Inhibin subunit beta e-related double-stranded oligonucleotide compositions and methods related thereto

Abstract

This disclosure provides inhibin subunit βE (INHBE)-related double-stranded oligonucleotides, compositions, and methods of using such double-stranded oligonucleotides and compositions for the prevention and / or treatment of various conditions, disorders, or diseases associated with INHBE expression. In some embodiments, the provided double-stranded oligonucleotides and compositions comprise nucleobase modifications, sugar modifications, internucleotide linking modifications, and / or patterns thereof, and have improved properties, activity, and / or selectivity. In some embodiments, the provided double-stranded oligonucleotides and compositions target INHBE.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 581,620, filed September 8, 2023, and U.S. Provisional Application No. 63 / 586,079, filed September 28, 2023, the contents of each of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] Among other things, the present disclosure provides double-stranded (ds) oligonucleotides, compositions, and methods thereof (e.g., methods of making, methods of using, etc.). In some embodiments, the provided technologies can be used to prevent and / or treat various conditions, disorders, or diseases associated with inhibin subunit beta E (INHBE) expression. BACKGROUND

[0004] Metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension) are increasingly of concern to the medical community. Typical treatments for such disorders and diseases involve targeted approaches and / or changes in diet and lifestyle, such as increased physical activity. In particular, treatments for obesity, a disorder involving excess fat in the body, require exercise and a healthy, balanced diet, thereby reducing body weight. However, there is a need for alternative treatment methods for metabolic diseases, particularly for reducing body weight.

[0005] Double-stranded (ds) oligonucleotides can be used for a variety of applications, such as therapeutic, diagnostic, and / or research applications. Inhibin subunit beta E (INHBE), which is primarily expressed in the liver, is associated with body mass index and insulin resistance. Thus, ds oligonucleotides targeting disorders or diseases associated with INHBE expression can be used to treat such conditions, including metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension). SUMMARY

[0006] In some embodiments, this disclosure provides ds oligonucleotides and compositions thereof that target INHBE and have significantly improved properties and / or high activity. Among other things, this disclosure provides techniques for designing, manufacturing, and utilizing such ds oligonucleotides and compositions. In particular, in some embodiments, this disclosure provides ds oligonucleotides comprising useful internucleotide linking patterns and / or sugar modification patterns, which, when combined with one or more other structural elements such as base sequences (or portions thereof), nucleobase modifications (and their patterns), additional chemical motifs, etc., can provide ds oligonucleotides and compositions thereof that target INHBE and have high activity and / or desired properties, including but not limited to effectively and efficiently reducing the expression, level, and / or activity of INHBE transcripts and products encoded therefrom. In some embodiments, the ds oligonucleotides and compositions that target INHBE reduce the level of INHBE transcripts and can be used to treat and / or prevent INHBE-related conditions, disorders, or diseases, including but not limited to metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension).

[0007] In some embodiments, ds oligonucleotides targeting INHBE can mediate INHBE knockdown, wherein the level, expression, and / or activity of INHBE or its products are reduced. In some embodiments, ds oligonucleotides targeting INHBE can mediate pan-specific knockdown of INHBE, wherein the level, expression, and / or activity of multiple or all INHBE alleles are reduced. In some embodiments, the ds oligonucleotides targeting INHBE have a base sequence complementary to a common sequence in multiple or all INHBE alleles.

[0008] In some embodiments, such structural elements include one or more of the following: (1) chemical modifications (e.g., modifications to sugar, base, and / or nucleotide linkages) and their patterns; and (2) stereochemical alterations (e.g., stereochemistry of backbone chiral nucleotide linkages) and their patterns. In some embodiments, one or more such structural elements may be independently present in one or both oligonucleotides of the ds oligonucleotide. In some embodiments, properties and / or activities affected by such structural elements include, but are not limited to, involvement in or guidance of the expression, activity, or reduction of levels of genes or their gene products, for example, mediated by RNA interference (RNAi interference).

[0009] In some embodiments, this disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also known as dsRNAi agents) having controlled structural elements provide unexpected properties and / or activities.

[0010] In some embodiments, the dsRNAi agent is capable of directing INHBE (inhibin subunit βE)-specific RNA interference to induce lipolysis while preserving muscle mass. This dsRNAi agent comprises a guide strand and a transit strand, wherein:

[0011] a) The guide strand is complementary to or substantially complementary to the INHBE target RNA sequence;

[0012] b) The bootstrap chain contains:

[0013] i. Sp-configured uncharged nucleotide linkage between the +3 nucleotide relative to the 5' terminal nucleotide and the immediately adjacent downstream (+4) nucleotide;

[0014] ii. An Rp-configured, uncharged nucleotide bond between a +10 nucleotide and its immediate downstream (+11) nucleotide;

[0015] iii. Sp-configured phosphate ester nucleotide linkages between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide; and / or

[0016] iv. Linkage between phosphate thioester nucleotides in Rp, Sp, or alternating configurations between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide;

[0017] c) The leading chain further includes 5' phosphate modification;

[0018] d) The transit chain contains one or more chiral nucleotides in the Rp or Sp configuration linked together; and

[0019] e) The guide strand and the guest strand each have an independent length of 15-49 nucleotides.

[0020] In alternative embodiments, this disclosure covers the recognition that stereochemistry (e.g., stereochemistry of the chiral center of the backbone) can unexpectedly preserve or improve the properties of ds oligonucleotides. For example, but not limited to, this disclosure partially relates to ds oligonucleotides comprising one or more of the following:

[0021] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0022] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0023] (3) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0024] (4) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0025] (5) A leading chain comprising one or more skeletal phosphate chiral centers, the skeletal phosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal phosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal phosphate chiral center is in an Rp or Sp configuration;

[0026] (6) The guide chain contains one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the immediately adjacent upstream (N-3) nucleotide (i.e. in the 5' direction);

[0027] (7) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification;

[0028] (8) A transit chain in combination with one or more of the aforementioned guiding chains, wherein the transit chain contains one or more skeletal chiral centers of an Rp or Sp configuration; and

[0029] (9) A transit chain in combination with one or more of the above-mentioned leading chains, the transit chain comprising a sp-configured skeletal phosphate chiral center between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (i.e. in the 3' direction) (+2) nucleotide and between the 3' terminal nucleotide and the penultimate (N-1) nucleotide.

[0030] The ds oligonucleotide further comprises one or more of the following:

[0031] (1) A guiding chain in which one or both of the 5' and 3' terminal dinucleotides are not linked by an internucleotide linking of Rp, Sp or stereorandom uncharged, i.e., the guiding chain contains one or more internucleotide links of Rp, Sp or stereorandom uncharged, said internucleotide links being downstream (i.e. in the 3' direction) of the linking of the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the linking of the 3' terminal dinucleotides.

[0032] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0033] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0034] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0035] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0036] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker. For example, a 2'-F modification. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0037] In some embodiments, this disclosure covers the recognition that stereochemistry, such as the stereochemistry of the chiral center at the 5' end of the leader chain (e.g., 5' phosphate modification, e.g., 5' triazole phosphate modification), can unexpectedly preserve or improve the properties of the ds oligonucleotides described herein. For example, but not limited to, this disclosure relates in part to ds oligonucleotides comprising a leader chain comprising: (1) a phosphate thioester chiral center in the Rp or Sp configuration; (2) an Rp, Sp, or stereorandom uncharged internucleotide linking, wherein the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged internucleotide linking comprises a 2' modification, e.g., 2' F; and (3) a 5' end modification, e.g., a 5' phosphate modification, e.g., a 5' triazole phosphate modification, selected from the following:

[0038] (a) 5' PO modification, for example but not limited to:

[0039] ;

[0040] (b) 5' VP modification, for example but not limited to:

[0041] ;

[0042] (c) 5' MeP modification, for example, but not limited to:

[0043] ;

[0044] (d) 5'PN and 5'triazole-P modifications, such as, but not limited to:

[0045] ;

[0046] The bases are selected from A, C, G, T, U, no bases, and modified nucleobases;

[0047] R 2' Selected from H, OH, O-alkyl, F, MOE, locked nucleic acid (LNA) bridges and bridging nucleic acid (BNA) bridges to 4' C, for example, but not limited to:

[0048] .

[0049] In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides incorporated into the guide chain are Rp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are Sp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are stereorandom uncharged nucleotide linkages. In some embodiments, the guide chain includes a Sp-configured skeletal phosphate thioester chiral center between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (i.e., in the 3' direction) (+2) nucleotide, and an Rp-configured skeletal phosphate thioester chiral center between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide.

[0050] In some embodiments, the leader chain includes a 5' terminal modification, such as a 5' phosphate modification, or a 5' triazole phosphate modification, selected from, but not limited to, 5' MeP and 5' triazole-P modifications. In some embodiments, the 5' terminal modification (e.g., 5' phosphate modification, or 5' triazole phosphate modification) is... In some embodiments, the 5' end modification (e.g., 5' phosphate modification, such as 5' triazole phosphate modification) is... In some embodiments, the 5' end modification (e.g., 5' phosphate modification, e.g., 5' triazole phosphate modification) is... In some embodiments, the bootstrap chain includes a 5' MeP modification. The 5' end (+1) nucleotide has a Sp-configured skeletal phosphate chiral center between the adjacent downstream (+2) nucleotide (i.e., in the 3' direction), and the +2 nucleotide has an Rp-configured skeletal phosphate chiral center between the adjacent downstream (+3) nucleotide.

[0051] In some other embodiments, this disclosure includes the recognition that stereochemistry, such as the stereochemistry of a chiral center at the 5' terminal nucleotide of the leader chain, can unexpectedly preserve or improve the properties of ds oligonucleotides, wherein the leader chain of the ds oligonucleotide also comprises a phosphate thioester chiral center of Rp or Sp configuration. For example, but not limited to, this disclosure relates in part to ds oligonucleotides comprising a leader chain comprising: (1) a phosphate thioester chiral center of Rp or Sp configuration; (2) an Rp, Sp, or stereorandom non-negatively charged internucleotide linking, wherein the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom non-negatively charged internucleotide linking comprises a 2' modification, such as 2' F; and (3) a 5' terminal modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification, selected from the following:

[0052] (a) 5' PO nucleotides, such as, but not limited to:

[0053] ;

[0054] (b) 5' VP nucleotides, such as, but not limited to:

[0055] ;

[0056] (c) 5' MeP nucleotides, such as, but not limited to:

[0057] ;

[0058] (d) 5' PN and 5' triazole-P nucleotides, such as, but not limited to:

[0059] , and ;

[0060] (e) 5'-free VP and 5'-free MeP nucleotides, such as, but not limited to:

[0061] and .

[0062] In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides incorporated into the leader chain are Rp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are Sp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are stereorandom uncharged nucleotide linkages.

[0063] In some embodiments, this disclosure includes recognizing non-naturally occurring internucleotide linkages, such as neutral nucleotide linkages, which in some embodiments can be used to link one or more molecules to the double-stranded oligonucleotides described herein. In some embodiments, such linked molecules can facilitate the targeting and / or delivery of the double-stranded oligonucleotide. For example, but not limited to, such linked molecules include lipophilic molecules. In some embodiments, the linked molecule is a molecule containing one or more GalNAc moieties. In some embodiments, the linked molecule is a receptor. In some embodiments, the linked molecule is a receptor ligand.

[0064] In some embodiments, this disclosure provides techniques for incorporating a variety of additional chemical moieties into ds oligonucleotides. In some embodiments, this disclosure provides reagents and methods, for example, for introducing additional chemical moieties via nucleobases (e.g., via sites optionally covalently linked to nucleobases via linkers).

[0065] In some embodiments, this disclosure provides techniques for achieving allele-specific repression, such as ds oligonucleotide compositions and methods thereof, wherein a transcript of an allele of a specific target gene is selectively knocked down relative to at least another allele of the same gene.

[0066] Among other things, this disclosure provides structural elements, techniques, and / or features that can be incorporated into ds oligonucleotides and impart or modulate one or more properties thereto (e.g., relative to other identical ds oligonucleotides lacking the relevant techniques or features). In some embodiments, this disclosure demonstrates that one or more of the provided techniques and / or features can be usefully incorporated into ds oligonucleotides of various sequences.

[0067] In some embodiments, this disclosure demonstrates that certain provided structural elements, techniques, and / or features are particularly useful for ds oligonucleotides involved in and / or directing RNAi mechanisms (e.g., RNAi agents). However, in any case, the teachings of this disclosure are not limited to ds oligonucleotides involved in or acting via any particular biochemical mechanism. In some embodiments, this disclosure relates to any ds oligonucleotide that can be used for any purpose, acts via any mechanism, and comprises any sequence, structure, or form (or part thereof) described herein. In some embodiments, this disclosure provides ds oligonucleotides that can be used for any purpose, acts via any mechanism, and comprises any sequence, structure, or form (or part thereof) described herein, including one or more of the following:

[0068] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0069] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0070] (3) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0071] (4) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0072] (5) A leading chain comprising one or more skeletal phosphate chiral centers, the skeletal phosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal phosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal phosphate chiral center is in an Rp or Sp configuration;

[0073] (6) The guide chain contains one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the immediately adjacent upstream (N-3) nucleotide (i.e. in the 5' direction);

[0074] (7) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification;

[0075] The ds oligonucleotide further comprises one or more of the following:

[0076] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more Rp, Sp or stereorandom non-negatively charged internucleotide links, said internucleotide links being downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0077] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0078] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0079] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0080] (5) A transit chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream of the central nucleotide of the transit chain, i.e., in the 3' direction, and

[0081] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker. For example, a 2' F modification. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0082] In some embodiments, the provided ds oligonucleotides may participate in (e.g., guide) RNAi mechanisms.

[0083] In some embodiments, the guide chain includes a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide, and one or more of the following:

[0084] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0085] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0086] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0087] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0088] (5) A transit chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream of the central nucleotide of the transit chain, i.e., in the 3' direction, and

[0089] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is approximately 1 to 49.

[0090] In some embodiments, this disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also known as dsRNAi agents) having controlled structural elements provide unexpected properties and / or activities.

[0091] In some embodiments, the guide chain comprises a skeletal phosphate chiral center of Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide, and one or more of the following:

[0092] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0093] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0094] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0095] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0096] (5) A transit chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream of the central nucleotide of the transit chain, i.e., in the 3' direction, and

[0097] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0098] In some embodiments, the guide chain comprises one or more chiral skeletal phosphate thioester centers of Rp or Sp configuration upstream of the Sp-configured backbone chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding upstream (N-2) nucleotide, and one or more of the following:

[0099] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0100] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0101] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0102] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0103] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0104] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0105] In some embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom uncharged nucleotide links between the second (+2) and third (+3) nucleotides of the guide strand relative to the 5' terminal nucleotide, and an internucleotide link to the penultimate 3' (N-1) nucleotide, and one or more of the following:

[0106] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0107] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0108] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0109] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0110] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0111] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0112] In some embodiments, the guide chain includes a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide, and one or more of the following:

[0113] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0114] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0115] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0116] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0117] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0118] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises one or more skeletal phosphate thioester chiral centers in the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0119] In some embodiments, the guide chain comprises a skeletal phosphate chiral center of Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide, and one or more of the following:

[0120] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0121] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0122] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0123] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0124] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0125] The ds oligonucleotide further comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, and the guest chain comprises one or more skeletal chiral centers of the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0126] In some embodiments, the guide chain comprises one or more skeletal phosphate thioester chiral centers of Rp or Sp configuration upstream of the Sp-configured skeletal chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding upstream (N-2) nucleotide, and one or more of the following:

[0127] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0128] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0129] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0130] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0131] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0132] The ds oligonucleotide further comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, and the guest chain comprises one or more skeletal chiral centers of the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0133] In some embodiments, the guide chain comprises one or more skeletal phosphate thioester chiral centers of Rp or Sp configuration, said skeletal phosphate thioester chiral centers being located between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the (+2) nucleotide and the immediately adjacent downstream (+3) nucleotide, and one or more of the following:

[0134] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0135] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0136] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0137] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0138] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0139] The ds oligonucleotide further comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, and the guest chain comprises one or more skeletal chiral centers of the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0140] In some embodiments, the guide chain comprises one or more Rp, Sp, or stereorandom uncharged nucleotide links between any two adjacent nucleotides between the second (+2) nucleotide of the guide chain relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide chain, wherein N is the 3' terminal nucleotide, the guide chain comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged nucleotide link, and the guest chain comprises one or more skeletal chiral centers in the Rp or Sp configuration. In some embodiments, the one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the guide chain are Rp uncharged nucleotide links. In some embodiments, the one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are stereorandom uncharged nucleotide linkages.

[0141] In some embodiments, the guide chain includes a sp-configured phosphate thioester chiral center in the backbone between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide. The guide chain includes a 2' modification, such as a 2' F modification, of the 3' nucleotide of nucleotide pairs linked by Rp, Sp, or stereorandom uncharged internucleotide links. The guest chain includes 0-n Rp, Sp, or stereorandom uncharged internucleotide links (where n is about 1 to 49) and one or more Rp or Sp-configured backbone chiral centers. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links incorporated into the guide chain are Rp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links are Sp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are stereorandom uncharged nucleotide linkages.

[0142] In some embodiments, the guide chain includes a backbone phosphate thioester chiral center in Rp, Sp, or alternating configurations between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide. The guide chain includes a 2' modification, such as a 2' F modification, of the 3' nucleotide of nucleotide pairs linked by Rp, Sp, or stereorandom uncharged nucleotide interlinkings. The guest chain includes 0-n Rp, Sp, or stereorandom uncharged nucleotide interlinkings (where n is about 1 to 49) and one or more backbone chiral centers in Rp or Sp configurations. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide interlinkings incorporated into the guide chain are Rp uncharged nucleotide interlinkings. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide interlinkings are Sp uncharged nucleotide interlinkings. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are stereorandom uncharged nucleotide linkages.

[0143] In some embodiments, the guide chain contains one or more chiral phosphate thioester backbones with an Rp or Sp configuration upstream of the Sp-configured skeletal phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding upstream (N-2) nucleotide. The guide chain contains a 2' modification, such as a 2' F modification, of the 3' nucleotide of nucleotide pairs linked by Rp, Sp, or stereorandom uncharged internucleotide links. The guest chain contains 0-n Rp, Sp, or stereorandom uncharged internucleotide links (where n is about 1 to 49) and one or more chiral backbones with an Rp or Sp configuration. In some embodiments, the one or more Rp, Sp, or stereorandom uncharged internucleotide links incorporated into the guide chain are Rp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are Sp uncharged nucleotide linkages.

[0144] In some embodiments, the guide chain contains one or more Rp, Sp, or stereorandom uncharged nucleotide links between any two adjacent nucleotides between the second (+2) nucleotide of the guide chain relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide chain, where N is the 3' terminal nucleotide. The guide chain contains a 2' modification, such as a 2' F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged nucleotide link. The guest chain contains 0-n Rp, Sp, or stereorandom uncharged nucleotide links (where n is about 1 to 49) and one or more skeletal chiral centers in the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the guide chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are stereorandom uncharged nucleotide linkages.

[0145] In some embodiments, the provided ds oligonucleotides can participate in exon skipping mechanisms. In some embodiments, the provided ds oligonucleotides can be aptamers. In some embodiments, the provided ds oligonucleotides can bind to proteins, small molecules, nucleic acids, or cells and inhibit their function. In some embodiments, the provided ds oligonucleotides can participate in the formation of triple helices with double-stranded nucleic acids in cells. In some embodiments, the provided ds oligonucleotides can bind to genomic (e.g., chromosome) nucleic acids. In some embodiments, the provided ds oligonucleotides can bind to genomic (e.g., chromosome) nucleic acids, thereby preventing or reducing nucleic acid expression (e.g., by preventing or reducing transcription, enhancing transcription, modifying, etc.). In some embodiments, the provided ds oligonucleotides can bind to DNA quadruplexes. In some embodiments, the provided ds oligonucleotides can be immunomodulatory. In some embodiments, the provided ds oligonucleotides can be immunostimulatory. In some embodiments, the provided oligonucleotides can be immunostimulatory and may contain a CpG sequence. In some embodiments, the provided ds oligonucleotides can be immunostimulatory and may contain a CpG sequence and may be used as adjuvants. In some embodiments, the provided ds oligonucleotides may be immunostimulatory and may contain a CpG sequence, and may be used as adjuvants for treating diseases (e.g., infectious diseases or cancer). In some embodiments, the provided ds oligonucleotides may be therapeutic. In some embodiments, the provided ds oligonucleotides may be non-therapeutic. In some embodiments, the provided ds oligonucleotides may be therapeutic or non-therapeutic. In some embodiments, the provided ds oligonucleotides may be used for therapeutic, diagnostic, research, and / or nanomaterial applications. In some embodiments, the provided ds oligonucleotides may be used for experimental purposes. In some embodiments, the provided ds oligonucleotides may be used for experimental purposes, e.g., as probes in microarrays. In some embodiments, the provided ds oligonucleotides may participate in more than one biological mechanism; in some such embodiments, for example, the provided ds oligonucleotides may participate in RNAi and RNase H mechanisms.

