the intemucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the intemucleoside linkages of the gap region of the sugar motif. Likewise, such sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Unless otherwise indicated, all modifications are independent of nucleobase sequence.

Certain Populations of Modified Oligonucleotides

Populations of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be stereorandom populations or chirally enriched populations. All of the chiral centers of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides of the population. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for b-D ribosyl sugar moieties, and all of the phosphorothioate intemucleoside linkages are stereorandom. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for both b-D ribosyl sugar moieties and at least one, particular phosphorothioate intemucleoside linkage in a particular stereochemical configuration.

Nucleobase Sequence

In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. 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 such 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 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.

Certain Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds, which 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 moieties and a conjugate linker which 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 are attached to the 3’ and/or 5’ end of oligonucleotides. 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 ’-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.

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 ah, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et ah, Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et ah, Ann. NY. Acad. Sci., 1992, 660, 306-309; Manoharan et ah, Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et ak, Niicl. Acids Res., 1992, 20, 533- 538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et ak, EMBO J, 1991,

10, 1111-1118; Kabanov et ak, FEBS Lett., 1990, 259, 327-330; Svinarchuk et ak, Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac -glycerol or triethyl-ammonium l,2-di-0-hexadecyl-rac-glycero-3- H-phosphonate (Manoharan et ak, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et ak, Nucl. Acids Res.,

1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et ak, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid a palmityl moiety (Mishra et ak, Biochim.

Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et ak, J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et ak, Molecular Therapy Nucleic Acids , 2015, 4, e220; and Nishina ct ak. Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620). Other targeting groups are described in WO/2017/053995, hereby incorporated by reference.

Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, 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, fingolimod, 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.

In certain embodiments, a conjugate moiety is selected from among: cholesterol, C10-C26 saturated fatty acid, CIO- C26 unsaturated fatty acid, C10-C26 alkyl, triglyceride, tocopherol, or cholic acid. In certain embodiments, a conjugate moiety is C16 alkyl.

Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain oligomeric compounds, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide 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 parent 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 react with particular site on a parent compound and the other is selected to react with 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, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif.

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-methyl cytosine, 4-N-benzoyl-5-methyl cytosine, 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 an oligomeric 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 an oligomeric 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 oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. 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 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, the 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 2'-deoxy nucleoside that is attached to either the 3' or 5'-terminal nucleoside of an oligonucleotide by a phosphate intemucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2'- deoxyadenosine.

Certain Terminal Groups

In certain embodiments, oligomeric compounds comprise one or more terminal groups. In certain such embodiments, oligomeric compounds comprise a stabilized 5’-phophate. Stabilized 5 ’-phosphates include, but are not limited to 5’-phosphanates, including, but not limited to 5’-vinylphosphonates. In certain embodiments, terminal groups comprise one or more abasic nucleosides and/or inverted nucleosides. In certain embodiments, terminal groups comprise one or more 2’-linked nucleosides. In certain such embodiments, the 2 ’-linked nucleoside is an abasic nucleoside.

Oligomeric Duplexes

In certain embodiments, oligomeric compounds described herein comprise an oligonucleotide, having a nucleobase sequence complementary to that of a target nucleic acid. In certain embodiments, an oligomeric compound is paired with a second oligomeric compound to form an oligomeric duplex. Such oligomeric duplexes comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound.

In certain embodiments, the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a modified or unmodified oligonucleotide and optionally a conjugate group and (2) a second modified or unmodified oligonucleotide and optionally a conjugate group. Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group. The oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary overhanging nucleosides.

Antisense Activity

In certain embodiments, oligomeric compounds and oligomeric duplexes are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric compounds and oligomeric duplexes are antisense compounds. In certain embodiments, antisense compounds have antisense activity when they increase the amount or activity of a target nucleic acid by 25% or more in the standard cell assay. In certain embodiments, antisense compounds selectively affect one or more target nucleic acid. Such antisense 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 significant undesired antisense activity.

In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain antisense compounds 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, described herein are antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. In certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.

In certain antisense activities, an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds result in cleavage of the target nucleic acid by Argonaute. Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double- stranded (siRNA) or single -stranded (ssRNA).

In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in an increase in the amount or activity of a target nucleic acid.

Use of oligomeric compounds is an effective means for modulating the expression of one or more specific gene products and is uniquely useful in a number of therapeutic, diagnostic, and research applications. Provided herein are oligomeric compounds useful for modulating gene expression via antisense mechanisms of action, including antisense mechanisms based on target occupancy. In certain embodiments, the oligomeric compounds provided herein modulate splicing of a target gene.