[0146] In some embodiments, the provided ds oligonucleotides target INHBE targets (e.g., INHBE target sequences, INHBE target RNA, INHBE target mRNA, INHBE target precursor mRNA, INHBE target genes, etc.). INHBE target genes are genes for which the expression and / or activity of one or more INHBE gene products (e.g., INHBE RNA and / or protein products) associated with them is intended to be altered. In some embodiments, the INHBE target genes are intended to be inhibited. Therefore, when the ds oligonucleotides described herein act on INHBE target genes, the presence and / or activity of one or more INHBE gene products are altered in the presence of the ds oligonucleotides compared to the absence of the ds oligonucleotides.

[0147] In some embodiments, an INHBE target is a specific INHBE allele from which the expression and / or activity of one or more associated products (e.g., INHBE RNA and / or protein products) is to be altered. In some embodiments, an INHBE target allele is an allele whose presence and / or expression are associated (e.g., related to) the presence, incidence, and / or severity of one or more INHBE-related diseases and / or conditions. Alternatively or additionally, in some embodiments, an INHBE target allele is an allele from which alterations in the level and / or activity of one or more INHBE gene products are associated with improvements in one or more aspects of INHBE-related diseases and / or conditions (e.g., delayed onset, reduced severity, response to other therapies, etc.).

[0148] In some embodiments, for example, when the presence and / or activity of a particular INHBE allele (INHBE disease-associated allele) is associated with the presence, incidence, and / or severity of one or more disorders, diseases, and / or conditions (e.g., relevant), and different INHBE alleles are present but not so relevant, or have a low degree of correlation (e.g., showing a less significant or statistically insignificant correlation), the ds oligonucleotides and methods described herein can preferentially or specifically target the relevant allele relative to the one or more low- or unrelated alleles, thereby mediating allele-specific repression.

[0149] In some embodiments, the INHBE target sequence is the INHBE sequence to which the oligonucleotide binds as described herein. In some embodiments, the INHBE target sequence is identical to or precisely complementary to the INHBE sequence of the provided oligonucleotide or its consecutive residues (e.g., the provided oligonucleotide includes an INHBE target-binding sequence that is identical to or precisely complementary to the INHBE target sequence). In some embodiments, the INHBE target-binding sequence is precisely complementary to the INHBE target sequence of an INHBE transcript (e.g., precursor mRNA, mRNA, etc.). The INHBE target-binding sequence / target sequence can have various lengths to provide an oligonucleotide with the desired activity and / or properties. In some embodiments, the INHBE target binding sequence / target sequence comprises 5-50 bases (e.g., 10-40, 15-30, 15-25, 16-25, 17-25, 18-25, 19-25, 20-25, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, minor differences / mismatches between the oligonucleotide (relevant portion) and its target sequence are tolerated, including but not limited to the 5' and / or 3' end regions of the INHBE target and / or oligonucleotide sequences. In some embodiments, the INHBE target sequence is present within the INHBE target gene. In some embodiments, the INHBE target sequence is present in INHBE transcripts (e.g., mRNA and / or precursor mRNA) generated from the INHBE target gene.

[0150] In some embodiments, the INHBE target sequence includes one or more allelic sites (i.e., locations within the INHBE target gene where allelic variations occur). In some embodiments, the allelic site is a mutation. In some embodiments, the allelic site is an SNP. In some such embodiments, the provided oligonucleotide preferentially or specifically binds to one allele relative to one or more other alleles. In some embodiments, the provided oligonucleotide preferentially binds to disease-associated alleles. For example, in some embodiments, the oligonucleotide provided herein (or its target-binding sequence portion) has a sequence that is completely or at least partially identical to, or precisely complementary to, a specific allelic version of the INHBE target sequence.

[0151] In some embodiments, the oligonucleotides (or their target-binding sequence portions thereof) provided herein have a sequence that is identical to or precisely complementary to the INHBE target sequence of an allele containing a disease-related allele or an allele containing a disease-related allele. In some embodiments, the oligonucleotides provided herein have an INHBE target-binding sequence that is precisely complementary to the INHBE target sequence of an INHBE transcript containing an allele (in some embodiments, a disease-related allele), wherein the allele is a mutation. In some embodiments, the oligonucleotides provided herein have an INHBE target-binding sequence that is precisely complementary to the INHBE target sequence of an INHBE transcript containing an allele (in some embodiments, a disease-related allele), wherein the allele is a SNP. In some embodiments, the sequence is any sequence disclosed herein.

[0152] Unless otherwise stated, all sequences (including, but not limited to, base sequences and chemical, modification and / or stereochemical patterns) are presented in 5' to 3' order, where the 5' terminal nucleotide is marked as "+1" and the 3' terminal nucleotide is marked by the number of nucleotides in the complete sequence or "N", where the penultimate nucleotide is marked as "N-1", and so on.

[0153] In some embodiments, this disclosure provides compositions and methods related to oligonucleotides that are specific to the INHBE target and have any form, structural element, or base sequence of any oligonucleotide disclosed herein.

[0154] In some embodiments, this disclosure provides compositions and methods associated with oligonucleotides that are specific to INHBE targets and have or contain the base sequence of any oligonucleotide disclosed herein, or a region of at least 15 consecutive nucleotides of the base sequence of any oligonucleotide disclosed herein, wherein the first nucleotide of the base sequence or the first nucleotide of the at least 15 consecutive nucleotides may optionally be replaced by T or DNA T.

[0155] In some embodiments, this disclosure provides compositions and methods for RNA interference directed by RNAi agents (also known as RNAi oligonucleotides). In some embodiments, the oligonucleotides of such compositions may have the form, structural elements, or base sequences of the oligonucleotides disclosed herein.

[0156] In some embodiments, this disclosure provides compositions and methods for INHBE target gene RNA knockdown mediated by RNase H guided by oligonucleotides (e.g., antisense oligonucleotides).

[0157] The provided oligonucleotides and oligonucleotide compositions may have any form, structural element, or base sequence of any oligonucleotide disclosed herein. In some embodiments, the structural element is a 5' end structure, a 5' end region, a 5' nucleotide, a seed region, a post-seed region, a 3' end region, a 3' terminal dinucleotide, a 3' cap, or any portion of these structures, GC content, long GC extension and / or any modification, chemical, stereochemical, modification pattern, chemical or stereochemical, or chemical motif (e.g., including but not limited to a targeting motif, a lipid motif, a GalNAc motif, a carbohydrate motif, etc.), any component, or any combination of any of the foregoing.

[0158] In some embodiments, this disclosure provides compositions of oligonucleotides and methods of using them.

[0159] In some embodiments, this disclosure provides compositions and methods of using oligonucleotides that can direct RNA interference and RNase H-mediated knockdown of INHBE target gene RNA. In some embodiments, the oligonucleotides of such compositions may have the form, structural elements, or base sequences of the oligonucleotides disclosed herein.

[0160] In some embodiments, oligonucleotides that direct specific events or activities are involved in specific events or activities, such as a decrease in the expression, level, or activity of a target gene or its gene product. In some embodiments, an oligonucleotide is considered to "direct" a specific event or activity when its presence in a system in which the event or activity may occur is associated with an increased detectable occurrence, frequency, intensity, and / or level of the event or activity.

[0161] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of an oligonucleotide as described herein, such as: a base sequence (or a portion of at least 15 consecutive bases thereof); a pattern of internucleotide bonding (or a portion of at least 5 consecutive nucleotide bonding thereof); a stereochemical pattern of internucleotide bonding (or a portion of at least 5 consecutive nucleotide bonding thereof); a 5' end structure; a 5' end region; a first region; a second region; and a 3' end region (which may be a 3' terminal dinucleotide and / or a 3' cap); and optional additional chemical portions; and, in some embodiments, at least one structural element comprises a chiral-controlled chiral center. In some embodiments, the 3' terminal dinucleotide may comprise a total of two nucleotides. In some embodiments, the oligonucleotide further comprises a chemical portion selected as a non-limiting example of a targeting portion, a carbohydrate portion, a GalNAc portion, a lipid portion, and any other chemical portion described herein or known in the art. In some embodiments, the portion binding APGR is a GalNAc portion, or a variant, derivative, or modified form thereof, as described herein and / or known in the art. In some embodiments, the oligonucleotide is an RNAi agent. In some embodiments, the first region is a seed region. In some embodiments, the second region is the post-seed region.

[0162] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of the RNAi agent described herein, such as a 5' end structure; a 5' end region; a seed region; a post-seed region (the region between the seed region and the 3' end region); and a 3' end region (which may be a 3' terminal dinucleotide and / or a 3' cap); and optional additional chemical moieties; and, in some embodiments, at least one structural element comprises a chiral-controlled chiral center. In some embodiments, the 3' terminal dinucleotide may comprise a total of two nucleotides. In some embodiments, the oligonucleotide further comprises a chemical moieties selected as non-limiting examples of a targeting moieties, carbohydrate moieties, GalNAc moieties, and lipid moieties. In some embodiments, the APGR-binding moiety is any GalNAc, or a variant, derivative, or modification thereof, as described herein or known in the art.

[0163] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of an oligonucleotide as described herein, such as a 5' end structure, a 5' end region, a first region, a second region, a 3' end region, and optionally additional chemical moieties, wherein at least one structural element comprises a chiral-controlled chiral center. In some embodiments, the oligonucleotide comprises a sequence span of at least 5 nucleotides without 2'-modification. In some embodiments, the oligonucleotide further comprises additional chemical moieties selected as non-limiting examples, including a targeting moieties, carbohydrate moieties, GalNAc moieties, and lipid moieties. In some embodiments, the provided oligonucleotide is capable of directing RNA interference. In some embodiments, the provided oligonucleotide is capable of directing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotide is capable of directing both RNA interference and RNase H-mediated knockdown. In some embodiments, the first region is a seed region. In some embodiments, the second region is a post-seed region.

[0164] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of an RNAi agent, such as a 5' end structure, a 5' end region, a seed region, a post-seed region, and a 3' end region, as well as optional additional chemical moieties, wherein at least one structural element comprises a chiral center controlled by chirality; and, in some embodiments, the oligonucleotide is also capable of directing RNase H-mediated knockdown of target gene RNA. In some embodiments, the oligonucleotide comprises a sequence segment of at least five 2'-deoxynucleotides in total. In some embodiments, the oligonucleotide further comprises a chemical moieties selected as non-limiting examples of a targeting moieties, carbohydrate moieties, GalNAc moieties, and lipid moieties, and any other additional chemical moieties described herein.

[0165] In some embodiments, this disclosure demonstrates that oligonucleotide properties can be modulated by chemical modification. In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more nucleotide linkages, sugars, and / or base modifications. In some embodiments, this disclosure provides an oligonucleotide composition capable of directing RNA interference and comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more nucleotide linkages, and / or one or more sugars, and / or one or more base modifications. In some embodiments, the oligonucleotide or oligonucleotide composition is also capable of directing RNase H-mediated knockdown of INHBE target gene RNA. In some embodiments, this disclosure demonstrates that the properties of oligonucleotides (e.g., activity, toxicity, etc.) can be modulated by chemical modification of sugars, nucleobases, and / or nucleotide linkages. In some embodiments, this disclosure provides oligonucleotide compositions comprising a variety of oligonucleotides having a common base sequence and including one or more modified internucleotide links (or “non-natural internucleotide links” found in natural DNA and RNA, which may be used in place of the natural phosphate ester internucleotide link (−OP(O)(OH)O−, which may be in salt form (−OP(O)(O)) at physiological pH found in natural DNA and RNA). −The oligonucleotide contains one or more modified sugar moieties and / or one or more natural phosphate esters. In some embodiments, the provided oligonucleotide may contain two or more types of modified internucleotide links. In some embodiments, the provided oligonucleotide contains uncharged internucleotide links. In some embodiments, the uncharged internucleotide links are neutral internucleotide links. In some embodiments, the neutral internucleotide links contain cyclic guanidine moieties. Such moieties may optionally be substituted. In some embodiments, the provided oligonucleotide contains a neutral internucleotide link and another internucleotide link that is not a neutral backbone. In some embodiments, the provided oligonucleotide contains a neutral internucleotide link and a phosphate thioester internucleotide link. In some embodiments, the provided oligonucleotide composition comprising multiple oligonucleotides is chiral controlled, and the levels of the multiple oligonucleotides in the composition are controlled or predetermined, and the multiple oligonucleotides share a common stereochemical configuration at one or more chiral internucleotide links. For example, in some embodiments, multiple oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral nucleotide linkages (each independently Rp or Sp); in some embodiments, multiple oligonucleotides share a common stereochemical configuration at each chiral nucleotide linkage. In some embodiments, chiral nucleotide linkages in which controlled levels of oligonucleotides in the composition share a common stereochemical configuration (independently Rp or Sp configuration) are referred to as chiral-controlled nucleotide linkages. In some embodiments, the modified nucleotide linkages are nucleotide linkages that are uncharged (neutral or cationic) at a specific pH (e.g., human physiological pH (approximately 7.4), pH of the delivery site (e.g., organelle, cell, tissue, organ, organism, etc.)). These linkages are predominantly (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc.; in some embodiments, at least 30%; in some embodiments, at least 40%; in some embodiments, at least 50%; in some embodiments, at least 60%; in some embodiments, at least 70%; in some embodiments, at least 80%; in some embodiments, at least 90%; in some embodiments, at least 99%; etc.) in neutral or cationic form (with anionic form (e.g., −O−P(O)(O − -O− (the anionic form of natural phosphate ester bonded), −O−P(O)(S −Compared to (anionic form of thiophosphate bond), etc., the modified internucleotide bonds are present. In some embodiments, the modified internucleotide bonds are neutral internucleotide bonds because they are mainly present in a neutral form at pH 7.4. In some embodiments, the modified internucleotide bonds are cationic internucleotide bonds because they are mainly present in a cationic form at pH 7.4. In some embodiments, the pH is the physiological pH of humans (about 7.4). In some embodiments, the modified internucleotide bonds are neutral internucleotide bonds because at least 90% of the internucleotide bonds are present in a neutral form in aqueous solution at pH 7.4. In some embodiments, the modified internucleotide bonds are neutral internucleotide bonds because at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the internucleotide bonds are present in a neutral form in aqueous solution of the oligonucleotide. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is at least 99%. In some embodiments, the non-negatively charged internucleotide linkages, such as neutral internucleotide linkages, do not have a pKa less than 8, 9, 10, 11, 12, 13, or 14 when in their neutral form. In some embodiments, the pKa of the internucleotide linkages in this disclosure can be represented by the pKa of the CH3−internucleotide linkage −CH3 (i.e., replacing the two nucleoside units linked by the internucleotide linkage with two −CH3 groups). Without wishing to be bound by any particular theory, in at least some cases, neutral internucleotide linkages in oligonucleotides can provide improved properties and / or activities compared to equivalent nucleic acids that do not contain neutral internucleotide linkages, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosome escape, and / or improved nuclear uptake, etc.

[0166] In some embodiments, the uncharged nucleotide linkages have, for example, structures of the formulas In-1, In-2, In-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, as described in US9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107, US 2019 / 0127733, US 10450568, US 2019 / 0077817, US 2019 / 0249173, US As described in WO 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612, etc. In some embodiments, the non-negatively charged internucleotide linking comprises a cyclic guanidine moiety. In some embodiments, the modified internucleotide linking comprising a cyclic guanidine moiety has the following structure: In some embodiments, the neutral nucleotide linking comprising the cyclic guanidine moiety is chiral controlled. In some embodiments, this disclosure relates to compositions comprising oligonucleotides containing at least one neutral nucleotide linking and at least one phosphate thioester nucleotide linking.

[0167] In some embodiments, this disclosure relates to compositions comprising oligonucleotides comprising at least one neutral nucleotide link and at least one phosphate thioester nucleotide link, wherein the phosphate thioester nucleotide link is a chiral-controlled nucleotide link of the Sp configuration.

[0168] In some embodiments, this disclosure relates to compositions comprising oligonucleotides comprising at least one neutral nucleotide link and at least one phosphate thioester nucleotide link, wherein the phosphate thioester is an Rp-configuration chiral-controlled nucleotide link.

[0169] In some embodiments, this disclosure relates to compositions comprising oligonucleotides, the oligonucleotides comprising at least one Tmg group ( The neutral nucleotides are linked together and at least one thiophosphate.

[0170] In some embodiments, the internucleotide linkages in each oligonucleotide are independently selected from natural phosphate linkages, thiophosphate linkages, and uncharged internucleotide linkages (e.g., n001, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n055). In some embodiments, the internucleotide linkages in each oligonucleotide are independently selected from natural phosphate linkages, thiophosphate linkages, and neutral nucleotide linkages (e.g., n001, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n055).

[0171] In some embodiments, this disclosure relates to compositions comprising oligonucleotides comprising at least one neutral nucleotide linker comprising a Tmg group and at least one thiophosphate ester, wherein the thiophosphate ester is a chiral-controlled nucleotide linker of the Sp configuration.

[0172] In some embodiments, this disclosure relates to compositions comprising oligonucleotides comprising at least one neutral nucleotide linker selected from those comprising a Tmg group and at least one thiophosphate ester, wherein the thiophosphate ester is a chiral-controlled nucleotide linker with an Rp configuration.

[0173] Various types of nucleotide linkages differ in properties. Without being bound by any theory, this disclosure states that native phosphate ester linkages (phosphodiester nucleotide linkages) are anionic and may be unstable when used alone in vivo without further chemical modification; thiophosphate nucleotide linkages are anionic, generally more stable in vivo than native phosphate ester linkages, and are generally more hydrophobic; neutral nucleotide linkages, such as the neutral nucleotide linkages containing a cyclic guanidine moiety exemplified in this disclosure, are neutral at physiological pH, may be more stable in vivo than native phosphate ester linkages, and are more hydrophobic.

[0174] In some embodiments, the chiral-controlled internucleotide linkages are neutral at physiological pH, chiral-controlled, in vivo stable, hydrophobic, and can increase endosome escape.

[0175] In some embodiments, the provided oligonucleotide includes one or more regions, such as block region, wing region, core region, 5' end region, 3' end region, middle region, seed region, post-seed region, etc. In some embodiments, the regions (e.g., block regions, wing regions, core regions, 5' end regions, 3' end regions, intermediate regions, etc.) comprise, for example, uncharged nucleotide linkages of the formula In-1, In-2, In-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., as described in US 9394333, US 9744183, US 9605019, US 9598458, US9982257, US 10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107, US 2019 / 0127733, US 10450568, US As described in WO 2019 / 0077817, US 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612. In some embodiments, the region comprises a neutral nucleotide link. In some embodiments, the region comprises a nucleotide link containing a cyclic guanidine. In some embodiments, the region comprises a nucleotide link that includes a cyclic guanidine moiety. In some embodiments, the region includes a structure Nucleotide-to-nucleotide linkages. In some embodiments, such nucleotide-to-nucleotide linkages are chiral controlled.

[0176] In some embodiments, the nucleotide is a natural nucleotide. In some embodiments, the nucleotide is a modified nucleotide. In some embodiments, the nucleotide is a nucleotide analog. In some embodiments, the base is a modified base. In some embodiments, the base is a protected nucleobase, such as a protected nucleobase used in oligonucleotide synthesis. In some embodiments, the base is a base analog. In some embodiments, the sugar is a modified sugar. In some embodiments, the sugar is a sugar analog. In some embodiments, the internucleotide link is a modified internucleotide link. In some embodiments, the nucleotide comprises a base, a sugar, and an internucleotide link, wherein each of the base, sugar, and internucleotide link is independently and optionally naturally occurring or non-naturally occurring. In some embodiments, the nucleoside comprises a base and a sugar, wherein each of the base and sugar is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2'-deoxy) and RNA (2'-OH) nucleotides; and those containing one or more modifications at the internucleotide, sugar, and / or internucleotide link. Non-limiting examples of sugars include ribose and deoxyribose; and ribose and deoxyribose with 2'-modifications, including but not limited to 2'-F, LNA, 2'-OMe, and 2'-MOE modifications. In some embodiments, the internucleotide linkage is a phosphorus-free portion used to link two natural or non-natural sugars.

[0177] In some embodiments, the composition comprises two or more of the following polymers: a first plurality of oligonucleotides and / or a second plurality of oligonucleotides, wherein the first and second plurality of oligonucleotides can independently guide the knockdown of the same or different targets via RNA interference and / or RNase H-mediated knockdown.

[0178] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides sharing the following:

[0179] 1) Common base sequence;

[0180] 2) Common skeleton bonding mode;

[0181] 3) Common stereochemistry, independently consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 chiral nucleotides linked together (“chiral-controlled nucleotide bonds”); the composition is chiral-controlled, and the level of the first plurality of oligonucleotides in the composition is predetermined.

[0182] In some embodiments, the oligonucleotide composition comprising multiple oligonucleotides (e.g., a first plurality of oligonucleotides) is chiral-controlled because the plurality of oligonucleotides independently share a common stereochemistry at one or more chiral nucleotide linking sites. In some embodiments, the plurality of oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral nucleotide linking sites, each of which is independently Rp or Sp. In some embodiments, the plurality of oligonucleotides share a common stereochemical configuration at each chiral nucleotide linking site. In some embodiments, chiral nucleotide linking where a predetermined level of oligonucleotides in the composition shares a common stereochemical configuration (independently Rp or Sp) is referred to as chiral-controlled nucleotide linking.

[0183] In some embodiments, the provided composition contains a predetermined level of oligonucleotides (e.g., a first plurality of oligonucleotides of certain exemplary compositions) comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral-controlled internucleotide bonds.

[0184] In some embodiments, at least 5 nucleotides are chirally controlled; in some embodiments, at least 10 nucleotides are chirally controlled; in some embodiments, at least 15 nucleotides are chirally controlled; and in some embodiments, each chiral nucleotide is chirally controlled.

[0185] In some embodiments, 1%–100% of the chiral nucleotide linkages are chiral controlled. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the chiral nucleotide linkages are chiral controlled.

[0186] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides sharing the following:

[0187] 1) Common base sequence;

[0188] 2) Common skeleton bonding mode; and

[0189] 3) A common backbone chiral center pattern, wherein the composition is a substantially pure formulation of oligonucleotides because the predetermined levels of oligonucleotides in the composition have a common base sequence and length, a common backbone bonding pattern, and a common backbone chiral center pattern. In some embodiments, the common backbone chiral center pattern comprises at least one internucleotide bond containing a chiral center with chiral control. In some embodiments, the predetermined levels of oligonucleotides are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides belonging to or comprising a common base sequence in the provided composition. In some embodiments, all oligonucleotides belonging to or comprising a common base sequence in the provided composition are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition that belong to or comprise a common base sequence, base modification, sugar modification, and / or modified nucleotide interlinking. In some embodiments, all oligonucleotides belonging to or comprising a common base sequence, base modification, sugar modification, and / or modified nucleotide interlinking in the provided composition are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition.In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition that belong to or contain a common base sequence, base modification pattern, sugar modification pattern, and / or modified internucleotide linking pattern. In some embodiments, all oligonucleotides belonging to or comprising a common base sequence, base modification pattern, sugar modification pattern and / or modified internucleotide linking pattern in the provided composition are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, and / or a common modified internucleotide linking pattern. In some embodiments, all oligonucleotides sharing a common base sequence, a common base modification pattern, a common sugar modification pattern, and / or a common modified internucleotide linking pattern in the provided composition are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level is 1%-100%. In some embodiments, the predetermined level is at least 1%. In some embodiments, the predetermined level is at least 5%. In some embodiments, the predetermined level is at least 10%. In some embodiments, the predetermined level is at least 20%. In some embodiments, the predetermined level is at least 30%. In some embodiments, the predetermined level is at least 40%. In some embodiments, the predetermined level is at least 50%. In some embodiments, the predetermined level is at least 60%. In some embodiments, the predetermined level is at least 10%. In some embodiments, the predetermined level is at least 70%. In some embodiments, the predetermined level is at least 80%. In some embodiments, the predetermined level is at least 90%.In some embodiments, the predetermined level is at least 5*(1 / 2 g), where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least 10*(1 / 2 g), where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least 100*(1 / 2 g), where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.80) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.80) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.80) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.85) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.90) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.95) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.96) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.97) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.98) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, the predetermined level is at least (0.99) g, where g is the number of chiral-controlled internucleotide bonds. In some embodiments, to determine the level of oligonucleotides having g chiral-controlled internucleotide linkages in the composition, the product of the diastereomeric purity of each of the g chiral-controlled internucleotide linkages is used as the level: (diastereomeric purity of chiral-controlled internucleotide linkage 1) * (diastereomeric purity of chiral-controlled internucleotide linkage 2) * … * (diastereomeric purity of chiral-controlled internucleotide linkage g), wherein the diastereomeric purity of each chiral-controlled internucleotide linkage is independently represented by the diastereomeric purity of a dimer containing the same internucleotide linkage and the nucleosides flanking the internucleotide linkage, and prepared according to a method similar to that used for oligonucleotides (e.g., similar or preferably identical oligonucleotide preparation cycles, including similar or preferably identical reagents and reaction conditions). In some embodiments, the level and / or diastereomeric purity of the oligonucleotides can be determined by analytical methods, such as chromatography, spectroscopy, spectrophotometry, or any combination thereof. Among other things, this disclosure covers the understanding that stereorandom oligonucleotide formulations contain multiple different chemical entities that differ from each other, for example, in the stereochemical structure (or stereochemistry) of individual skeletal chiral centers within the oligonucleotide chain.Stereorandom oligonucleotide formulations provide uncontrolled compositions comprising undetermined levels of oligonucleotide stereoisomers without controlling the stereochemistry of the backbone chiral centers. Although these stereoisomers may have the same base sequence and / or chemical modifications, they are distinct chemical entities, at least due to their different backbone stereochemistry, and as demonstrated herein, they can possess different properties, such as sensitivity to nucleases, activity, distribution, etc. In some embodiments, a particular stereoisomer can be defined, for example, by its base sequence, its length, its backbone bonding pattern, and its backbone chiral center pattern. In some embodiments, this disclosure demonstrates that improvements in properties and activity achieved by controlling the stereochemistry within the oligonucleotide can be comparable to, or even better than, those achieved by using chemical modifications.

[0190] Among other things, this disclosure covers the understanding that stereorandom oligonucleotide formulations contain multiple distinct chemical entities that differ from each other, for example, in the stereochemical structure (or stereochemistry) of individual chiral centers within the oligonucleotide chain. Without controlling the stereochemistry of the chiral centers, stereorandom oligonucleotide formulations provide uncontrolled compositions containing undetermined levels of oligonucleotide stereoisomers. Although these stereoisomers may have the same base sequence and / or chemical modifications, they are distinct chemical entities, at least due to their different chiral stereochemistry, and as demonstrated herein, they can possess different properties, such as sensitivity to nucleases, activity, distribution, etc. In some embodiments, a particular stereoisomer can be defined, for example, by its base sequence, its length, its skeletal bonding pattern, and its chiral center pattern. In some embodiments, this disclosure demonstrates that improvements in properties and activity achieved by controlling the stereochemistry within the oligonucleotide can be comparable to, or even better than, those achieved by using chemical modifications.

[0191] In some embodiments, ds oligonucleotides targeting INHBE or compositions of ds oligonucleotides targeting INHBE can be used to prevent or treat INHBE-related conditions, disorders, or diseases in subjects of need. In some embodiments, this disclosure provides a method for preventing or treating INHBE-related conditions, disorders, or diseases, comprising administering a therapeutically effective amount of the provided ds oligonucleotide to a subject suffering from or affected by the condition, disorder, or disease, or a pharmaceutical composition that can deliver or contain a therapeutically effective amount of the provided ds oligonucleotide. In some embodiments, this disclosure provides pharmaceutical compositions comprising the provided ds oligonucleotide targeting INHBE and a pharmaceutically acceptable carrier. In some embodiments, the oligonucleotide in the pharmaceutical composition is in one or more pharmaceutically acceptable salt forms, such as sodium salts, ammonium salts, etc.

[0192] In some embodiments, oligonucleotides or oligonucleotide compositions may be used to manufacture medicines for the prevention or treatment of INHBE-related conditions, disorders, or diseases, such as metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension), in subjects in need.

[0193] The provided techniques (e.g., oligonucleotides, compositions, methods, etc.) can be used to prevent and / or treat various conditions, disorders, or diseases associated with INHBE. In some embodiments, the condition, disorder, or disease is a metabolic disorder, such as metabolic syndrome, or a related disease, such as obesity, cardiovascular disease, diabetes, or hypertension. Detailed Implementation

[0194] The technology of this disclosure can be more readily understood by referring to the following detailed description of certain embodiments.

[0195] Definitions

[0196] Unless otherwise indicated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th edition, edited by Smith, MB, and March, J., John Wiley & Sons, New York: 2001.

[0197] As used herein, unless the context clearly specifies otherwise, in this disclosure, (i) the terms “a” or “an” are to be understood as “at least one”; (ii) the term “or” is to be understood as “and / or”; (iii) the terms “comprising” and “comprise” (whether or not used with “but not limited to”) are to be understood as covering each of the components or steps listed individually or together with one or more other components or steps; (iv) the term “another” is to be understood as meaning at least another / second or more; (v) the terms “about” and “approximately” are to be understood as allowing standard deviation, as would be understood by one of ordinary skill in the art; and (vi) wherever a range is provided, the endpoints are included.

[0198] Unless otherwise stated, the description of oligonucleotides and their elements (e.g., base sequence, sugar modification, internucleotide linkages, linkage phosphorus stereochemistry, their patterns, etc.) is from 5' to 3'. As those skilled in the art will understand, in some embodiments, oligonucleotides may be provided and / or used as salt forms, particularly pharmaceutically acceptable salt forms (e.g., sodium salts). Unless otherwise stated, oligonucleotides include multiple forms of oligonucleotides. As those skilled in the art will also understand, in some embodiments, individual oligonucleotides in a composition may be considered to have the same composition and / or structure, even in such compositions (e.g., liquid compositions), particularly, such oligonucleotides may be in different one or more salt forms at a given time (and, for example, in liquid compositions, they may be soluble and the oligonucleotide chain may be present in anionic form). For example, those skilled in the art will understand that, at a given pH, internucleotide linkages along an oligonucleotide chain may be in acid (H) form or one of a variety of possible salt forms (e.g., sodium salts or salts of different cations, depending on which ions may be present in the preparation or composition), and will understand that as long as their acid form (e.g., with H) is acceptable, + If all cations (if any) have the same composition and / or structure, such a single oligonucleotide can be properly considered to have the same composition and / or structure.

[0199] Analog: The term "analog" includes any chemical part that is structurally different from a reference chemical part or class of parts but is capable of performing at least one function of such reference chemical part or class of parts. As non-limiting examples, nucleotide analogs are structurally different from nucleotides but perform at least one function of nucleotides; nucleobase analogs are structurally different from nucleosides but perform at least one function of nucleosides; and so on.

[0200] Antisense: As used herein, the term "antisense" refers to an oligonucleotide or other nucleic acid having a base sequence that is complementary or substantially complementary to a target nucleic acid to which it can hybridize. In some embodiments, the target nucleic acid is a target gene mRNA. In some embodiments, hybridization is necessary for or results in an activity, such as a reduction in the level, expression, or activity of the target nucleic acid or its gene product. As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide complementary to the target nucleic acid. In some embodiments, an antisense oligonucleotide can direct a reduction in the level, expression, or activity of the target nucleic acid or its product. In some embodiments, an antisense oligonucleotide can direct a reduction in the level, expression, or activity of the target nucleic acid or its product through a mechanism involving RNA interference.

[0201] Chiral control: As used herein, “chiral control” refers to the stereochemical name for controlling the chiral linking phosphorus in a chiral internucleotide link within an oligonucleotide. As used herein, a chiral internucleotide link is an internucleotide link in which the linking phosphorus is chiral. In some embodiments, control is achieved by a chiral element absent in the sugar and base moieties of the oligonucleotide, for example, in some embodiments, by using one or more chiral auxiliaries during oligonucleotide preparation, which, as described in this disclosure, are typically part of a chiral phosphoramidite used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will understand that if conventional oligonucleotide synthesis is used to form chiral internucleotide links, such conventional oligonucleotide synthesis without the use of chiral auxiliaries cannot control the stereochemistry at the chiral internucleotide link. In some embodiments, the stereochemical designation of each chiral linking phosphorus in each chiral internucleotide link within the oligonucleotide is controlled.

[0202] Chiral-controlled oligonucleotide compositions: As used herein, the terms "chiral-controlled oligonucleotide composition," "chiral-controlled nucleic acid composition," etc., refer to compositions comprising multiple oligonucleotides (or nucleic acids) that share: 1) a common base sequence, 2) a common backbone linkage pattern, and 3) a common backbone phosphorus modification pattern, wherein the multiple oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral nucleotide linkages (chiral-controlled or stereodefined nucleotide linkages whose chiral linkage phosphorus is Rp or Sp in the composition ("stereodefined"), rather than a random mixture of Rp and Sp as in achiral-controlled nucleotide linkages). The levels of the multiple oligonucleotides (or nucleic acids) in the chiral-controlled oligonucleotide composition are predetermined / controlled (e.g., by chiral-controlled oligonucleotide preparation to stereoselectively form one or more chiral nucleotide linkages). In some embodiments, the chiral-controlled oligonucleotide composition comprises about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 3%) of all oligonucleotides. 0%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the oligonucleotides. In some embodiments, the chiral-controlled oligonucleotide composition comprises approximately 1%-100% (e.g., approximately 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%) of all oligonucleotides sharing a common base sequence, a common backbone bonding pattern, and a common backbone phosphorus modification pattern. Or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the multiple oligonucleotides.In some embodiments, the level is approximately 1%-100% (e.g., approximately 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%) of all oligonucleotides in the composition; or all oligonucleotides in the composition that share a common base sequence (e.g., multiple oligonucleotides or a base sequence of one type of oligonucleotide); or all oligonucleotides in the composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linking type pattern, and / or a common internucleotide linking modification pattern. 00%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 chiral nucleotides. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1%-100% of the chiral nucleotides. In some embodiments, multiple oligonucleotides (or nucleic acids) have the same composition (as understood by those skilled in the art, they may be present in one or more forms in some embodiments, such as acidic forms, salt forms, etc.). In some embodiments, the level of the multiple oligonucleotides (or nucleic acids) is about 1% to 100% of all oligonucleotides (or nucleic acids) in a composition sharing the same composition with the multiple oligonucleotides (or nucleic acids). In some embodiments, each chiral nucleotide link is a chiral-controlled nucleotide link, and the composition is a fully chiral-controlled oligonucleotide composition. In some embodiments, the multiple oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral-controlled nucleotide links have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% diastereomeric purity. In some embodiments, the chiral-controlled nucleotide links have at least 95% diastereomeric purity. In some embodiments, the chiral-controlled internucleotide linkages have at least 96% diastereomeric purity.In some embodiments, the chiral-controlled internucleotide linkage has at least 97% diastereomeric purity. In some embodiments, the chiral-controlled internucleotide linkage has at least 98% diastereomeric purity. In some embodiments, the chiral-controlled internucleotide linkage has at least 99% diastereomeric purity. In some embodiments, the percentage (e.g., at the level described herein) is or is at least (DS). nc DS is the diastereomeric purity as described in this disclosure (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or higher), and nc is the number of chiral-controlled internucleotide bonds as described in this disclosure (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, the percentage (e.g., the level as described herein) is or is at least (DS). nc Where DS is 95%-100%. For example, when DS is 99% and nc is 10, the percentage is 90% or at least 99%. 10≈ 0.90 = 90%). In some embodiments, the level of multiple oligonucleotides in the composition is expressed as the product of the diastereomeric purity of each chiral-controlled internucleotide linking strand in the oligonucleotide. In some embodiments, the diastereomeric purity of the internucleotide linking two nucleosides in an oligonucleotide (or nucleic acid) is expressed as the diastereomeric purity of the internucleotide linking a dimer linking the same two nucleosides, wherein comparable conditions are used, and in some cases, the same synthetic cycle conditions are used to prepare the dimer (e.g., for the linking of Nx and Ny in oligonucleotides ... NxNy ..., the dimer is NxNy). In some embodiments, not all chiral internucleotide links are chiral-controlled internucleotide links, and the composition is a partially chiral-controlled oligonucleotide composition. In some embodiments, as commonly observed in stereorandom oligonucleotide compositions (e.g., from conventional oligonucleotide synthesis, such as phosphoramidite methods, as understood by those skilled in the art), the non-chiral controlled internucleotide linkages have diastereomeric purities of less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%. In some embodiments, multiple oligonucleotides (or nucleic acids) have the same type. In some embodiments, the chiral controlled oligonucleotide composition comprises various oligonucleotide types or nucleic acid types at non-random or controlled levels. For example, in some embodiments, the chiral controlled oligonucleotide composition comprises one and no more than one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide composition is a composition of oligonucleotides of one oligonucleotide type comprising multiple oligonucleotides of that oligonucleotide type at non-random or controlled levels.

[0203] Nucleotide linkage: As used herein, the phrase “nucleotide linkage” generally refers to the linkage of nucleoside units of oligonucleotides or nucleic acids. In some embodiments, the nucleotide linkage is a phosphodiester linkage, as widely found in naturally occurring DNA and RNA molecules (the natural phosphodiester linkage (-OP(=O)(OH)O-), which, as understood by those skilled in the art, can exist in salt form). In some embodiments, the nucleotide linkage is a modified nucleotide linkage (not a natural phosphodiester linkage). In some embodiments, the nucleotide linkage is a “modified nucleotide linkage” in which at least one oxygen atom or -OH of the phosphodiester linkage is replaced by a different organic or inorganic moiety. In some embodiments, such an organic or inorganic moiety is selected from =S, =Se, =NR', –SR', –SeR', –N(R')2, B(R')3, –S–, –Se–, and –N(R')–, wherein each R' is independently as defined and described in this disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a thiophosphate linkage (or a thiophosphate diester linkage, i.e., -OP(=O)(SH)O-, which, as understood by those skilled in the art, may exist in salt form), or a thiophosphate triester linkage. In some embodiments, the modified internucleotide linkage is a thiophosphate linkage. In some embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) or PMO (diaminophosphate morpholino oligomer) linkage. In some embodiments, the modified internucleotide linkage is a negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage (e.g., n001 in some of the provided oligonucleotides). Those skilled in the art will understand that, due to the presence of an acidic or basic moiety in the linkage, the internucleotide linkage can exist as an anion or a cation at a given pH. In some embodiments, the modified nucleotide linkages are modified nucleotide linkages named s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, and s18, as described in WO 2017 / 210647.

[0204] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment (e.g., in a test tube or reaction vessel, in a cell culture, etc.) rather than in a living organism (e.g., an animal, plant, and / or microorganism).

[0205] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., an animal, plant, and / or microorganism).

[0206] Linked phosphorus: As defined herein, the phrase “linked phosphorus” is used to indicate that the specific phosphorus atom mentioned is a phosphorus atom present in an internucleotide link, corresponding to the phosphorus atom in a phosphodiester nucleotide link, such as those present in naturally occurring DNA and RNA. In some embodiments, the linked phosphorus atom is located in a modified internucleotide link, wherein each oxygen atom of the phosphodiester link is optionally and independently replaced by an organic or inorganic portion. In some embodiments, the linked phosphorus atom is P of Formula I as described herein. In some embodiments, the linked phosphorus atom is chiral. In some embodiments, the linked phosphorus atom is achiral (e.g., as in natural phosphate ester links).

[0207] Linker: The terms "linker," "connecting part," etc., refer to any chemical part that links one chemical part to another. As will be understood by those skilled in the art, a linker can be divalent, trivalent, or higher, depending on the number of chemical parts it links. In some embodiments, a linker is a part that links one oligonucleotide to another oligonucleotide in a polymer. In some embodiments, a linker is optionally located between a terminal nucleoside and a solid support or between a terminal nucleoside and another nucleoside, nucleotide, or nucleic acid. In some embodiments, in oligonucleotides, a linker links a chemical part (e.g., a targeting part, a lipid part, a carbohydrate part, etc.) to an oligonucleotide chain (e.g., via its 5' end, 3' end, nucleobase, sugar, internucleotide bond, etc.).