In certain embodiments, an antisense compound is complementary to a region of an HBB pre- mRNA. In certain embodiments, a modified oligonucleotide modulates splicing of a pre-mRNA. In certain embodiments, a modified oligonucleotide modulates splicing of an HBB pre-mRNA. In certain such embodiments, the HBB pre-mRNA is transcribed from a mutant variant of HBB. In certain embodiments, the mutant variant comprises an aberrant splice site. In certain embodiments, the aberrant splice site of the mutant variant comprises a mutation that induces a cryptic 5’ splice site. In certain embodiments, a modified oligonucleotide increases wildtype HBB mRNA. In certain embodiments, a modified oligonucleotide increases the production of b-globin protein.

Splice Modulating Activity

In certain embodiments, hybridization of a compound disclosed herein to an HBB RNA results in modulating the splicing of the HBB RNA. In certain embodiments, modulating the splicing of HBB RNA alters splicing of an HBB pre-mRNA. In certain embodiments, the HBB pre-mRNA is encoded by a HBB gene having the IVS-2-745 mutation. In certain embodiments, modulating the splicing of the HBB pre- mRNA encoded by the HBB gene having the IVS-2-745 mutation decreases the amount of mutant HBB RNA in the cell or the subject. In certain embodiments, modulating the splicing of the HBB pre-mRNA encoded by the HBB gene having the IVS-2-745 mutation increases the amount of wildtype HBB mRNA in the cell or the subject. In certain embodiments, hybridization of an antisense compound to the HBB RNA results in inhibition of a binding interaction between the HBB RNA and a protein or other nucleic acid. In certain embodiments, hybridization of an antisense compound to the HBB RNA results in alteration of translation of the HBB RNA. In certain embodiments, hybridization of an antisense compound to the HBB RNA results in exon skipping. In certain embodiments, hybridization of an antisense compound to the HBB RNA results in an increase or a reduction in the amount or activity of the HBB RNA. In certain embodiments, hybridization of an antisense compound complementary to the HBB RNA results in alteration of splicing, leading to the omission of an exon in an HBB mRNA. This alteration of a splice site may be referred to, for example, as splice-switching, or splice skipping, and the alteration of a splice site that leads to the omission of an exon may be referred to as exon skipping, or exon (number) skipping. In certain embodiments, the alteration of a splice site, or exon skipping, may result in elimination of a premature stop codon. In certain embodiments, the alteration of a splice site, or exon skipping, may result in elimination of a frame-shift; in certain embodiments the elimination of a frame-shift may result in elimination of a premature stop codon.

In some embodiments, modulating the splicing of HBB RNA, comprises contacting an HBB RNA in a cell with a splice-switching oligonucleotide. In certain embodiments, the splice -switching oligonucleotide is an oligonucleotide that reverses aberrant splicing in a pre-mRNA. In certain embodiments, aberrant splicing is caused by an IVS-2-745 (OG) mutation in HBB. In certain embodiments, reversing aberrant splicing caused by the IVS-2-745 (OG) mutation results in an increase in the amount of wildtype HBB mRNA in a cell, and in some instances an increase in an amount of b-globin in the cell. In some embodiments, splice switching oligonucleotides are stable and are RNase H resistant. In some embodiments, splice switching oligonucleotides are safe and have low toxicity. In some embodiments, splice switching oligonucleotides are freely taken up by a cell.

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 subject.

Certain Target Nucleic Acids

In certain embodiments, oligomeric compounds 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: a mature RNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is a mature RNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid has a disease-associated mutation. In certain embodiments, the target nucleic acid is the RNA transcriptional product of a retrogene. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long non-coding RNA, a short non-coding RNA, an intronic RNA molecule.

Complementaritv/Mismatches to the Target Nucleic Acid

It is possible to introduce mismatch bases without eliminating activity. For example, Gautschi et al (./ Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti tumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase oligonucleotides, and a 28 and 42 nucleobase oligonucleotides comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase oligonucleotides.

In certain embodiments, oligonucleotides are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid. In certain embodiments, the region of full complementarity is from 6 to 20, 10 to 18, or 18 to 20 nucleobases in length.

In certain embodiments, oligonucleotides comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain embodiments selectivity of the oligonucleotide is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5’-end of the gap region. In certain embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3 ’-end of the gap region. In certain embodiments, the mismatch is at position 1, 2, 3, or 4 from the 5 ’-end of the wing region. In certain embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3 ’-end of the wing region.

HBB

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide comprising a region that is complementary to an equal length portion of a target nucleic acid, wherein the target nucleic acid is an HBB nucleic acid. In certain embodiments, the HBB nucleic acid is pre-mRNA. In certain embodiments, the HBB nucleic acid is represented by the nucleobase sequence set forth in SEQ ID NO: 1 (the complement of GENBANK Accession No. NT_009237.18 truncated from nucleotides 5186000 to 5189000). In certain embodiments, the target nucleic acid comprises the nucleobase sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 is identical to SEQ ID NO: 1 aside from a guanine at position 1939 of SEQ ID NO: 2, which corresponds to position 745 of intron 2. In contrast, there is a cytosine at position 1939 of SEQ ID NO:l.

In certain embodiments, contacting a cell with an oligomeric compound complementary to an equal length portion of SEQ ID NO: 1 or SEQ ID NO: 2 increases wildtype HBB mRNA, and in certain embodiments increases b-globin in the cell. In certain embodiments, wildtype HBB mRNA is an mRNA encoded by the HBB gene that can be translated into a full-length, wildtype b-globin protein. In certain embodiments, wildtype HBB mRNA is represented by the nucleobase sequence set forth in SEQ ID NO: 3 (NM_000518.5). In certain embodiments, contacting a cell with an oligomeric compound complementary to an equal length portion of SEQ ID NO: 1 or SEQ ID NO: 2 decreases mutant HBB RNA. In certain embodiments, the mutant HBB RNA is encoded by the HBB gene, wherein the HBB gene comprises the IVS- 2-745 (OG) mutation. In certain embodiments, the mutant HBB RNA is selected from pre-mRNA, mRNA, and a combination thereof.

Certain Target Nucleic Acids in Certain Tissues or Biological Fluids

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid, wherein the target nucleic acid is expressed in a pharmacologically relevant tissue or cells. In certain embodiments, the pharmacologically relevant tissue is selected from blood, bone marrow, brain, macrophages, liver, kidney, and lung. In certain embodiments, the pharmacologically relevant tissue is blood. In certain embodiments, the pharmacologically relevant cells are erythroblasts, neurons, macrophages, alveolar cells, endometrial cells, hepatocytes, mesangial cells, and epithelial cells. In certain embodiments, the pharmacologically relevant cells are erythroblasts. In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid, wherein the target nucleic acid is expressed in a blood cell or progenitor thereof.

Certain Compositions

Compound No 671991

In certain embodiments, Compound No. 671991 is a modified oligonucleotide having a sequence of (from 5’ to 3’) CTTTAGAATGGTGCAAAG (SEQ ID NO: 58), wherein each nucleoside is a 2 -0- methoxyethyl nucleoside, and each of the intemucleoside linkages is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, Compound No. 671991 is represented by the following chemical notation: mCes Tes Tes Tes Aes Ges Aes Aes Tes Ges Ges Tes Ges mCes Aes Aes Aes Ge (SEQ ID NO: 58); wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2 -b-ϋ-MOE sugar moiety, and s = a phosphorothioate intemucleoside linkage. In certain embodiments, Compound No. 671991 is represented by the following chemical structure:

(SEQ ID NO: 58).

Structure 1. Compound No. 671991

In certain embodiments, the sodium salt of Compound No. 671991 is represented by the following chemical structure:

(SEQ ID NO: 58). Structure 2. The sodium salt of Compound No. 671991

Compound No 671992

In certain embodiments, Compound No. 671992 is a modified oligonucleotide having a sequence of (from 5’ to 3’) TTCTTTAGAATGGTGCAA (SEQ ID NO: 59), wherein each nucleoside is a 2 -0- methoxyethyl nucleoside, and each of the intemucleoside linkages is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, Compound No. 671992 is represented by the following chemical notation: Tes Tes mCes Tes Tes Tes Aes Ges Aes Aes Tes Ges Ges Tes Ges mCes Aes Ae (SEQ ID NO: 59); wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2 -b-ϋ-MOE sugar moiety, and s = a phosphorothioate intemucleoside linkage. In certain embodiments, Compound No. 671992 is described by the following chemical structure

(SEQ ID NO: 59).