[0208] Modified nucleobases: The terms "modified nucleobases," "modified bases," etc., refer to a chemical portion that is chemically different from a nucleobase but capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase comprising a modification. In some embodiments, a modified nucleobase is capable of having at least one function of a nucleobase, for example, forming a portion in a polymer capable of pairing with a nucleic acid base comprising at least a complementary base sequence. In some embodiments, a modified nucleobase is a substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, in the context of oligonucleotides, a modified nucleobase refers to a nucleobase that is not A, T, C, G, or U.

[0209] Modified nucleosides: The term "modified nucleosides" refers to a moiety derived from or chemically similar to a natural nucleoside, but containing chemical modifications that distinguish it from a natural nucleoside. Non-limiting examples of modified nucleosides include those containing modifications at the bases and / or sugars. Non-limiting examples of modified nucleosides include those having a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include baseless nucleosides (which lack a nucleobase). In some embodiments, modified nucleosides are capable of having at least one function of a nucleoside, for example, forming a moiety in a polymer capable of pairing with nucleic acid bases comprising at least a complementary base sequence.

[0210] Modified nucleotides: The term "modified nucleotide" includes any chemical portion that differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, modified nucleotides comprise modifications at sugar, base, and / or internucleotide links. In some embodiments, modified nucleotides comprise modified sugars, modified nucleobases, and / or modified internucleotide links. In some embodiments, modified nucleotides are capable of having at least one function of a nucleotide, for example, forming subunits in polymers capable of pairing with nucleic acid bases comprising at least a complementary base sequence.

[0211] Modified sugars: The term "modified sugar" refers to a portion that can replace a sugar. Modified sugars mimic the spatial arrangement, electronic properties, or some other physicochemical properties of sugars. In some embodiments, as described in this disclosure, the modified sugar is a substituted ribose or deoxyribose. In some embodiments, the modified sugar contains a 2'-modification. Examples of useful 2'-modifications are widely used in the art and described herein. In some embodiments, the 2'-modification is 2'-OR, where R is an optionally substituted C 1-10 Aliphatic group. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the modified sugar is a bicyclic sugar (e.g., the sugar used in LNA, BNA, etc.). In some embodiments, in the case of oligonucleotides, the modified sugar is a sugar of ribose or deoxyribose that is not typically found in natural RNA or DNA.

[0212] Nucleic Acids: As used herein, the term “nucleic acid” includes any nucleotide and its polymers. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA) or combinations thereof) of any length. These terms refer to the primary structure of a molecule and include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include analogs of RNA or DNA as equivalents, which contain modified nucleotides and / or modified polynucleotides (such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides). These terms cover polynucleotides or oligonucleotides (RNA) and polydeoxynucleotides or oligodeoxynucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleotides and / or modified nucleotides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate ester bridges and / or modified internucleotide bonds. This term covers nucleic acids containing any combination of nucleosides, modified nucleosides, sugars, modified sugars, phosphate bridges, or modified internucleotide bonds. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both a ribose and a deoxyribose moiety, and nucleic acids containing both a ribose moiety and a modified ribose moiety. Unless otherwise specified, the prefix "poly-" refers to nucleic acids containing 2 to approximately 10,000 nucleotide monomer units, and the prefix "oligo-" refers to nucleic acids containing 2 to approximately 200 nucleotide monomer units.

[0213] Nucleotides: The term "nucleotide" refers to the portion of a nucleic acid involved in hydrogen bonding, which binds one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleotides are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, naturally occurring nucleotides are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, naturally occurring nucleotides are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, nucleotides comprise a heteroaryl ring, wherein the ring atom is nitrogen, and when in a nucleoside, the nitrogen is partially bonded to the sugar. In some embodiments, nucleotides comprise a heterocycle, wherein the ring atom is nitrogen, and when in a nucleoside, the nitrogen is partially bonded to the sugar. In some embodiments, the nucleotide is a "modified nucleotide," that is, a nucleotide other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleotide is a substituted A, T, C, G, or U. In some embodiments, the modified nucleotide is a substituted tautomer of A, T, C, G, or U. In some embodiments, the modified nucleotide is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleotide mimics the spatial arrangement, electronic properties, or some other physicochemical properties of nucleotides and retains the hydrogen-bonded, sequence-specific binding of one nucleic acid strand to another. In some embodiments, the modified nucleotide can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting unwinding behavior, recognition by intracellular enzymes, or activity of oligonucleotide duplexes. As used herein, the term "nucleobase" also encompasses structural analogs of naturally occurring nucleotides or nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, "nucleobase" refers to a nucleobase unit (e.g., A, T, C, G, or U) in an oligonucleotide or nucleic acid.

[0214] Nucleoside: The term "nucleoside" refers to the portion in which a nucleobase or a modified nucleobase is covalently bonded to a sugar or a modified sugar. In some embodiments, the nucleoside is a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

[0215] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more internucleotide bonds (e.g., phosphate ester bonds in native DNA and RNA). Naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, but it should be understood that naturally occurring and non-naturally occurring base analogues are also included. Naturally occurring sugars are pentose sugars, namely deoxyribose (which forms DNA) or ribose (which forms RNA), but it should be understood that naturally occurring and non-naturally occurring sugar analogues are also included. Nucleotides are linked by internucleotide bonds to form nucleic acids, or polynucleotides. Many internucleotide bonds are known in the art (e.g., but not limited to phosphate esters, thiophosphate esters, borane phosphate esters, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphate triesters, thiophosphates, H-phosphonates, aminophosphates, boron phosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, as described herein. In some embodiments, a natural nucleotide comprises naturally occurring bases, sugars, and nucleotide-to-nucleotide bonds. As used herein, the term "nucleotide" also encompasses structural analogs used in place of natural nucleotides or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, "nucleotide" refers to an oligonucleotide or a nucleotide unit in a nucleic acid.

[0216] Oligonucleotides: The term “oligonucleotide” refers to polymers or oligomers of nucleotides and can contain any combination of natural and non-natural nucleobases, sugars, and internucleotide bonds.

[0217] Oligonucleotides can be single-stranded or double-stranded. Single-stranded oligonucleotides can have double-stranded regions (formed by the two parts of a single-stranded oligonucleotide), and double-stranded oligonucleotides containing two oligonucleotide chains can have single-stranded regions, such as regions where the two oligonucleotide chains are not complementary to each other. Example oligonucleotides include, but are not limited to, structural genes, genes containing control and termination regions, self-replicating systems (such as viral DNA or plasmid DNA), single-stranded and double-stranded RNAi reagents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, ultramicroRNAs, aptamers, antimicroRNAs, microRNA antagonists, Ul adaptors, triple-stranded oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0218] The oligonucleotides disclosed herein can have various lengths. In specific embodiments, the length of the oligonucleotide can be from about 2 to about 200 nucleotides. In several related embodiments, the length of the (single-stranded, double-stranded, or triple-stranded) oligonucleotide can range from about 4 to about 10 nucleotides, from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, and from about 20 to about 30 nucleotides. In some embodiments, the length of the oligonucleotide is from about 9 to about 39 nucleotides. In some embodiments, the length of the oligonucleotide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the oligonucleotide is at least 19 nucleotides. In some embodiments, the length of the oligonucleotide is at least 20 nucleotides. In some embodiments, the length of the oligonucleotide is at least 25 nucleotides. In some embodiments, the length of the oligonucleotide is at least 30 nucleotides. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 18 nucleotides. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 21 nucleotides. In some embodiments, each nucleotide counted in the length of the oligonucleotide independently comprises A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or optionally substituted tautomers of A, T, C, G, or U.

[0219] Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define a specific base sequence, backbone linkage pattern (i.e., internucleotide linkage pattern, such as phosphate ester, thiophosphate ester, trithiophosphate ester, etc.), backbone chiral center pattern [i.e., phosphorus linkage stereochemistry pattern (Rp / Sp)], and backbone phosphorus modification pattern (e.g., “−XLR” in Formula I as described herein). 1Oligonucleotides of the "type" designation. In some embodiments, oligonucleotides of the common designated "type" are structurally identical to each other.

[0220] Those skilled in the art will understand that the synthetic methods of this disclosure provide a degree of control during the synthesis of oligonucleotide chains, allowing for the prior design and / or selection of each nucleotide unit of the oligonucleotide chain to have a specific stereochemistry at the phosphorus-linked site and / or to have a specific modification at the phosphorus-linked site, and / or to have a specific base, and / or to have a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the phosphorus-linked site. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the phosphorus-linked site. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of having one or more structural features. In some embodiments, this disclosure provides compositions comprising a plurality of oligonucleotide molecules or compositions thereof (e.g., chiral-controlled oligonucleotide compositions). In some embodiments, all such molecules belong to the same type (i.e., are structurally identical to each other). However, in some embodiments, the provided compositions comprise a plurality of oligonucleotides of different types (typically in predetermined relative amounts).

[0221] Optionally substituted: As described herein, compounds of this disclosure (e.g., oligonucleotides) may contain optionally substituted and / or substituted moieties. Generally, the term “substituted,” whether or not preceded by the term “optionally,” means that one or more hydrogens of the specified moieties are replaced by suitable substituents. Unless otherwise indicated, an “optionally substituted” group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure can be substituted by more than one substituent selected from the specified group. In some embodiments, the optionally substituted group is unsubstituted. Combinations of substituents contemplated in this disclosure are preferably combinations that result in the formation of stable or chemically viable compounds. As used herein, the term “stable” means that the compound does not undergo substantial change when subjected to conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for one or more purposes disclosed herein. Some substituents are described below.

[0222] A suitable monovalent substituent on a substituted atom (e.g., a suitable carbon atom) is independently a halogen; –(CH2) 0– 4R°;–(CH2) 0–4 OR°;−O(CH2) 0-4 R o –O–(CH2) 0–4C(O)OR°; –(CH2) 0–4 CH(OR°)2;–(CH2) 0–4 Ph, which can be replaced by R°; −(CH2) 0–4 O(CH2) 0–1 Ph, which can be substituted by R°; –CH=CHPh, which can be substituted by R°; –(CH2) 0–4 O(CH2) 0–1 -pyridyl group, which can be substituted by R°; –NO2; -CN; –N3; -(CH2) 0–4 N(R°)2;–(CH2) 0–4 N(R°)C(O)R°; –N(R°)C(S)R°; −(CH2) 0–4 N(R°)C(O)NR°2; −N(R°)C(S)NR°2; –(CH2) 0–4 N(R°)C(O)OR°; –N(R°)N(R°)C(O)R°; −N(R°)N(R°)C(O)NR°2; −N(R°)N(R°)C(O)OR°; –(CH2) 0–4 C(O)R°; –C(S)R°; –(CH2) 0–4 C(O)OR°;−(CH2) 0–4 C(O)SR°;-(CH2) 0–4 C(O)OSiR°3;–(CH2) 0–4 OC(O)R°;–OC(O)(CH2) 0– 4SR°, −SC(S)SR°; −(CH2) 0–4 SC(O)R°;–(CH2) 0–4 C(O)NR°2; –C(S)NR°2; –C(S)SR°; –(CH2) 0–4 OC(O)NR°2; -C(O)N(OR°)R°; –C(O)C(O)R°; –C(O)CH2C(O)R°; −C(NOR°)R°; -(CH2) 0–4 SSR°;–(CH2) 0–4 S(O)2R°;–(CH2) 0–4 S(O)₂OR°;–(CH₂) 0–4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0–4S(O)R°; –N(R°)S(O)2NR°2; –N(R°)S(O)2R°; –N(OR°)R°; −C(NH)NR°2; –Si(R°)3; –OSi(R°)3; −B(R°)2; −OB( R°)2; −OB(OR°)2; −P(R°)2; −P(OR°)2; −P(R°)(OR°); −OP(R°)2; −OP(OR°)2; −OP(R°)(OR°); −P(O)(R°)2; −P( O)(OR°)2; −OP(O)(R°)2; −OP(O)(OR°)2; −OP(O)(OR°)(SR°); −SP(O)(R°)2; −SP(O)(OR°)2; −N(R°)P(O)(R°) 2; −N(R°)P(O)(OR°)2; −P(R°)2[B(R°)3]; −P(OR°)2[B(R°)3]; −OP(R°)2[B(R°)3]; −OP(OR°)2[B(R°)3]; –(C 1–4 (straight-chain or branched alkylene)O–N(R°)2; or –(C 1–4 (linear or branched alkylene)C(O)O–N(R°)2, wherein each R° may be substituted as defined herein and independently be hydrogen, C 1–20 Aliphatic group, C having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus. 1–20 Heteroaliphatic group, −CH2−(C 6-14 Aryl), –O(CH2) 0–1 (C 6-14 aryl), −CH2-(5-14 membered heteroaryl ring), 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the above definition, two independently occurring R° together with the atoms between them to form a 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

[0223] Suitable monovalent substituents on R° (or a ring formed by two independently occurring R° and the atoms in between) are independently halogens, –(CH2). 0–2 R ● –(halogenated R) ● ), –(CH2) 0–2 OH, –(CH2) 0–2 OR ● –(CH2) 0–2 CH(OR ● )2;−O(halogenated R ●–CN, –N3, –(CH2) 0–2 C(O)R ● –(CH2) 0–2 C(O)OH, –(CH2) 0–2 C(O)OR ● –(CH2) 0–2 SR ● –(CH2) 0–2 SH, –(CH2) 0–2 NH2、–(CH2) 0–2 NHR ● –(CH2) 0–2 NR ● 2, –NO2, –SiR ● 3、−OSiR ● 3. -C(O)SR ● 、 –(C 1–4 (straight-chain or branched alkylene)C(O)OR ● Or –SSR ● , where each R ● None of them were replaced, or if "halogen" is preceding them, they were replaced by one or more halogens, and were independently selected from C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph and 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on the saturated carbon atom at R° include =O and =S.

[0224] For example, suitable divalent substituents on suitable carbon atoms are independently the following: =O, =S, =NNR. * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * 、 –O(C(R) * 2)) 2–3 O – or –S(C(R) * 2)) 2–3 S–, where each independently occurring R * Selected from: hydrogen, with C substituents as defined below. 1–6 Aliphatic groups, and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents bonded to the ortho-substituted carbon of the "optionally substituted" group include: –O(CR * 2) 2–3O–, where each independently occurring R * Selected from hydrogen, C can be substituted as defined below. 1–6 Aliphatic, and 5 to 6 saturated, partially unsaturated and aryl rings with 0 to 4 independently selected heteroatoms selected from nitrogen, oxygen and sulfur.

[0225] In R * Suitable substituents on the aliphatic group are independently halogens, -R ● -(halogenated R) ● –OH, –OR ● –O (halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R λ It is either unsubstituted or, in the case of being preceded by "halogenated," substituted with only one or more halogens, and is independently C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0226] In some embodiments, a suitable substituent on the nitrogen atom is independently –R † –NR † 2. –C(O)R † –C(O)OR † –C(O)C(O)R † –C(O)CH2C(O)R † –S(O)2R † 、−S(O)2NR † 2. –C(S)NR † 2. –C(NH)NR † 2 or –N(R) † )S(O)2R † ; where each R † Independently, it is hydrogen, which can be defined as a substituted C-molecule as follows. 1–6 Aliphatic group, unsubstituted –OPh or a 5–6 membered saturated ring, partially unsaturated ring or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or, despite the above definition, two independently occurring R groups. † Together with one or more atoms in between, they form unsubstituted 3–12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic rings with 0–4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0227] R† Suitable substituents on the aliphatic group are independently halogens, -R ● -(halogenated R) ● –OH, –OR ● –O (halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ● It is either unsubstituted or, in the case of being preceded by "halogenated," substituted with only one or more halogens, and is independently C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0228] Oral administration: As used herein, the phrases “oral administration” and “administered orally” have the meaning as understood in the art, referring to the oral administration of a compound or composition.

[0229] Parenteral administration: As used herein, the phrases “parenteral administration” and “administered parenterally” have the meaning as understood in the art, referring to a mode of administration other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0230] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. As defined herein, the term “partially unsaturated” is intended to cover rings having multiple unsaturated sites, but not to include aryl or heteroaryl moiety.

[0231] Pharmaceutical Composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen, showing a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual, and systemic absorption), pills, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension or sustained-release formulation; topical administration, such as as a cream, ointment, sustained-release patch, or spray applied to the skin, lungs, or mouth; vaginal or rectal administration, such as as a suppository, cream, or foam; sublingual; ocular; transdermal; or nasal, pulmonary, and other mucosal surfaces.

[0232] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” means, to a reasonable extent of medical judgment, compounds, materials, compositions and / or dosage forms that are suitable for use in human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0233] Pharmaceutically acceptable carriers: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or mediator, such as a liquid or solid filler, diluent, excipient, or solvent-encapsulating material, which participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and is harmless to the patient. Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; astragalus powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0234] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salt" means a salt of such compounds suitable for use in a pharmaceutical context, i.e., a salt suitable for contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and commensurate with a reasonable benefit / risk ratio, within reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are amino-containing salts formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disaccharide, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. In some embodiments, the provided compound (e.g., an oligonucleotide) comprises one or more acidic groups, and the pharmaceutically acceptable salt is an alkali metal salt, an alkaline earth metal salt, or an ammonium salt (e.g., an ammonium salt of N(R)3, wherein each R is independently defined and described in this disclosure). Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, the pharmaceutically acceptable salt suitably includes non-toxic ammonium, quaternary ammonium, and amine cations formed using balancing ions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, alkyl groups having 1 to 6 carbon atoms, sulfonates, and arylsulfonates.In some embodiments, the provided compounds comprise more than one acidic group; for example, oligonucleotides may comprise two or more acidic groups (e.g., natural phosphate ester linkages and / or modified nucleotide linkages). In some embodiments, pharmaceutically acceptable salts (or generally, salts) of such compounds comprise two or more cations that may be identical or different. In some embodiments, in pharmaceutically acceptable salts (or generally, salts), all ionizable hydrogen atoms in the acidic groups (e.g., in aqueous solutions with pKa not exceeding about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, not exceeding about 7; in some embodiments, not exceeding about 6; in some embodiments, not exceeding about 5; in some embodiments, not exceeding about 4; in some embodiments, not exceeding about 3) are replaced by cations. In some embodiments, each thiophosphate and phosphate ester group exists independently in its salt form (e.g., if a sodium salt, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In some embodiments, each thiophosphate and phosphate ester nucleotide linker exists independently in its salt form (e.g., -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively, if sodium salts). In some embodiments, pharmaceutically acceptable salts are sodium salts of oligonucleotides. In some embodiments, pharmaceutically acceptable salts are sodium salts of oligonucleotides, wherein each acidic phosphate and modified phosphate ester group (e.g., thiophosphate, phosphate, etc.) (if any) exists in salt form (all in sodium salt form).

[0235] Protecting Groups: As used herein, the term “protecting group” refers to those well-known in the art and included in detail in *Protecting Groups in Organic Synthesis*, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999 (the entire contents of which are incorporated herein by reference). Also included are those protecting groups described below that are particularly applicable to nucleoside and nucleotide chemistry: *Current Protocols in Nucleic Acid Chemistry*, edited by Serge L. Beaucage et al., 06 / 2012, Chapter 2 (the entire contents of which are incorporated herein by reference). Suitable amino protecting groups include, but are not limited to, those described herein and / or hereinafter: WO 2018 / 022473, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 223073, WO2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951 and / or WO 2019 / 075357 or U.S. Provisional Patent Applications 62 / 825766 and 62 / 911339, the description of the protecting group of each of which is independently incorporated herein by reference.

[0236] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from or derived from a source intended for use. In some embodiments, the source intended for use is a biological or environmental source. In some embodiments, the source intended for use may be or comprises cells or organisms such as microorganisms, plants, or animals (e.g., humans). In some embodiments, the source intended for use is or comprises biological tissues or body fluids. In some embodiments, biological tissues or body fluids may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chyme, prostatic fluid, endolymph, exudate, excretion, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum, semen, serum, smegma, phlegm, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomitus, and / or combinations or one or more components thereof. In some embodiments, the biological fluid may be or comprise intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cell permeation fluid. In some embodiments, the biological fluid may be or comprise plant exudate. In some embodiments, biological tissues or samples may be obtained, for example, by aspiration, biopsy (e.g., fine-needle or tissue biopsy), swabs (e.g., oral, nasal, skin, or vaginal swabs), scraping, surgery, washing, or irrigation (e.g., bronchoalveolar, catheter, nasal, eye, oral, uterine, vaginal, or other washing or irrigation). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a “primary sample” obtained directly from the intended source by any suitable method. In some embodiments, as the context will clearly show, the term “sample” refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, filtration using a semi-permeable membrane. Such “processed samples” may contain, for example, nucleic acids or proteins, which are extracted from the sample or obtained by subjecting the primary sample to one or more techniques such as nucleic acid amplification or reverse transcription, separation and / or purification of certain components.

[0237] Subject: As used herein, the term "subject" or "test subject" means, for example, any organism that administers the provided compound (e.g., the provided oligonucleotide) or composition according to this disclosure for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject is a human. In some embodiments, the subject may have and / or be susceptible to diseases, disorders, and / or conditions.

[0238] Essentially: As used herein, the term “essentially” refers to a qualitative condition that exhibits all or nearly all of the range or extent of the feature or property of interest. A base sequence substantially complementary to the second sequence is not identical to the second sequence, but is substantially identical or nearly identical to it. Furthermore, those skilled in the art of biology and / or chemistry will understand that biological and / or chemical phenomena, if any, rarely reach completion and / or proceed to completion or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to capture the potential incompleteness inherent in many biological and / or chemical phenomena.

[0239] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in a closed and / or open form. In some embodiments, a sugar is a monosaccharide. In some embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexapyranose moieties. As used herein, the term "sugar" also encompasses structural analogs used to replace conventional sugar molecules, such as diols, polymers forming the backbone of nucleic acid analogs, diol nucleic acids ("GNAs"), etc. As used herein, the term "sugar" also encompasses structural analogs used to replace natural nucleotides or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, a sugar is a modified ribose or deoxyribose, for example, 2'-modified, 5'-modified, etc. As described herein, in some embodiments, when used with oligonucleotides and / or nucleic acids, modified sugars can provide one or more desired properties, activities, etc. In some embodiments, a sugar is an optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to a sugar unit in an oligonucleotide or nucleic acid.

[0240] Susceptible: An individual "susceptible" to a disease, disorder, and / or condition is an individual who has a higher risk of developing the disease, disorder, and / or condition than the general public. In some embodiments, an individual susceptible to a disease, disorder, and / or condition has a predisposition to develop the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may not be diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0241] Therapeutic agent: As used herein, the term "therapeutic agent" generally refers to any agent that, when administered to a subject, elicits a desired effect (e.g., a desired biological, clinical, or pharmaceutical effect). In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a group of subjects who have and / or are susceptible to a disease, disorder, or condition. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population may be defined by one or more criteria, such as age group, sex, genetic background, or pre-existing clinical condition prior to receiving the therapy. In some embodiments, when administered to a subject in an effective amount, a therapeutic agent is a substance that reduces, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition in the subject. In some embodiments, a "therapeutic agent" is an agent that has been or requires approval by a government agency before it can be marketed and administered to humans. In some embodiments, a "therapeutic agent" is an agent that requires a prescription for administration to humans. In some embodiments, the therapeutic agent is a provided compound, such as a provided oligonucleotide.

[0242] Therapeutic Effective Amount: As used herein, the term "therapeutic effective amount" means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject who has or is susceptible to such a disease, disorder, and / or condition. As will be understood by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, and the target cells or tissues. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, reduces, inhibits, prevents, delays, reduces, and / or reduces the incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0243] Treatment: As used herein, the term "treatment" means any method used to partially or completely relieve, improve, reduce, suppress, prevent, delay the onset of, reduce the severity of, and / or decrease the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who only exhibit early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing a pathology associated with the disease, disorder, and / or condition.

[0244] Unsaturated: As used herein, the term “unsaturated” means a portion having one or more unsaturated units.

[0245] Wild-type: As used herein, the term "wild-type" has its meaning as understood in the art, referring to an entity having the structure and / or activity found in nature in a "normal" state or background (as opposed to mutants, diseased, altered, etc.). Those skilled in the art will understand that wild-type genes and polypeptides typically exist in many different forms (e.g., alleles).

[0246] As those skilled in the art will understand, the methods and compositions described herein involving the provided compounds (e.g., ds oligonucleotides) are generally also applicable to pharmaceutically acceptable salts of such compounds.

[0247] Description of Certain Embodiments

[0248] Double-stranded oligonucleotides are useful tools for a variety of applications. For example, ds oligonucleotides targeting INHBE (e.g., NCBI gene ID: 345275 for human INHBE and related sequences from other organisms) can be used for therapeutic, diagnostic, and research applications, including the treatment of various INHBE-related conditions, disorders, and diseases, including but not limited to metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension). The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. Thus, a variety of synthetic counterparts have been developed to circumvent these drawbacks and / or further improve various properties and activities. These synthetic counterparts include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., which in particular make these molecules less susceptible to degradation and improve other properties and / or activities of the oligonucleotides. From a structural perspective, modifications to the internucleotide bonds can introduce chirality and / or change charge, and certain properties may be affected by the configuration of the linking phosphorus atoms of the oligonucleotide. For example, the chirality and / or charge of backbone-linked atoms particularly affect binding affinity, sequence-specific binding to complementary RNA, stability to nucleases, cleavage of target nucleic acids, delivery, and pharmacokinetics.

[0249] In some embodiments, the dsRNAi agent is capable of directing INHBE (inhibin subunit βE)-specific RNA interference to induce lipolysis while preserving muscle mass. This dsRNAi agent comprises a guide strand and a transit strand, wherein:

[0250] c) The guide strand is complementary to or substantially complementary to the INHBE target RNA sequence;

[0251] d) The bootstrap chain contains:

[0252] i. Sp-configured uncharged nucleotide linkage between the +3 nucleotide relative to the 5' terminal nucleotide and the immediately adjacent downstream (+4) nucleotide;

[0253] ii. An Rp-configured, uncharged nucleotide bond between a +10 nucleotide and its immediate downstream (+11) nucleotide;

[0254] iii. Sp-configured phosphate ester nucleotide linkages between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide; and / or

[0255] iv. Linkage between phosphate thioester nucleotides in Rp, Sp, or alternating configurations between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide;

[0256] c) The leading chain further includes 5' phosphate modification;

[0257] d) The transit chain contains one or more chiral nucleotides in the Rp or Sp configuration linked together; and

[0258] e) The guide strand and the guest strand each have an independent length of 15-49 nucleotides.

[0259] In alternative embodiments, the ds oligonucleotide targeting INHBE (e.g., dsRNAi agents) comprises one or more of the following:

[0260] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0261] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0262] (3) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0263] (4) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0264] (5) A leading chain comprising one or more skeletal phosphate chiral centers, the skeletal phosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal phosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal phosphate chiral center is in an Rp or Sp configuration;

[0265] (6) The guide chain contains one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the immediately adjacent upstream (N-3) nucleotide (i.e. in the 5' direction);

[0266] (7) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification;

[0267] The ds oligonucleotide further comprises one or more of the following:

[0268] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0269] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0270] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0271] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0272] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0273] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker. For example, a 2' F modification. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0274] In some embodiments, the ds oligonucleotide targeting INHBE comprises: (1) a phosphate thioester chiral center in the form of Rp or Sp; (2) an Rp, Sp, or stereorandom uncharged internucleotide linker, wherein the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged internucleotide linker contains a 2' modification, such as 2' F; and (3) a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification, selected from the following:

[0275] (a) 5' PO modification, for example but not limited to:

[0276] ;

[0277] (b) 5' VP modification, for example but not limited to:

[0278] ;

[0279] (c) 5' MeP modification, for example, but not limited to:

[0280] ;

[0281] (d) 5'PN and 5'triazole-P modifications, such as, but not limited to:

[0282] ;

[0283] The bases are selected from A, C, G, T, U, no bases, and modified nucleobases;

[0284] R 2' Selected from H, OH, O-alkyl, F, MOE, locked nucleic acid (LNA) bridges and bridging nucleic acid (BNA) bridges to 4' C, for example, but not limited to:

[0285] In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides incorporated into the guide chain are Rp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are Sp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are stereorandom uncharged nucleotide linkages. In some embodiments, the guide chain comprises a Sp-configured skeletal phosphate thioester chiral center between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (i.e., in the 3' direction) (+2) nucleotide, and an Rp-configured skeletal phosphate thioester chiral center between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide.

[0286] In some embodiments, the leader chain includes a 5' terminal modification, such as a 5' phosphate modification, or a 5' triazole phosphate modification, selected from, but not limited to, 5' MeP and 5' triazole-P modifications. In some embodiments, the leader chain includes a 5' MeP modification. The 5' end (+1) nucleotide has a Sp-configured skeletal phosphate chiral center between the adjacent downstream (+2) nucleotide (i.e., in the 3' direction), and the +2 nucleotide has an Rp-configured skeletal phosphate chiral center between the adjacent downstream (+3) nucleotide.

[0287] In some embodiments, the ds oligonucleotide targeting INHBE comprises: (1) a phosphate thioester chiral center in the form of Rp or Sp; (2) an Rp, Sp, or stereorandom uncharged internucleotide linker, wherein the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged internucleotide linker contains a 2' modification, such as 2' F; and (3) a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification, selected from the following:

[0288] (a) 5' PO nucleotides, such as, but not limited to:

[0289] ;

[0290] (b) 5' VP nucleotides, such as, but not limited to:

[0291] ;

[0292] (c) 5' MeP nucleotides, such as, but not limited to:

[0293] ;

[0294] (d) 5' PN and 5' triazole-P nucleotides, such as, but not limited to:

[0295] , and ;

[0296] (e) 5'-free VP and 5'-free MeP nucleotides, such as, but not limited to:

[0297] and In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides incorporated into the leader chain are Rp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are Sp uncharged nucleotide linkages. In some embodiments, the internucleotide linkages of one or more Rp, Sp, or stereorandom uncharged nucleotides are stereorandom uncharged nucleotide linkages.

[0298] In some embodiments, the ds oligonucleotide targeting INHBE comprises one or more of the following:

[0299] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0300] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0301] (3) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0302] (4) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0303] (5) A leading chain comprising one or more skeletal phosphate chiral centers, the skeletal phosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal phosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal phosphate chiral center is in an Rp or Sp configuration;

[0304] (6) The guide chain contains one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the immediately adjacent upstream (N-3) nucleotide (i.e. in the 5' direction);

[0305] (7) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification;

[0306] The ds oligonucleotide further comprises one or more of the following:

[0307] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0308] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0309] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0310] (4) A transit chain in which one or more Rp, Sp or stereorandom non-negatively charged nucleotides are bonded upstream of the central nucleotide of the transit chain, i.e. in the 5' direction.

[0311] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0312] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker. For example, a 2' F modification. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0313] In some embodiments, the ds oligonucleotide targeting INHBE comprises non-naturally occurring internucleotide links, such as neutral internucleotide links, which in some embodiments can be used to link one or more molecules to the double-stranded oligonucleotide described herein. In some embodiments, such linked molecules can facilitate the targeting and / or delivery of the double-stranded oligonucleotide. For example, but not limited to, such linked molecules include lipophilic molecules. In some embodiments, the linked molecule is a molecule containing one or more GalNac moieties. In some embodiments, the linked molecule is a receptor. In some embodiments, the linked molecule is a receptor ligand.

[0314] In some embodiments, this disclosure provides techniques (e.g., compounds, methods, etc.) for improving the stability of oligonucleotides while maintaining or enhancing their activity, including compositions of oligonucleotides with improved stability.

[0315] In some embodiments, this disclosure provides techniques for incorporating a variety of additional chemical moieties into ds oligonucleotides. In some embodiments, this disclosure provides reagents and methods, for example, for introducing additional chemical moieties via nucleobases (e.g., via sites optionally covalently linked to nucleobases via linkers).

[0316] In some embodiments, this disclosure provides techniques for achieving allele-specific repression, such as ds oligonucleotide compositions and methods thereof, wherein a transcript of an allele of a specific target gene is selectively knocked down relative to at least another allele of the same gene.

[0317] Among other things, this disclosure provides structural elements, techniques, and / or features that can be incorporated into ds oligonucleotides and impart or modulate one or more properties thereto (e.g., relative to other identical ds oligonucleotides lacking the relevant techniques or features). In some embodiments, this disclosure demonstrates that one or more of the provided techniques and / or features can be usefully incorporated into ds oligonucleotides of various sequences.

[0318] In some embodiments, this disclosure demonstrates that certain structural elements, techniques, and / or features provided are particularly useful for ds oligonucleotides that participate in and / or guide RNAi mechanisms (e.g., RNAi agents). However, in any case, the teachings of this disclosure are not limited to ds oligonucleotides that participate in or act via any particular biochemical mechanism.

[0319] In some embodiments, this disclosure relates to any ds oligonucleotide that can be used for any purpose, works through any mechanism, and comprises any sequence, structure, or form (or part thereof) described herein.

[0320] In some embodiments, the ds oligonucleotide targeting INHBE comprises a guide chain consisting of an internucleotide linking a Sp-configured backbone of a +3 nucleotide to an immediately adjacent downstream (+4) nucleotide (i.e., in the 3' direction) without a negative charge; and one or more of the following:

[0321] (1) The guide chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides linked between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e., in the 3' direction);

[0322] (2) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0323] (3) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0324] (4) A leading chain comprising one or more skeletal thiophosphate chiral centers, the skeletal thiophosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal thiophosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal thiophosphate chiral center is in an Rp or Sp configuration;

[0325] (5) A guide chain comprising one or more chiral phosphate thioester backbones in the Rp or Sp configuration between an N-2 nucleotide and an adjacent upstream (N-3) nucleotide (i.e., in the 5' direction); (1) A guide chain comprising an inter-nucleotide link in the Sp configuration between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e., in the 3' direction).

[0326] (6) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification.

[0327] In some embodiments, the ds oligonucleotide targeting INHBE comprises a guide chain consisting of an Rp-configured backbone of a non-negatively charged nucleotide linking a +10 nucleotide to an immediately adjacent downstream (+11) nucleotide (i.e., in the 3' direction); and one or more of the following:

[0328] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0329] (2) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0330] (3) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0331] (4) A leading chain comprising one or more skeletal thiophosphate chiral centers, the skeletal thiophosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal thiophosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal thiophosphate chiral center is in an Rp or Sp configuration;

[0332] (5) The guide chain contains one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the adjacent upstream (N-3) nucleotide (i.e. in the 5' direction);

[0333] (6) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification.

[0334] In some embodiments, the ds oligonucleotide targeting INHBE comprises a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide, and one or more of the following:

[0335] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0336] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0337] (3) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0338] (4) A leading chain comprising one or more skeletal thiophosphate chiral centers, the skeletal thiophosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal thiophosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal thiophosphate chiral center is in an Rp or Sp configuration;

[0339] (5) The guide chain contains one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the adjacent upstream (N-3) nucleotide (i.e. in the 5' direction);

[0340] (6) A leading chain containing a 5' end modification, such as a 5' phosphate modification, such as a 5' triazole phosphate modification;

[0341] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0342] In some embodiments, the ds oligonucleotide targeting INHBE comprises a skeletal phosphate thioester chiral center in an Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide, and one or more of the following:

[0343] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0344] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0345] (3) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0346] (4) A leading chain comprising one or more skeletal thiophosphate chiral centers, the skeletal thiophosphate chiral centers being upstream (i.e. in the 5' direction) of a Sp-configured skeletal thiophosphate chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, wherein the upstream skeletal thiophosphate chiral center is in an Rp or Sp configuration;

[0347] (5) A guide chain comprising one or more chiral phosphate thioester backbones in the Rp or Sp configuration between the N-2 nucleotide and the immediately preceding upstream (N-3) nucleotide (i.e., in the 5' direction), and

[0348] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0349] In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE comprising one or more chiral phosphate thioester backbones in an Rp or Sp configuration, the chiral phosphate thioester backbones being upstream of the Sp configuration chiral phosphate thioester backbone between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately preceding upstream (N-2) nucleotide, and one or more of the following:

[0350] (1) The leading chain, which consists of an Sp-configured backbone of non-negatively charged nucleotides between a +3 nucleotide and an adjacent downstream (+4) nucleotide (i.e. in the 3' direction);

[0351] (2) The leading chain, which consists of an Rp-configured backbone of non-negatively charged nucleotides bonded between a +10 nucleotide and an adjacent downstream (+11) nucleotide (i.e. in the 3' direction);

[0352] (3) The leading strand contains a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide in the 5' direction.

[0353] (4) The leading strand contains a skeletal phosphate chiral center in Rp, Sp or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide in the 3' direction, and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0354] (5) A guide strand comprising one or more chiral phosphate thioester centers of Rp or Sp configuration between the N-2 nucleotide and the immediately preceding upstream (N-3) nucleotide (i.e., in the 5' direction); and

[0355] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0356] In some embodiments, the guide chain comprises one or more sp-uncharged nucleotide bonds occurring between a +3 nucleotide and an immediately adjacent downstream (+4) nucleotide (i.e., in the 3' direction), and one or more of the following:

[0357] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0358] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0359] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0360] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0361] (5) A passer chain in which one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked downstream of the central nucleotide of the passer chain, i.e. in the 3' direction.

[0362] In some embodiments, the guide chain comprises one or more Rp-neutral nucleotide bonds occurring between a +10 nucleotide and an immediately adjacent downstream (+11) nucleotide (i.e., in the 3' direction), and one or more of the following:

[0363] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0364] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0365] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0366] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0367] (5) A passer chain in which one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked downstream of the central nucleotide of the passer chain, i.e. in the 3' direction.

[0368] In some embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom uncharged nucleotide links between the second (+2) and third (+3) nucleotides of the guide strand relative to the 5' terminal nucleotide, and an internucleotide link to the penultimate 3' (N-1) nucleotide, and one or more of the following:

[0369] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0370] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0371] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0372] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0373] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0374] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises 0-n Rp, Sp, or stereorandom uncharged nucleotide links, where n is about 1 to 49. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader chain or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0375] In some embodiments, the guide chain comprises a sp-configured phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide, and one or more of the following:

[0376] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0377] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0378] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0379] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0380] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0381] The ds oligonucleotide further comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, such as a 2' F modification, and the guest chain comprises one or more skeletal phosphate thioester chiral centers in the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0382] In some embodiments, the guide chain comprises a skeletal phosphate chiral center in an Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide, and one or more of the following:

[0383] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0384] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0385] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0386] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0387] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0388] The ds oligonucleotide further comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged internucleotide linker, and the guest chain comprises one or more skeletal chiral centers of the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links incorporated into the leader or guest chain are Rp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links are Sp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links are stereorandom uncharged internucleotide links. In some embodiments disclosed herein, the skeletal phosphate ester chiral center is in the Rp configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide. In some embodiments described herein, the chiral center of the phosphate thioester backbone is in an Sp configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide. In some embodiments, the chiral center of the phosphate thioester backbone is in an Rp and Sp configuration, respectively, between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide. In some embodiments described herein, the chiral center of the phosphate thioester backbone is in an Sp and Rp configuration, respectively, between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide.

[0389] In some embodiments, the guide chain comprises one or more skeletal phosphate thioester chiral centers of Rp or Sp configuration upstream of the Sp-configured skeletal chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding upstream (N-2) nucleotide, and one or more of the following:

[0390] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0391] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0392] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0393] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0394] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0395] The ds oligonucleotide further comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, and the guest chain comprises one or more skeletal chiral centers of the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0396] In some embodiments, the guide chain comprises one or more skeletal phosphate thioester chiral centers of Rp or Sp configuration, said skeletal phosphate thioester chiral centers being located between a 5' terminal (+1) nucleotide and an immediately adjacent downstream (+2) nucleotide, and between a (+2) nucleotide and an immediately adjacent downstream (+3) nucleotide, and between one or both of the following: (a) a (+3) nucleotide and a (+4) nucleotide; and (b) a (+5) nucleotide and a (+6) nucleotide, and one or more of the following:

[0397] (1) A guide chain in which one or both of the 5' and 3' terminal dinucleotides are not connected by a non-negatively charged internucleotide link, i.e., the guide chain contains one or more non-negatively charged internucleotide links downstream (i.e. in the 3' direction) of the link between the 5' terminal dinucleotides and / or upstream (i.e. in the 5' direction) of the link between the 3' terminal dinucleotides.