Structure 3. Compound No. 671992 In certain embodiments, the sodium salt of Compound No. 671992 is represented by the following chemical structure:

(SEQ ID NO: 59). Structure 4. The sodium salt of Compound No. 671992

Compound No 671993

In certain embodiments, Compound No. 671993 is a modified oligonucleotide having a sequence of (from 5’ to 3’) TATTCTTTAGAATGGTGC (SEQ ID NO: 60), wherein each nucleoside is a 2 -0- methoxyethyl nucleoside, and each of the intemucleoside linkages is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine. In certain embodiments, Compound No. 671993 is represented by the following chemical notation: Tes Aes Tes Tes mCes Tes Tes Tes Aes Ges Aes Aes Tes Ges Ges Tes Ges mCe (SEQ ID NO: 60); wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2’- -D-MOE sugar moiety, and s = a phosphorothioate intemucleoside linkage. In certain embodiments, Compound No. 671993 is represented by the following chemical structure:

(SEQ ID NO: 60).

Structure 5. Compound No. 671993 In certain embodiments, the sodium salt of Compound No. 671993 is represented by the following chemical structure:

(SEQ ID NO: 60). Structure 6. The sodium salt of Compound No. 671993 Certain Pharmaceutical Compositions

In certain embodiments, described herein are pharmaceutical compositions comprising one or more oligomeric compounds. In certain embodiments, the one or more oligomeric compounds each consists of a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises or 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 or consists of one or more 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, the sterile PBS is pharmaceutical grade PBS. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

In certain embodiments, a pharmaceutical composition comprises a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists essentially of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

In certain embodiments, pharmaceutical compositions comprise one or more oligomeric compound and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, oligomeric compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions comprising an oligomeric compound encompass any pharmaceutically acceptable salts of the oligomeric compound, esters of the oligomeric compound, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising oligomeric compounds comprising one or more oligonucleotide, upon administration to a subject, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligomeric 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. In certain embodiments, prodrugs comprise one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligomeric compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Under certain conditions, certain compounds disclosed herein are shown in the form of a free acid. Although such compounds may be drawn or described in protonated (free acid) form, aqueous solutions of such compounds may exist in equilibrium among an ionized (anion) form, and in association with a cation (salt form). For example, a phosphate linkage of an oligonucleotide in aqueous solution exists in equilibrium among free acid, anion, and salt forms. Unless otherwise indicated, compounds described herein are intended to include all such forms. Moreover, oligonucleotides have several such linkages, each of which is in equilibrium. Thus, oligonucleotides in solution exist in an ensemble of forms at multiple positions, all at equilibrium. The term “oligonucleotide” is intended to include all such forms. Drawn structures necessarily depict a single form. Nevertheless, unless otherwise indicated, such drawings are likewise intended to include corresponding forms. Herein, a structure depicting the free acid of a compound followed by the term “or salts thereof’ expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with a cation. In certain instances, one or more specific cation is identified.

In certain embodiments, oligomeric compounds disclosed herein are in a form of a sodium salt. In certain embodiments, oligomeric compounds disclosed herein are in a form of a potassium salt. In certain embodiments, oligomeric compounds disclosed herein are in aqueous solution with sodium. In certain embodiments, oligomeric compounds are in aqueous solution with potassium. In certain embodiments, oligomeric compounds are in PBS. In certain embodiments, oligomeric compounds are in water. In certain such embodiments, the pH of the solution is adjusted with NaOH and/or HC1 to achieve a desired pH.

Nonlimiting disclosure and incorporation by reference

Each of the literature and patent publications listed herein is incorporated by reference in its entirety.

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

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 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.

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 (S), 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 included all such possible isomers, including their stereorandom and optically pure forms, unless specified otherwise. Likewise, all tautomeric forms of the compounds herein are also included unless otherwise indicated. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.

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 ¾, ¹³ C or ¹⁴ C in place of ¹² C, ¹⁵ N in place of ¹⁴ N, ¹⁷ 0 or ¹⁸ 0 in place of ¹⁶ 0, 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 illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated. Example 1: Effects of modified oligonucleotides complementary to HBB RNA in vitro

Modified oligonucleotides complementary to SEQ ID NO: 1 (the complement of GENBANK Accession No. NT_009237.18 truncated from nucleotides 5186000 to 5189000) or SEQ ID NO: 4 were designed and tested for their effect on mutant HBB RNA and wildtype HBB mRNA expression in a murine erythroleukemia cell line that stably expresses human mutant HBB RNA (MEL IVS-2-745 cells). These modified oligonucleotides are listed in Tables 1 and 2. Tables 1 and 2 correspond to two separate plates assayed in a single experiment. “Start site” indicates the 5 ’-most nucleoside of SEQ ID NO: 1 or SEQ ID NO: 4 to which the modified oligonucleotide is complementary. “Stop site” indicates the 3 ’-most nucleoside of SEQ ID NO: 1 or SEQ ID NO: 4 to which the modified oligonucleotide is complementary. Modified oligonucleotides are 18 nucleosides in length, and each nucleoside is a 2’-MOE modified nucleoside and each intemucleoside linkage is a phosphorothioate intemucleoside linkage. All cytosine residues are 5- methylcytosines.