[0398] (2) A guide strand, wherein one or more Rp, Sp or stereorandom non-negatively charged nucleotides are linked between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

[0399] (3) The leader chain, wherein the internucleotide bonding of Rp, Sp or stereorandom non-negatively charged nucleotides occurs between the third (+3) and fourth (+4) nucleotides of the leader chain relative to the 5' terminal nucleotide and / or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide;

[0400] (4) A transit chain in which one or more Rp, Sp, or stereorandomly neutral nucleotides are linked upstream of the central nucleotide of the transit chain, i.e., in the 5' direction; and

[0401] (5) A transient chain in which one or more Rp, Sp, or stereorandom uncharged nucleotides are linked downstream, i.e., in the 3' direction relative to the central nucleotide of the transient chain, and

[0402] The ds oligonucleotide further comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom uncharged nucleotide linker, and the guest chain comprises one or more skeletal chiral centers of the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the leader or guest chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are stereorandom uncharged nucleotide links.

[0403] In some embodiments, the leader chain comprises one or more Rp, Sp, or stereorandom uncharged nucleotide interlinkings between any two adjacent nucleotides between the second (+2) nucleotide of the leader chain relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the leader chain, wherein N is the 3' terminal nucleotide, the leader chain comprises a 2' modification, such as a 2' F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged nucleotide interlinking, and the guest chain comprises one or more skeletal chiral centers in the Rp or Sp configuration.

[0404] In some embodiments, the guide chain contains a skeletal phosphate thioester chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide. The guide chain contains a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotide linker. The guest chain contains 0 to n Rp, Sp, or stereorandom non-negatively charged internucleotide links (where n is about 1 to 49) and one or more skeletal chiral centers in the Rp or Sp configuration.

[0405] In some embodiments, the guide chain includes a skeletal phosphate thioester chiral center in Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide. The guide chain includes a 2' modification, such as a 2' F modification, of the 3' nucleotide of nucleotide pairs linked by Rp, Sp, or stereorandom uncharged internucleotide links. The guest chain includes 0-n Rp, Sp, or stereorandom uncharged internucleotide links (where n is about 1 to 49) and one or more skeletal chiral centers in Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links incorporated into the guide chain or guest chain are Rp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged internucleotide links are Sp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are stereorandom uncharged nucleotide linkages.

[0406] In some embodiments, the guide chain contains one or more chiral phosphate thioester backbones with an Rp or Sp configuration upstream of the Sp-configured skeletal phosphate thioester chiral center between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding upstream (N-2) nucleotide. The guide chain contains a 2' modification, such as a 2' F modification, of the 3' nucleotide of nucleotide pairs linked by Rp, Sp, or stereorandom uncharged internucleotide links. The guest chain contains 0-n Rp, Sp, or stereorandom uncharged internucleotide links (where n is about 1 to 49) and one or more chiral backbones with an Rp or Sp configuration. In some embodiments, the one or more Rp, Sp, or stereorandom uncharged internucleotide links incorporated into the guide chain are Rp uncharged internucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are Sp uncharged nucleotide linkages.

[0407] In some embodiments, the guide chain contains one or more Rp, Sp, or stereorandom uncharged nucleotide links between any two adjacent nucleotides between the second (+2) nucleotide of the guide chain relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide chain, where N is the 3' terminal nucleotide. The guide chain contains a 2' modification, such as a 2' F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or stereorandom uncharged nucleotide link. The guest chain contains 0-n Rp, Sp, or stereorandom uncharged nucleotide links (where n is about 1 to 49) and one or more skeletal chiral centers in the Rp or Sp configuration. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links incorporated into the guide chain are Rp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide links are Sp uncharged nucleotide links. In some embodiments, one or more Rp, Sp, or stereorandom uncharged nucleotide linkages are stereorandom uncharged nucleotide linkages.

[0408] In some embodiments, the internucleotide linkages of the oligonucleotide comprise or consist of 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chiral-controlled internucleotide linkages. In some embodiments, this disclosure provides dsRNAi oligonucleotide compositions wherein the dsRNAi oligonucleotide comprises at least one chiral-controlled internucleotide linkage. In some embodiments, this disclosure provides dsRNAi oligonucleotide compositions wherein the dsRNAi oligonucleotide is stereorandom or chiral-controlled. In some embodiments, in the dsRNAi oligonucleotide, at least one internucleotide linkage is stereorandom, and at least one internucleotide linkage is chiral-controlled.

[0409] In some embodiments, the internucleotide linkages of the oligonucleotides comprise or consist of one or more electrically neutral internucleotide linkages.

[0410] INHBE

[0411] In some embodiments, INHBE refers to a gene or its gene product (including, but not limited to, nucleic acids, including but not limited to, DNA or RNA, transcripts, and proteins encoded therefrom) from any species; it may be derived from any form of INHBE, such as wild-type or mutant alleles. In some embodiments, it refers to a gene and its product in humans. In some embodiments, it refers to a gene and its product in non-human primates. Various INHBE sequences, including their variants, from humans, mice, rats, monkeys, etc., are readily available to those skilled in the art. In some embodiments, INHBE is a human or mouse INHBE, which is wild-type or mutant. INHBEs have been reported to have many functions. Various techniques, such as assays, cells, animal models, etc., have also been reported and can be used to characterize and / or evaluate the provided techniques (e.g., oligonucleotides, compositions, methods, etc.) according to this disclosure.

[0412] In some embodiments, the INHBE gene, transcript (e.g., pre- or post-splicing mRNA), or protein variant or isotype contains a mutation. In some embodiments, the INHBE gene, transcript, or protein is a transcribed or translated product of a variant or isotype of the spliced ​​protein.

[0413] INHBE-Related Condition, Disorder, or Disease

[0414] Various conditions, disorders, or diseases have been reported to be associated with INHBE. Generally, a disease, disorder, or disease is associated with INHBE if the presence, level, activity, and / or form of INHBE and / or its products (e.g., transcripts, encoded proteins, etc.) are associated with the incidence and / or susceptibility to the disease, disorder, or disease (e.g., in relevant populations). In some embodiments, conditions, disorders, or diseases associated with INHBE can be treated and / or prevented by reducing the expression, level, and / or activity of INHBE transcripts and / or proteins.

[0415] Various conditions, disorders, or diseases associated with INHBE are reported. In some embodiments, INHBE-related conditions, disorders, or diseases are metabolic syndromes such as heart disease, type 2 and type 1 diabetes, kidney disease, and obesity. In some embodiments, INHBE-related conditions, disorders, or diseases are type 2 diabetes. In some embodiments, INHBE-related conditions, disorders, or diseases are obesity.

[0416] Among other things, the provided techniques may be used to treat or prevent conditions, disorders, or diseases associated with INHBE, including but not limited to metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension). In some embodiments, this disclosure relates to the use of ds oligonucleotides or compositions thereof targeting INHBE in the treatment of INHBE-related disorders, diseases, or conditions, including but not limited to metabolic disorders (e.g., metabolic syndrome) and related diseases (e.g., obesity, cardiovascular disease, diabetes, and hypertension).

[0417] In some embodiments, treatment or prevention using the provided technology reduces the rate of INHBE production and reduces, stops, or reverses INHBE accumulation. In some embodiments, treatment or prevention using the provided technology increases the rate of weight loss or otherwise allows for weight control.

[0418] As will be understood by those skilled in the art, the mechanisms, genotypes, symptoms, biomarkers, etc. of such conditions, disorders, or diseases can be utilized in accordance with this disclosure to characterize / evaluate the provided technology.

[0419] Double-Stranded Oligonucleotide

[0420] Among other things, this disclosure provides ds oligonucleotides with various designs that may comprise various nucleobases and their patterns, sugars and their patterns, nucleotide linkages and their patterns, and / or additional chemical moieties and their patterns as described in this disclosure. In some embodiments, the provided ds oligonucleotides targeting INHBE can direct a reduction in the expression, level, and / or activity of one or more of the INHBE gene and / or its products (e.g., transcripts, mRNA, proteins, etc.). In some embodiments, the provided ds oligonucleotides targeting INHBE can direct a reduction in the expression, level, and / or activity of one or more of the INHBE gene and / or its products in the cells of a subject or patient. In some embodiments, cells normally express INHBE or produce INHBE protein. In some embodiments, the provided ds oligonucleotide targeting INHBE can direct the reduction of expression, level, and / or activity of INHBE target genes or gene products, and has the following base sequence, which consists of the base sequence of the ds oligonucleotide targeting INHBE disclosed herein, contains the base sequence of the ds oligonucleotide targeting INHBE disclosed herein, or contains a portion of the base sequence of the ds oligonucleotide targeting INHBE disclosed herein (e.g., a sequence segment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive bases), wherein each T can be independently substituted with U, and vice versa, and the ds oligonucleotide contains at least one non-naturally occurring modification of bases, sugars, and / or internucleotide bonds.

[0421] In some embodiments, ds oligonucleotides targeting INHBE can direct a reduction in the expression, level, and / or activity of a target gene (e.g., an INHBE target gene) or its product. In some embodiments, ds oligonucleotides targeting INHBE can direct a reduction in the expression, level, and / or activity of an INHBE target gene or its product via RNase H-mediated knockdown. In some embodiments, ds oligonucleotides targeting INHBE can direct a reduction in the expression, level, and / or activity of an INHBE target gene or its product by spatially blocking translation after binding to INHBE target gene mRNA, and / or by altering or interfering with mRNA splicing. However, this disclosure is not limited to any particular mechanism. In some embodiments, this disclosure provides oligonucleotides, compositions, methods, etc., that can be manipulated via double-stranded RNA interference.

[0422] In some embodiments, ds oligonucleotides targeting INHBE can mediate a decrease in the expression, level, and / or activity of INHBE. In some embodiments, ds oligonucleotides targeting INHBE can mediate a decrease in the level of INHBE protein. In some embodiments, ds oligonucleotides targeting INHBE can mediate a decrease in the level of INHBE protein.

[0423] In some embodiments, ds oligonucleotides targeting INHBE can mediate a reduction in INHBE expression, levels, and / or activity via mechanisms involving mRNA degradation.

[0424] In some embodiments, ds oligonucleotides targeting INHBE can mediate the reduction in expression, level, and / or activity of more than one INHBE allele.

[0425] In some embodiments, this disclosure relates to a method for treating INHBE-related diseases, disorders, or conditions (where INHBE is expressed), the method comprising the step of administering a therapeutically effective amount of a ds oligonucleotide targeting INHBE capable of mediating a reduction in the expression, level, and / or activity of INHBE. In some embodiments, multiple forms of INHBE (e.g., alleles) may be present, and the provided techniques can reduce the expression, level, and / or activity of two or more or all of the forms and their products.

[0426] In some embodiments, this disclosure relates to a method for treating INHBE-related diseases, disorders, or conditions, the method comprising the step of administering a therapeutic amount of a ds oligonucleotide targeting INHBE capable of mediating a reduction in the expression, level, and / or activity of INHBE.

[0427] In some embodiments, ds oligonucleotides targeting INHBE can mediate a reduction in the expression, level, and / or activity of INHBE via mechanisms involving splicing regulation (e.g., exon skipping).

[0428] In some embodiments, the ds oligonucleotide targeting INHBE comprises the structural elements described herein (e.g., in Table 1) or a portion thereof. In some embodiments, the ds oligonucleotide targeting INHBE comprises the base sequence described herein (or a portion thereof) (where each T may be independently substituted with U, and vice versa), a chemical modification or a chemical modification pattern (or a portion thereof), and / or the form described herein or a portion thereof. In some embodiments, the ds oligonucleotide targeting INHBE has a base sequence comprising the base sequence described herein (or a portion thereof) (where each T may be independently substituted with U), a chemical modification pattern (or a portion thereof), and / or the form of an oligonucleotide disclosed herein (e.g., in Table 1 or otherwise disclosed herein). In some embodiments, such oligonucleotides, such as the ds oligonucleotide targeting INHBE, reduce the expression, level, and / or activity of a gene (e.g., the INHBE gene) or its gene product.

[0429] Among other things, the ds oligonucleotide targeting INHBE can hybridize with its target nucleic acid (e.g., precursor mRNA, mature mRNA, etc.). For example, in some embodiments, the ds oligonucleotide targeting INHBE can hybridize with INHBE nucleic acid derived from a DNA strand (any strand of the INHBE gene). In some embodiments, the ds oligonucleotide targeting INHBE can hybridize with INHBE transcripts. In some embodiments, the ds oligonucleotide targeting INHBE can hybridize with INHBE nucleic acid at any stage of RNA processing (including but not limited to precursor mRNA or mature mRNA). In some embodiments, the ds oligonucleotide targeting INHBE can hybridize with any element of the INHBE nucleic acid or its complementary strand, including but not limited to: promoter regions, enhancer regions, transcription termination regions, translation initiation signals, translation termination signals, coding regions, non-coding regions, exons, introns, intron / exon or exon / intron junctions, 5' UTRs, or 3' UTRs. In some embodiments, the ds oligonucleotide targeting INHBE can hybridize with a target having no more than two mismatches with it. In some embodiments, the ds oligonucleotide targeting INHBE may hybridize with a target having no more than one mismatch with it. In some embodiments, the ds oligonucleotide targeting INHBE may hybridize with a target that does not have a mismatch (e.g., when all CG and / or AT / U bases are paired).

[0430] In some embodiments, the oligonucleotide may hybridize with two or more transcript variants. In some embodiments, the ds oligonucleotide targeting INHBE may hybridize with two or more or all INHBE transcript variants. In some embodiments, the ds oligonucleotide targeting INHBE may hybridize with two or more or all INHBE transcript variants derived from the sense strand.

[0431] In some embodiments, the INHBE target of the ds oligonucleotide targeting INHBE is INHBE RNA, which is not mRNA.

[0432] In some embodiments, the oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) contains one or more isotopes at increased levels. In some embodiments, the oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) is labeled, for example, with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the oligonucleotide in the provided composition (e.g., a ds oligonucleotide targeting INHBE) (e.g., oligonucleotides of various compositions) comprises base modifications, sugar modifications, and / or internucleotide linking modifications, wherein the oligonucleotide contains enriched levels of deuterium. In some embodiments, the oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) is labeled with deuterium at one or more positions (using −2 H substitution− 1 H). In some embodiments, one or more of the oligonucleotide chain or any portion (e.g., a targeting portion, etc.) conjugated to the oligonucleotide chain. 1 H was 2 H substitution. Such oligonucleotides can be used in the compositions and methods described herein.

[0433] In some embodiments, this disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides are:

[0434] 1) It has a common base sequence complementary to the target sequence in the transcript (e.g., the INHBE target sequence); and

[0435] 2) Contains one or more modified sugar moieties and / or modified nucleotide inter-linkings.

[0436] In some embodiments, ds oligonucleotides targeting INHBE that share a common base sequence may have the same nucleoside modification pattern, such as sugar modification, base modification, etc. In some embodiments, the nucleoside modification pattern may be represented by a combination of position and modification. In some embodiments, the backbone linkage pattern includes the position and type of linkage between each nucleotide (e.g., phosphate ester, thiophosphate ester, substituted thiophosphate ester, etc.).

[0437] In some embodiments, for example, the oligonucleotides in the provided composition are of the same oligonucleotide type. In some embodiments, oligonucleotides of one oligonucleotide type have a common sugar modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have a common base modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have a common nucleoside modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have the same composition. In some embodiments, oligonucleotides of one oligonucleotide type are identical. In some embodiments, the multiple oligonucleotides are identical. In some embodiments, the multiple oligonucleotides share the same composition.

[0438] In some embodiments, as illustrated herein, the ds oligonucleotide targeting INHBE is chiral controlled and comprises one or more chiral-controlled internucleotide bonds. In some embodiments, the ds oligonucleotide targeting INHBE is stereochemically pure. In some embodiments, the ds oligonucleotide targeting INHBE is substantially separate from other stereoisomers.

[0439] In some embodiments, the ds oligonucleotide targeting INHBE comprises one or more modified nucleobases, one or more modified sugars, and / or one or more modified internucleotide bonds.

[0440] In some embodiments, the ds oligonucleotide targeting INHBE comprises one or more modified sugars. In some embodiments, the oligonucleotide of this disclosure comprises one or more modified nucleobases. According to this disclosure, various modifications may be introduced into the sugars and / or nucleobases. For example, in some embodiments, the modifications are those described in US 9006198. In some embodiments, the modifications are described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107, US 2019 / 0127733, US10450568, US 2019 / 0077817, US 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056, WO2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO Modifications in WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612 and / or WO 2020 / 191252, each of which involves sugar, base and nucleotide linkage modifications, are incorporated herein by reference independently.

[0441] As used in this disclosure, in some embodiments, "one or more" is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, "one or more" is one. In some embodiments, "one or more" is two. In some embodiments, "one or more" is three. In some embodiments, "one or more" is four. In some embodiments, "one or more" is five. In some embodiments, "one or more" is six. In some embodiments, "one or more" is seven. In some embodiments, "one or more" is eight. In some embodiments, "one or more" is nine. In some embodiments, "one or more" is ten. In some embodiments, "one or more" is at least one. In some embodiments, "one or more" is at least two. In some embodiments, "one or more" is at least three. In some embodiments, "one or more" is at least four. In some embodiments, "one or more" is at least five. In some embodiments, "one or more" means at least six. In some embodiments, "one or more" means at least seven. In some embodiments, "one or more" means at least eight. In some embodiments, "one or more" means at least nine. In some embodiments, "one or more" means at least ten.

[0442] As used in this disclosure, in some embodiments, "at least one" is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, "at least one" is one. In some embodiments, "at least one" is two. In some embodiments, "at least one" is three. In some embodiments, "at least one" is four. In some embodiments, "at least one" is five. In some embodiments, "at least one" is six. In some embodiments, "at least one" is seven. In some embodiments, "at least one" is eight. In some embodiments, "at least one" is nine. In some embodiments, "at least one" is ten.

[0443] In some embodiments, the ds oligonucleotide targeting INHBE is or comprises the ds oligonucleotide targeting INHBE described in Table 1.

[0444] As demonstrated in this disclosure, in some embodiments, the provided oligonucleotide (e.g., ds oligonucleotide targeting INHBE) is characterized in that, when it is contacted with the transcript in a knockdown system, it achieves knockdown of its target (e.g., INHBE transcript targeting ds oligonucleotide targeting INHBE).

[0445] In some embodiments, ds oligonucleotides are provided in salt form. In some embodiments, ds oligonucleotides are provided in salt form, which contain negatively charged internucleotide linkages (e.g., phosphate thioester linkages, native phosphate linkages, etc.) present as salts. In some embodiments, ds oligonucleotides are provided in pharmaceutically acceptable salt form. In some embodiments, ds oligonucleotides are provided in metal salt form. In some embodiments, oligonucleotides are provided in sodium salt form. In some embodiments, ds oligonucleotides are provided in metal salt form, such as sodium salt, wherein each negatively charged internucleotide linkage is independently in salt form (e.g., for sodium salts, for phosphate thioester linkages it is −O−P(O)(SNa)−O−, for native phosphate linkages it is −O−P(O)(ONa)−O−, etc.).

[0446] Double-Stranded Oligonucleotide Base Sequence

[0447] In some embodiments, the ds oligonucleotide targeting INHBE comprises the base sequence described herein or a portion thereof having 0-5 (e.g., 0, 1, 2, 3, 4, or 5) mismatches (e.g., 5-50, 5-40, 5-30, 5-20, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, or a sequence segment of at least 10 or at least 15 consecutive nucleobases), wherein each T may be independently substituted with U, and vice versa. In some embodiments, the ds oligonucleotide targeting INHBE comprises the base sequence described herein or a portion thereof, wherein the portion is a sequence segment of at least 10 consecutive nucleobases, or a sequence segment of at least 15 consecutive nucleobases having 1-5 mismatches. In some embodiments, the ds oligonucleotide targeting INHBE comprises the base sequence described herein or a portion thereof, wherein the portion is a sequence segment of at least 10 consecutive nucleobases, or a sequence segment of at least 10 consecutive nucleobases having 1-5 mismatches, wherein each T may be independently substituted with U, and vice versa. In some embodiments, the oligonucleotide's base sequence comprises or consists of 10-50 (e.g., about or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45; in some embodiments, at least 15; in some embodiments, at least 16; in some embodiments, at least 17; in some embodiments, at least 18; in some embodiments, at least 19; in some embodiments, at least 20; in some embodiments, at least 21; in some embodiments, at least 22; in some embodiments, at least 23; in some embodiments, at least 24; in some embodiments, at least 25) consecutive bases, the base sequence being identical or complementary to the base sequence of the INHBE gene or its transcript (e.g., mRNA) (e.g., in an intron).