Compound Nos. 18076 and 18078, which are not complementary to SEQ ID NO: 1 or SEQ ID NO:

4, were included as controls. Compound Nos. 18076 and 18078 are gapmers, wherein the central gap segment consists of nine 2’- -D-deoxyribonucleosides, the 3 ’-wing consists of five 2’-MOE modified nucleosides, and the 5 ’-wing consists of six five 2’-MOE modified nucleosides. Each intemucleoside linkage is a phosphorothioate intemucleoside linkage. Compound Nos. 18076 and 18078 contain a mix of 5- methylcytosines and non-methylated cytosines. The non-methylated cytosines are underlined. ‘N/A’ indicates that the modified oligonucleotide is not 100% complementary to that particular gene sequence.

Human MEL cells that stably express human mutant HBB RNA (MEL IVS-2-745) were cultured at a density of 300,000 cells per well, and treated with modified oligonucleotides at a concentration of 7,000 nM using electroporation for a treatment period of 24 hours. At the end of the treatment period, total RNA was isolated from the cells and human HBB mRNA levels were measured by quantitative real-time PCR.

Wildtype human HBB mRNA levels were measured with human HBB primer probe set RTS4553 (forward sequence CACCTTTGCCACACTGAGTGA, designated herein as SEQ ID NO: 6; reverse sequence GCCCAGGAGCCTGAAGTTCT, designated herein as SEQ ID NO: 7; probe sequence CACTGTGACAAGCTGCACGTGGATCC, designated herein as SEQ ID NO: 8). Mutant human HBB RNA levels were measured with human HBB primer-probe set RTS4500 (forward sequence GCTCACCTGGACAACCTCAAG, designated herein as SEQ ID NO: 9; reverse sequence TCATTATTGCCCCTGAAGTTCTC, designated herein as SEQ ID NO: 10; probe sequence CACCTTTGCCACACTGAGTGAGCTGC, designated herein as SEQ ID NO: 11). Both wildtype and mutant human HBB RNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 1 as percent human HBB RNA levels relative to untreated control cells (%UTC). As shown in Table 1, modified oligonucleotides complementary to human HBB increased the amount of wildtype human HBB mRNA and decreased the amount of mutant human HBB RNA I these cells.

Table 1. Modified oligonucleotides with uniform MOE sugars and uniform PS internucleoside linkages complementary to human HBB

Table 2. Modified oligonucleotides with uniform MOE sugars and uniform PS internucleoside linkages complementary to human HBB

Example 2: Effect of modified oligonucleotides on human HBB mRNA expression in vitro, multiple doses

Modified oligonucleotides described in Example 1 were tested at various doses in MEL IVS-2-745 cells. Cultured MEL IVS-2-745 cells at a density of 30,000 cells per well were transfected using electroporation with 625 nM, 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM of modified oligonucleotide, as specified in the tables below. After a treatment period of approximately 24 hours, total RNA was isolated from the cells and HBB mRNA levels were measured by quantitative real-time PCR. Wildtype human HBB mRNA levels were measured with human HBB primer probe set RTS4553 as described above. Mutant human HBB RNA levels were measured with human HBB primer probe set RTS4500, as described above. Human HBB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Tables 3 and 4 present results as percent change in the amount of wildtype human HBB mRNA relative to untreated control (%UTC), as well as the corresponding fold increase in wildtype human HBB mRNA. Tables 5 and 6 present results as percent change in the amount of mutant human HBB RNA relative to untreated control (%UTC), as well as the corresponding IC50 values. Results presented in Tables 3 and 5 were obtained from a single experiment. Results presented in Tables 4 and 6 were obtained from separate experiments carried out under similar conditions.

Table 3. Dose-dependent modulation of human wildtype HBB mRNA in MEL IVS-2-745 cells

Table 4. Dose-dependent modulation of human wildtype HBB mRNA in MEL IVS-2-745 cells

Table 5. Dose-dependent modulation of human mutant HBB RNA in MEL IVS-2-745 cells

Table 6. Dose-dependent modulation of human mutant HBB RNA in MEL IVS-2-745 cells