[0448] As will be understood by those skilled in the art, the base sequence of the ds oligonucleotide targeting INHBE typically has sufficient length and complementarity with its target (e.g., RNA transcript (e.g., precursor mRNA, mature mRNA, etc.)) to mediate target-specific knockdown. In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE has sufficient length and identity with the INHBE transcript target to mediate target-specific knockdown. In some embodiments, the ds oligonucleotide targeting INHBE is complementary to a portion of the INHBE transcript (the INHBE transcript target sequence). In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE has 90% or higher identity with the base sequences of the oligonucleotides disclosed in Table 1, wherein each T can be independently substituted with U, and vice versa. In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE has 95% or higher identity with the base sequences of the oligonucleotides disclosed in Table 1, wherein each T can be independently substituted with U, and vice versa. In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE comprises a continuous sequence segment of 15 or more bases of the oligonucleotides disclosed in Table 1, wherein each T may be independently substituted with U, and vice versa, except that one or more bases within the sequence segment are baseless (e.g., a nucleobase is absent in the nucleotide). In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE comprises a continuous sequence segment of 19 or more bases of the ds oligonucleotides targeting INHBE disclosed herein, except that one or more bases within the sequence segment are baseless (e.g., a nucleobase is absent in the nucleotide). In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE comprises a continuous sequence segment of 19 or more bases of the oligonucleotides disclosed herein, wherein each T may be independently substituted with U, and vice versa, except for a difference of one or two bases at the 5' end and / or 3' end of the base sequence.

[0449] In some embodiments, this disclosure relates to oligonucleotides having a base sequence comprising the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently substituted with U and vice versa.

[0450] In some embodiments, this disclosure relates to an oligonucleotide having a base sequence of at least 15 consecutive bases comprising any oligonucleotide disclosed herein, wherein each T may be independently substituted with U and vice versa.

[0451] In some embodiments, this disclosure relates to oligonucleotides having a base sequence that is at least 90% identical to that of any oligonucleotide disclosed herein, wherein each T may be independently substituted with U and vice versa.

[0452] In some embodiments, this disclosure relates to oligonucleotides having a base sequence that is at least 95% identical to that of any oligonucleotide disclosed herein, wherein each T may be independently substituted with U and vice versa.

[0453] In some embodiments, the ds oligonucleotides targeting INHBE are selected from Table 1.

[0454] In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE is complementary to the base sequence of the INHBE transcript or a portion thereof.

[0455] In some embodiments, the base sequence of the ds oligonucleotide targeting INHBE is complementary to a portion of the INHBE nucleic acid sequence (e.g., the INHBE gene sequence, INHBE transcript, INHBE mRNA sequence, etc.). In some embodiments, the ds oligonucleotide targeting INHBE is identical to a portion of the INHBE nucleic acid sequence (e.g., the INHBE gene sequence, INHBE transcript, INHBE mRNA sequence, etc.). In some embodiments, the base sequence of such a portion is characteristic of INHBE because no other genomic or transcriptomic sequence in the system contains a sequence identical to that portion. In some embodiments, no other genomic or transcriptomic sequence in the system contains a sequence that differs from such a portion by no more than one nucleobase. In some embodiments, no other genomic or transcriptomic sequence in the system contains a sequence that differs from such a portion by no more than two nucleobases. In some embodiments, a portion of the gene complementary to the oligonucleotide is referred to as the target sequence of the oligonucleotide. In some embodiments, the system is or comprises cells, samples, tissues, organs, or species. For example, for oligonucleotides targeting human INHBE, in many embodiments the relevant species is human. In some embodiments, such as when characterizing and / or evaluating cross-species activity and / or properties, the system may be or contain multiple species. In some embodiments, such a portion is in an exon. In some embodiments, such a portion is in an intron. In some embodiments, such a portion spans both an intron and an exon. In some embodiments, such a portion spans two exons. In some embodiments, such a portion is in the 5'-UTR region. In some embodiments, such a portion is in the 3'-UTR region.

[0456] In some embodiments, the ds oligonucleotide targeting INHBE targets two or more or all of the INHBE alleles (if multiple alleles are present in the relevant system). In some embodiments, the oligonucleotide reduces the expression, level, and / or activity of wild-type INHBE and mutant INHBE and / or their transcripts and / or products.

[0457] In some embodiments, the base sequence of the provided oligonucleotide is fully complementary to both the human and non-human primate (NHP) INHBE target sequences. In some embodiments, such sequences may be particularly useful because they can be readily evaluated in human and non-human primates.

[0458] In some embodiments, the ds oligonucleotide targeting INHBE comprises the base sequence described in the table or a portion thereof (where each T may be independently replaced by U and vice versa), and / or the sugar, nucleobase and / or nucleotide linking modifications and / or patterns described in Table 1, and / or other chemical motifs described in Table 1 (in addition to the oligonucleotide chain, there may be, for example, target motifs, lipid motifs, carbohydrate motifs, etc.).

[0459] In some embodiments, as those skilled in the art will understand from the context of use, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used with respect to base matching between the base sequence of an oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) and a target sequence (e.g., an INHBE target sequence). It should be noted that substituting T with U or vice versa generally does not change the amount of complementarity. As used herein, an oligonucleotide that is “substantially complementary” to a target sequence is largely or mostly complementary, but not 100% complementary. In some embodiments, a substantially complementary sequence (e.g., a ds oligonucleotide targeting INHBE) has 1, 2, 3, 4, or 5 mismatches when aligned with a target sequence. In some embodiments, a ds oligonucleotide targeting INHBE has a base sequence that is substantially complementary to the complementary sequence of the ds oligonucleotide targeting INHBE disclosed herein. As will be understood by those skilled in the art, in some embodiments, for an oligonucleotide to perform its function (e.g., knock down a target nucleic acid), the sequence of the oligonucleotide does not need to be 100% complementary to its target. Typically, when complementarity is determined, A and T (or U) are complementary nucleobases, while C and G are complementary nucleobases.

[0460] In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE, which includes the sequence found in the oligonucleotides described in the table. In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE, which includes the sequence found in the oligonucleotides described in Table 1, wherein one or more Us are independently and optionally substituted with T, or vice versa. In some embodiments, the ds oligonucleotide targeting INHBE may contain at least one T and / or at least one U. In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE, which includes the sequence found in the oligonucleotides described in the table, wherein the sequence has more than 50% identity with the sequence of the oligonucleotides described in the table. In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE, which includes the sequence of the oligonucleotide disclosed in Table 1. In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE, the base sequence of which is the sequence of the oligonucleotides disclosed in Table 1, wherein each T can be independently substituted with U, and vice versa. In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE comprising the sequence found in the oligonucleotides in Table 1, wherein the oligonucleotide has the same backbone bonding pattern, backbone chiral center pattern, and / or backbone phosphorus modification pattern as the same oligonucleotides in Table 1 or another oligonucleotide.

[0461] Among other things, this disclosure presents a variety of ds oligonucleotides, each having a defined base sequence, in Table 1 and elsewhere. In some embodiments, this disclosure provides oligonucleotides whose base sequences are those of oligonucleotides disclosed herein (e.g., in Table 1 herein, where each T may be independently substituted with U, and vice versa), containing the base sequences of oligonucleotides disclosed herein, or containing a portion of the base sequences of oligonucleotides disclosed herein. In some embodiments, this disclosure provides oligonucleotides having base sequences that are those of oligonucleotides disclosed herein (e.g., in Table 1), or containing the base sequences of oligonucleotides disclosed herein, or containing a portion of the base sequences of oligonucleotides disclosed herein, where each T may be independently substituted with U, and vice versa, wherein the oligonucleotide further contains the chemical modifications, stereochemistry, form, additional chemical moieties (e.g., targeting moieties, lipid moieties, carbohydrate moieties, etc.) and / or another structural feature as described herein.

[0462] In some embodiments, the length of a “partial” (e.g., a portion of a base sequence or modification pattern) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units (e.g., for a base sequence, the length is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases). In some embodiments, the length of a “partial” of a base sequence is at least 5 bases. In some embodiments, the length of a “partial” of a base sequence is at least 10 bases. In some embodiments, the length of a “partial” of a base sequence is at least 15 bases. In some embodiments, the length of a “partial” of a base sequence is at least 16, 17, 18, 19, or 20 bases. In some embodiments, the length of a “partial” of a base sequence is at least 20 bases. In some embodiments, a portion of the base sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous / consecutive bases. In some embodiments, a portion of the base sequence is 15 or more contiguous / consecutive bases. In some embodiments, a portion of the base sequence is 16, 17, 18, 19 or 20 or more contiguous / consecutive bases. In some embodiments, a portion of the base sequence is 20 or more contiguous / consecutive bases.

[0463] In some embodiments, this disclosure provides an oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) whose base sequence is a subset of the oligonucleotide sequences in Table 1, wherein each T may be independently substituted with U, and vice versa. In some embodiments, this disclosure provides a ds oligonucleotide targeting INHBE having the sequence of the oligonucleotides in Table 1, wherein the oligonucleotide is capable of directing the reduction of expression, level, and / or activity of the INHBE gene or its gene product. As will be understood by those skilled in the art, in the provided base sequence, each U may optionally and independently be substituted with T, or vice versa, and the sequence may contain a mixture of U and T. In some embodiments, C may optionally and independently be substituted with 5mC.

[0464] In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides. In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0-3 mismatches. In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0-3 mismatches, wherein a sequence segment with 0 mismatches is complementary, and a sequence segment with one or more mismatches is substantially complementary—a non-limiting example. In some embodiments, bases constitute a characteristic portion of a nucleic acid (e.g., a gene), wherein this portion is identical or complementary to a portion of the nucleic acid or its transcript, but not identical or complementary to a portion of any other nucleic acid (e.g., a gene) or its transcript in the same genome. In some embodiments, a portion is a characteristic of human INHBE.

[0465] In some embodiments, as described herein, the provided oligonucleotides (e.g., ds oligonucleotides targeting INHBE) have a length of no more than about 49, 45, 40, 30, 35, 25, or 23 total nucleotides. In some embodiments where the sequence described herein begins with U or T at the 5' end, U may be omitted and / or substituted with another base. In some embodiments, the oligonucleotide has a base sequence that is, or comprises, a portion of, the base sequence of the oligonucleotides listed in the table, or a portion thereof, wherein each T may be independently substituted with U, and vice versa, having the form disclosed herein or a portion thereof.

[0466] In some embodiments, the oligonucleotide (e.g., ds oligonucleotide targeting INHBE) is stereorandom. In some embodiments, the ds oligonucleotide targeting INHBE is chiral controlled. In some embodiments, the ds oligonucleotide targeting INHBE is chiral pure (or "stereopure", "stereochemically pure"), wherein the oligonucleotide exists in a single stereoisomer (in many cases a single diastereoisomer (or "diastereomeric") form, since multiple chiral centers may be present in the oligonucleotide, for example at the phosphate, sugar carbon, etc.). As those skilled in the art will understand, a chiral pure oligonucleotide is separated from its other stereoisomers (to the extent that some impurities may be present because chemical and biological processes, selectivity and / or purification, etc., rarely (if any) achieve absolute completeness). In a chiral pure oligonucleotide, each chiral center is independently defined in terms of its configuration (for a chiral pure oligonucleotide, the internucleotide linkages are independently stereodefined or chiral controlled). In contrast to chiral and chiral pure oligonucleotides containing a chiral phosphorus-linked phosphorus, “racemic” (or “stereochemically random”, “chirally uncontrolled”) oligonucleotides containing a chiral phosphorus-linked phosphorus (e.g., from conventional phosphoramide oligonucleotide synthesis, where there is no stereochemical control in the coupling step and it is combined with conventional sulfidation (forming a stereochemically random inter-nucleotide link of thiophosphate nucleotides) refers to a variety of stereoisomers (typically a random mixture of diastereomers (or “diastereomers”) because there are multiple chiral centers in the oligonucleotide; e.g., from conventional oligonucleotide preparation using reagents that do not contain chiral elements other than those in the nucleoside and the phosphorus-linked phosphorus). For example, for A*A*A, where * is an inter-nucleotide link of thiophosphate nucleotides (which contains a chiral phosphorus-linked phosphorus), the racemic oligonucleotide formulation comprises four diastereomers [2]. 2 = 4, considering two chiral linked phosphorus groups, each of which can exist in one of two configurations (Sp or Rp): A*SA*SA, A*SA*RA, A*RA*SA, and A*RA*RA, where *S represents Sp (phosphothioester nucleotide linkage) and *R represents Rp (phosphothioester nucleotide linkage). For chiral pure oligonucleotides, such as A*SA*SA, it exists in a single stereoisomer and is distinct from other stereoisomers (e.g., diastereomeric A*SA*RA, A*RA*SA, and A*RA*RA).

[0467] In some embodiments, the ds oligonucleotide targeting INHBE comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom internucleotide links (a mixture of Rp and Sp-linked phosphorus at the internucleotide link, e.g., from conventional chiral-controlled oligonucleotide synthesis). In some embodiments, the ds oligonucleotide targeting INHBE comprises one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) chiral-controlled internucleotide links (Rp or Sp-linked phosphorus at the internucleotide link, e.g., from chiral-controlled oligonucleotide synthesis). In some embodiments, the internucleotide link is a phosphate thioester internucleotide link. In some embodiments, the internucleotide linkage is a stereorandom phosphate thioester nucleotide linkage. In some embodiments, the internucleotide linkage is a chiral controlled phosphate thioester nucleotide linkage.

[0468] In particular, this disclosure provides techniques for preparing chiral-controlled (in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, the oligonucleotides are stereochemically pure. In some embodiments, the oligonucleotides disclosed herein are about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. In some embodiments, the internucleotide linkages of the oligonucleotides comprise or consist of one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) chiral nucleotide linkages, each of which independently has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% diastereomeric purity. In some embodiments, the oligonucleotides disclosed herein (e.g., ds oligonucleotides targeting INHBE) have (DS) CIL The diastereomeric purity, wherein DS is the diastereomeric purity as described in this disclosure (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or higher), and CIL is the number of chiral-controlled internucleotide links (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, DS is 95%-100%. In some embodiments, each internucleotide link is independently chiral-controlled, and CIL is the number of chiral-controlled internucleotide links.

[0469] As examples, Table 1 below presents certain ds oligonucleotides targeting INHBE, which contain certain examples of base sequences, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide bonds and their patterns, linked phosphorus stereochemistry and their patterns, linkers and / or other chemical components. Among other things, oligonucleotides (such as those in Table 1) can be used to target INHBE transcripts, for example, to reduce the levels of INHBE transcripts and / or their products.

[0470] In some exemplary embodiments, the ds oligonucleotides targeting INHBE of this disclosure comprise the following base sequences, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide bonds and their patterns, linking phosphorus stereochemistry and its patterns, linkers and / or additional chemical motifs: DSR-0104068, DSR-0104099, DSR-0104072, DSR-0104075, DSR-0104083, DSR-0104091 and DSR-0104071. In some exemplary embodiments, the ds oligonucleotides targeting INHBE of this disclosure comprise the following base sequences, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linking phosphorus stereochemistry and its patterns, linkers, and / or additional chemical portions: DSR-0104068, DSR-0104099, DSR-0104072, DSR-0104075, DSR-0104083, DSR-0104091, and DSR-0104071. In some exemplary embodiments, the ds oligonucleotides targeting INHBE of this disclosure comprise a transit chain that comprises the base sequence of DSR-0104068, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linking phosphorus stereochemistry and its patterns, linkers, and / or additional chemical portions. In some exemplary embodiments, the ds oligonucleotides targeting INHBE of this disclosure include a guide chain comprising the base sequences of DSR-0104068, DSR-0104099, DSR-0104072, DSR-0104075, DSR-0104083, DSR-0104091 and DSR-0104071, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linking phosphorus stereochemistry and their patterns, linkers and / or additional chemical portions, and the base sequence of DSR-0104068, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linking phosphorus stereochemistry and their patterns, linkers and / or additional chemical portions.

[0471] In some exemplary embodiments, the ds oligonucleotide targeting INHBE of this disclosure comprises the base sequence of DSR-0104068, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and their patterns, linkers and / or other chemical portions comprising the base sequence, nucleobase modifications and their patterns, sugar modifications and their patterns.

[0472] In some exemplary embodiments, the ds oligonucleotides targeting INHBE disclosed herein include the base sequence of DSR-0104099, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and its patterns, linkers and / or other chemical components.

[0473] In some exemplary embodiments, the ds oligonucleotides targeting INHBE of this disclosure include the base sequence of DSR-0104072, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and its patterns, linkers and / or other chemical components.

[0474] In some exemplary embodiments, the ds oligonucleotides targeting INHBE disclosed herein include the base sequence of DSR-0104075, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and its patterns, linkers and / or other chemical components.

[0475] In some exemplary embodiments, the ds oligonucleotides targeting INHBE disclosed herein include the base sequence of DSR-0104083, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and its patterns, linkers and / or other chemical components.

[0476] In some exemplary embodiments, the ds oligonucleotides targeting INHBE disclosed herein include the base sequence of DSR-0104091, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and its patterns, linkers and / or other chemical components.

[0477] In some exemplary embodiments, the ds oligonucleotides targeting INHBE disclosed herein include the base sequence of DSR-0104071, nucleobase modifications and their patterns, sugar modifications and their patterns, internucleotide linkages and their patterns, linkage phosphorus stereochemistry and its patterns, linkers and / or other chemical components.

[0478] Table 1. Exemplary double-stranded oligonucleotides targeting INHBE.

[0479]

[0480] Table 1a. Examples of guide sequences targeting INHBE

[0481]

[0482] Table 1b. Examples of passenger sequences targeting INHBE

[0483]

[0484] Precautions:

[0485] This symbolic language is described in Zhang, T. et al., Chem. Inf. Model. 2012, 52, 10, 2796–2806 and Milton, J. et al., J. Chem. Inf. Model. 2017, 57, 6, 1233–1239. The descriptions, base sequences, and stereochemistry / linkings in Table 1 may be divided into multiple rows due to their length. Unless otherwise stated, all oligonucleotides in Table 1 are single-stranded. As will be understood by those skilled in the art, unless otherwise specified (e.g., with r, m, etc.), the nucleoside units are unmodified and contain unmodified nucleobases and 2'-deoxy sugars; unless otherwise indicated, the linkages are native phosphate ester linkages; and acidic / basic groups may exist independently in their salt form. If a sugar is not specified, it is a native DNA sugar; and if an internucleotide linkage is not specified, it is a native phosphate ester linkage. Parts and Modifications:

[0486] m: 2'-OMe;

[0487] f or fl2r: 2'-F;

[0488] O, PO, or p: Phosphodiester (phosphate ester). It can be a linker or a terminal group (or a component thereof), such as a linker to an oligonucleotide chain, an internucleotide link (natural phosphate ester link), etc. Phosphodiester is typically indicated by "O" in the stereochemistry / linking column and is typically not marked in the description column (if it is a terminal group, such as a 5' terminal group, it is indicated in the description and is typically not indicated in the stereochemistry / linking column); if the linker is not indicated in the description column, it is typically a phosphate diester unless otherwise indicated. Note that a phosphate ester linker (e.g., L001) to an oligonucleotide chain may not be marked in the description column but may be indicated by "O" in the stereochemistry / linking column;

[0489] *, PS: Phosphothiophosphate. It can be a terminal group (if it is a terminal group, such as a 5' terminal group, it is indicated in the description column and is typically not indicated in the stereochemistry / linking), or a link, such as a link between a linker (e.g., L001) and an oligonucleotide chain, an internucleotide link (phosphothiophosphate internucleotide linking), etc.

[0490] R, Rp, or Rsp: Thiophosphates in the Rp configuration. Note that *R in the description indicates a single thiophosphate bond in the Rp configuration;

[0491] S, Sp, or Ssp: Thiophosphates in the Sp configuration. Note that *S in the description indicates a single thiophosphate bond in the Sp configuration;

[0492] X: Stereo atactic thiophosphate or phosphoryl guanidine;

[0493] n001: ;

[0494] nX: cubic random n001;

[0495] nR or n001R or [n001R]: n001 in Rp configuration;

[0496] nS or n001S or [n001S]: n001 in Sp configuration;

[0497] n002: ;

[0498] nX: cubic random n002;

[0499] nR or n002R: n002 in Rp configuration;

[0500] nS or n002S: n002 in Sp configuration;

[0501] n003: ;

[0502] nX: cubic random n003;

[0503] nR or n003R: n003 in Rp configuration;

[0504] nS or n003S: n003 in Sp configuration;

[0505] n004: ;

[0506] nX: cubic random n004;

[0507] nR or n004R: n004 in Rp configuration;

[0508] nS or n004S: n004 in Sp configuration;

[0509] n006: ;

[0510] nX: cubic random n006;

[0511] nR or n006R: n006 in Rp configuration;

[0512] nS or n006S: n006 in the Sp configuration;

[0513] n008: ;

[0514] nX: cubic random n008;

[0515] nR or n008R: n008 in Rp configuration;

[0516] nS or n008S: n008 in Sp configuration;

[0517] n009: ;

[0518] nX: cubic random n009;

[0519] nR or n009R: n009 in Rp configuration;

[0520] nS or n009S: n009 in the Sp configuration;

[0521] n012: ;

[0522] nX: cubic random n012;

[0523] nR or n012R: n012 in Rp configuration;

[0524] nS or n012S: n012 in Sp configuration;

[0525] n020: ;

[0526] nX: cubic random n020;

[0527] nR or n020R: n020 in Rp configuration;

[0528] nS or n020S: n020 in Sp configuration;

[0529] n021: ;

[0530] nX: cubic random n021;

[0531] nR or n021R: n021 in Rp configuration;

[0532] nS or n021S: n021 in Sp configuration;

[0533] n025: ;

[0534] nX: cubic random n025;

[0535] nR or n025R: n025 in Rp configuration;

[0536] nS or n025S: n025 in Sp configuration;

[0537] n026: ;

[0538] nX: cubic random n026;

[0539] nR or n026R: n026 in Rp configuration;

[0540] nS or n026S: n026 in the Sp configuration;

[0541] n029: ;

[0542] nX: cubic random n029;

[0543] nR or n029R: n029 in the Rp configuration;

[0544] nS or n029S: n029 in Sp configuration;

[0545] n030: ;

[0546] nX: cubic random n030;

[0547] nR or n030R: n030 in Rp configuration;

[0548] nS or n030S: n030 in the Sp configuration;

[0549] n031: ;

[0550] nX: cubic random n031;

[0551] nR or n031R: n031 in Rp configuration;

[0552] nS or n031S: n031 in Sp configuration;

[0553] n033: ;

[0554] nX: cubic random n033;

[0555] nR or n033R: n033 in Rp configuration;

[0556] nS or n033S: n033 in Sp configuration;

[0557] n034: ;

[0558] nX: cubic random n034;

[0559] nR or n034R: n034 in Rp configuration;

[0560] nS or n034S: n034 in Sp configuration;

[0561] n035: ;

[0562] nX: cubic random n035;

[0563] nR or n035R: n035 in Rp configuration;

[0564] nS or n035S: n035 in the Sp configuration;

[0565] n036: ;

[0566] nX: cubic random n036;

[0567] nR or n036R: n036 in Rp configuration;

[0568] nS or n036S: n036 in the Sp configuration;

[0569] n037: ;

[0570] nX: cubic random n037;

[0571] nR or n037R: n037 in Rp configuration;

[0572] nS or n037S: n037 in the Sp configuration;

[0573] n039: ;

[0574] nX: cubic random n039;

[0575] nR or n039R: n039 in the Rp configuration;

[0576] nS or n039S: n039 in the Sp configuration;

[0577] n040: ;

[0578] nX: cubic random n040;

[0579] nR or n040R: n040 in Rp configuration;

[0580] nS or n040S: n040 in the Sp configuration;

[0581] n041: ;

[0582] nX: cubic random n041;

[0583] nR or n041R: n041 in Rp configuration;

[0584] nS or n041S: n041 in Sp configuration;

[0585] n043:

[0586] nX: cubic random n043;

[0587] nR or n043R: n043 in Rp configuration;

[0588] nS or n043S: n043 in the Sp configuration;

[0589] n045:

[0590] nX: cubic random n045;

[0591] nR or n045R: n045 in Rp configuration;

[0592] nS or n045S: n045 in the Sp configuration;

[0593] n046: ;

[0594] nX: cubic random n046;

[0595] nR or n046R: n046 in Rp configuration;

[0596] nS or n046S: n046 in the Sp configuration;

[0597] n047: ;

[0598] nX: cubic random n047;

[0599] nR or n047R: n047 in the Rp configuration;

[0600] nS or n047S: n047 in the Sp configuration;

[0601] n051: ;

[0602] nX: cubic random n051;

[0603] nR or n051R: n051 in Rp configuration;

[0604] nS or n051S: n051 in Sp configuration;

[0605] n052: ;

[0606] nX: cubic random n052;

[0607] nR or n052R: n052 in Rp configuration;

[0608] nS or n052S: n052 in Sp configuration;

[0609] n054: ;

[0610] nX: cubic random n054;

[0611] nR or n054R: n054 in Rp configuration;

[0612] nS or n054S: n054 in Sp configuration;

[0613] n055: ;

[0614] nX: cubic random n055;

[0615] nR or n055R: n055 in Rp configuration;

[0616] nS or n055S: n055 in Sp configuration;

[0617] n057:

[0618] nX: cubic random n057;

[0619] nR or n057R: n057 in Rp configuration;

[0620] nS or n057S: n057 in the Sp configuration;

[0621] n058:

[0622] nX: cubic random n058;

[0623] nR or n058R: n058 in Rp configuration;

[0624] nS or n058S: n058 in Sp configuration;

[0625] n060:

[0626] nX: stereoironic n060;

[0627] nR or n060R: n060 in Rp configuration;

[0628] nS or n060S: n060 in the Sp configuration;

[0629] n061:

[0630] nX: cubic random n061;

[0631] nR or n061R: n061 in Rp configuration;

[0632] nS or n061S: n061 in Sp configuration;

[0633] n062:

[0634] nX: cubic random n062;

[0635] nR or n062R: n062 in Rp configuration;

[0636] nS or n062S: n062 in the Sp configuration;

[0637] n065:

[0638] nX: cubic random n065;

[0639] nR or n065R: n065 in Rp configuration;

[0640] nS or n065S: n065 in the Sp configuration;

[0641] n066:

[0642] nX: cubic random n066;

[0643] nR or n066R: n066 in Rp configuration;

[0644] nS or n066S: n066 in the Sp configuration;

[0645] n068:

[0646] nX: cubic random n068;

[0647] nR or n068R: n068 in Rp configuration;

[0648] nS or n068S: n068 in the Sp configuration;

[0649] n069:

[0650] nX: cubic random n069;

[0651] nR or n069R: n069 in the Rp configuration;

[0652] nS or n069S: n069 in the Sp configuration;

[0653] n070:

[0654] nX: cubic random n070;

[0655] nR or n070R: n070 in Rp configuration;

[0656] nS or n070S: n070 in the Sp configuration;

[0657] n071:

[0658] nX: cubic random n071;

[0659] nR or n071R: n071 in Rp configuration;

[0660] nS or n071S: n071 in Sp configuration;

[0661] n072:

[0662] nX: cubic random n072;

[0663] nR or n072R: n072 in Rp configuration;

[0664] nS or n072S: n072 in Sp configuration;

[0665] n073:

[0666] nX: cubic irregularity n073;

[0667] nR or n073R: n073 in Rp configuration;

[0668] nS or n073S: n073 in the Sp configuration;

[0669] ;

[0670] nX: cubic random n076;

[0671] nR or n076R: n076 in Rp configuration;

[0672] nS or n076S: n076 in the Sp configuration;

[0673] ;

[0674] nX: cubic random n077;

[0675] nR or n077R: n077 in Rp configuration;

[0676] nS or n077S: n077 in the Sp configuration;

[0677] X: Stereo atactic thiophosphate or phosphoryl guanidine;

[0678] sm01n001: (For example, Asm01n001:) Gsm01n001: ;Tsm01n001: ;

[0679] Csm01n001: Usm01n001: );

[0680] sm01*n001: (For example, Asm01*n001:) ;Gsm01*n001: ;Tsm01*n001: ;Csm01*n001: Usm01*n001: );

[0681] L026 L027 ;mU ;

[0682] fU dT ;POdT or PO4-dT ;

[0683] PO5MRdT ;PO5MSdT ;

[0684] VPdT ;5mvpdT ;

[0685] 5mrpdT or p[Rm5d5m]T ;5mspdT ;

[0686] PNdT ;SPNdT ;

[0687] 5ptzdT Teo ;

[0688] n013: , where −C(O)− is bonded to nitrogen;

[0689] sm01n013: ;

[0690] That is, the internucleotide linkage of morpholine carbamate (sm01n013). ;

[0691] Gsm01n013: ;

[0692] Csm01n013: Usm01n013: ;Tsm01n013: m5Csm01n013: Mod001:

[0693] ;

[0694] Mod015:

[0695] ;

[0696] Mod020:

[0697] ;

[0698] Mod029:

[0699] ;

[0700] L001: −NH−(CH2)6-linker (C6 linker, C6 amine linker, or C6 amino linker), which is connected to Mod (e.g., Mod001) via −NH−, and in the case of WV-38061, to the 5' end of the oligonucleotide chain via a phosphate ester bond (O or PO). For example, in WV-38061, L001 is connected to Mod001 via –NH− (forming an amide group –C(O)−NH−) and to the oligonucleotide chain via a phosphate ester bond (O).

[0701] L010: In some embodiments, when L010 is present in the middle of an oligonucleotide, it is linked to the nucleotide as another sugar (e.g., DNA sugar), for example, its 5'-carbon is linked to another unit (e.g., the 3' of a sugar), and its 3'-carbon is independently linked to another unit (e.g., the 5'-carbon of a carbon), for example, via a link (e.g., phosphate link (O or PO) or thiophosphate link (which may be non-chirally controlled or chirally controlled (Sp or Rp))).

[0702] L012: −CH2CH2OCH2CH2OCH2CH2−. When L012 is present in the middle of an oligonucleotide, its two ends are independently bonded to internucleotide bonds (e.g., phosphate ester bonds (O or PO) or thiophosphate ester bonds (which may be non-chirally controlled or chirally controlled (Sp or Rp))).

[0703] L022: L022 is linked to the rest of the molecule via a phosphate ester, unless otherwise indicated;

[0704] L023: HO−(CH2)6−, where CH2 is connected to the rest of the molecule via phosphate unless otherwise indicated. For example, in WV-42644 (where OnRnRnRnRSSSSSSSSSSSSSSSSSSSSnRSSSSSnRSSnR O (This indicates the phosphate ester bond connecting L023 to the rest of the molecule).

[0705] L025: The -CH2- linker site serves as the C5 linker site for sugars (e.g., DNA sugars) and connects to another unit (e.g., the 3' end of the sugar), while the linker site on the loop serves as the C3 linker site and connects to another unit (e.g., the 5' carbon) via a bond (e.g., a phosphate ester bond (O or PO) or a thiophosphate bond (which may be non-chirally controlled or chirally controlled (Sp or Rp))). When L025 is at the 5' end without any modification, its -CH2- linker site is bonded to -OH. For example, L025 in various oligonucleotides has... The structure (which can exist in various salt forms) is attached to the 5'-carbon of the oligonucleotide chain via an indicated link (e.g., phosphate link (O or PO) or thiophosphate link (which may be non-chirally controlled or chirally controlled (Sp or Rp))).

[0706] L016: L016 is connected to the rest of the molecule via phosphate, unless otherwise stated; L016 combines with n001 to form L016n001, which has the structure .

[0707] Double-Stranded Oligonucleotide Length

[0708] As will be understood by those skilled in the art, ds oligonucleotides targeting INHBE can have various lengths to provide desired properties and / or activities for a variety of uses. Many techniques for evaluating, selecting, and / or optimizing oligonucleotide lengths are available in the art and can be used according to this disclosure. As demonstrated herein, in many embodiments, ds oligonucleotides targeting INHBE have an appropriate length to hybridize with their target and reduce the levels of their target and / or their encoded products. In some embodiments, the oligonucleotide is long enough to recognize the target nucleic acid (e.g., INHBE mRNA). In some embodiments, the oligonucleotide is long enough to distinguish the target nucleic acid from other nucleic acids (e.g., nucleic acids with a base sequence that is not INHBE) to reduce off-target effects. In some embodiments, ds oligonucleotides targeting INHBE are short enough to reduce the complexity of manufacturing or production and reduce product costs.

[0709] In some embodiments, the oligonucleotide has a base sequence length of about 10-500 nucleotides. In some embodiments, the base sequence length is about 10-500 nucleotides. In some embodiments, the base sequence length is about 10-50 nucleotides. In some embodiments, the base sequence length is about 15-50 nucleotides. In some embodiments, the base sequence length is about 15 to about 30 nucleotides. In some embodiments, the base sequence length is about 10 to about 25 nucleotides. In some embodiments, the base sequence length is about 15 to about 22 nucleotides. In some embodiments, the base sequence length is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the base sequence length is about 18 nucleotides. In some embodiments, the base sequence length is about 19 nucleotides. In some embodiments, the base sequence length is about 20 nucleotides. In some embodiments, the base sequence is about 21 nucleobases long. In some embodiments, the base sequence is about 22 nucleobases long. In some embodiments, the base sequence is about 23 nucleobases long. In some embodiments, the base sequence is about 24 nucleobases long. In some embodiments, the base sequence is about 25 nucleobases long. In some embodiments, each nucleobase is an optionally substituted A, T, C, G, U, or an optionally substituted tautomer of A, T, C, G, or U.

[0710] Double-Stranded Oligonucleotide Intemucleotide Linkage

[0711] In some embodiments, the ds oligonucleotide targeting INHBE comprises base modifications, sugar modifications, and / or internucleotide linking modifications. According to this disclosure, various internucleotide links can be used to link units containing nucleobases, such as nucleosides. In some embodiments, the ds oligonucleotide targeting INHBE comprises one or more modified internucleotide links and one or more natural phosphate ester links. As is well known to those skilled in the art, natural phosphate ester links are widely present in natural DNA and RNA molecules; they have a -OP(O)(OH)O- structure, link sugars in nucleosides of DNA and RNA, and can be in various salt forms, for example, at physiological pH (about 7.4), natural phosphate ester links are primarily in the form of -OP(O)(OH)O-. -The O- anion exists in its salt form. Modified nucleotide linkages or non-natural phosphate linkages are nucleotide linkages that are not natural phosphate linkages or their salt forms. Modified nucleotide linkages may also exist in their salt form depending on their structure. For example, as understood by those skilled in the art, thiophosphate nucleotide linkages having the structure -OP(O)(SH)O- can exist in various salt forms, such as at physiological pH (approximately 7.4), where the anion is -OP(O)(SH). - )O-.

[0712] In some embodiments, the oligonucleotide includes an internucleotide link that is a modified internucleotide link, such as a thiophosphate, dithiophosphate, methylphosphonate, aminophosphate, thiophosphate, 3'-thiophosphate, or 5'-thiophosphate.

[0713] In some embodiments, the modified internucleotide link is a chiral internucleotide link containing a chiral linking phosphorus. In some embodiments, the chiral internucleotide link is a phosphate thioester link. In some embodiments, the chiral internucleotide link is a negatively charged internucleotide link. In some embodiments, the chiral internucleotide link is a neutral internucleotide link. In some embodiments, the chiral internucleotide link is chiral controlled with respect to its chiral linking phosphorus. In some embodiments, the chiral internucleotide link is stereochemically pure with respect to its chiral linking phosphorus. In some embodiments, the chiral internucleotide link is not chiral controlled. In some embodiments, the skeletal chiral center pattern comprises or consists of the following: the position of the chiral internucleotide link (Rp or Sp) and the configuration of the linking phosphorus, and the position of the achiral internucleotide link (e.g., a native phosphate ester link).

[0714] In some embodiments, the internucleotide linking includes a P-modification, wherein the P-modification is a modification at the phosphorus site of the linking. In some embodiments, the modified internucleotide linking is a phosphorus-free portion used, for example, to link two sugars or two independently containing nucleotide moieties in a peptide nucleic acid (PNA).

[0715] In some embodiments, the ds oligonucleotide comprises modified internucleotide links, such as those having the structure of formula I, Ia, Ib, or Ic and described herein and / or hereinafter: WO 2018 / 022473, WO 2018 / 098264, WO2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612, wherein the internucleotide links of each of these (e.g., those of formula I, Ia, Ib, Ic, etc.) are independently incorporated herein by reference. In some embodiments, the modified nucleotide linkages are chiral nucleotide linkages. In some embodiments, the modified nucleotide linkages are phosphate thioester nucleotide linkages.

[0716] In some embodiments, the modified internucleotide links are negatively charged internucleotide links. In some embodiments, the provided ds oligonucleotides comprise one or more negatively charged internucleotide links. In some embodiments, the negatively charged internucleotide links are positively charged internucleotide links. In some embodiments, the negatively charged internucleotide links are neutral internucleotide links. In some embodiments, this disclosure provides ds oligonucleotides comprising one or more neutral internucleotide links. In some embodiments, the uncharged internucleotide linkages have structures of the formulas In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or their salts, as described herein and / or in the following references: US 9394333, US 9744183, US9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647、WO2018 / 098264、WO 2018 / 022473、WO 2018 / 223056、WO 2018 / 223073、WO 2018 / 223081、WO2018 / 237194、WO 2019 / 032607、WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612, the respective uncharged internucleotide bonds (e.g., those having the formula In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or their suitable salt forms) are independently incorporated herein by reference.

[0717] In some embodiments, non-negatively charged nucleotide inter-linking can improve delivery and / or activity (e.g., adenosine editing activity).

[0718] In some embodiments, the modified internucleotide link (e.g., an uncharged internucleotide link) comprises a optionally substituted triazole group. In some embodiments, the modified internucleotide link (e.g., an uncharged internucleotide link) comprises an optionally substituted alkynyl group. In some embodiments, the modified internucleotide link comprises a triazole or alkynyl moiety. In some embodiments, the triazole moiety (e.g., a triazole group) is optionally substituted. In some embodiments, the triazole moiety (e.g., a triazole group) is substituted. In some embodiments, the triazole moiety is unsubstituted. In some embodiments, the modified internucleotide link comprises an optionally substituted cyclic guanidine moiety. In some embodiments, the modified internucleotide link has… The structure is optionally chiral controlled, where R 1 It is −L−R', where L is L as described in this article. B And R' is as described herein. In some embodiments, each R 1 Each R' is independently R. In some embodiments, each R' is independently R. In some embodiments, two Rs are independent Rs. 1 They are R atoms and come together to form a ring as described herein. In some embodiments, the two R atoms on two different nitrogen atoms... 1 R are together and form a ring as described herein. In some embodiments, R 1 Independently, C is optionally substituted as described herein. 1-6 Aliphatic group. In some embodiments, R 1 It is a methyl group. In some embodiments, the two R' on the same nitrogen atom are R and together form a ring as described herein. In some embodiments, the modified nucleotide interlinking has The structure is optionally chiral controlled. In some embodiments, yes In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety and has the following structure: , or Where W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the internucleotide bonding without negative charge is stereochemically controlled.

[0719] In some embodiments, the uncharged or neutral internucleotide linkages are internucleotide linkages comprising a triazole moiety. In some embodiments, internucleotide linkages comprising a triazole moiety (e.g., optionally substituted triazole groups) have The structure. In some embodiments, the internucleotide linkages comprising the triazole moiety have The structure. In some embodiments, the internucleotide linkages comprising the triazole moiety have The formula is given, where W is O or S. In some embodiments, the internucleotide linkages comprising an alkyne moiety (e.g., optionally substituted alkynyl group) have... The formula is given, where W is O or S. In some embodiments, the internucleotide link (e.g., an uncharged internucleotide link, a neutral internucleotide link) includes a cyclic guanidine moiety. In some embodiments, the internucleotide link including the cyclic guanidine moiety moiety has... The structure. In some embodiments, the uncharged nucleotide linking or neutral nucleotide linking is or includes a selection from... , , or The structure is where W is O or S. In some embodiments, the internucleotide linkage, for example, an uncharged internucleotide linkage or a neutral internucleotide linkage, includes a cyclic guanidine moiety. In some embodiments, the internucleotide linkage including the cyclic guanidine moiety moiety has The structure. In some embodiments, uncharged nucleotide linkages or neutral nucleotide linkages are or include the structure. , where W is O or S.

[0720] In some embodiments, the internucleotide linking includes a Tmg group ( In some embodiments, the internucleotide linking includes a Tmg group and has The structure (“Tmg nucleotide linkage”). In some embodiments, neutral nucleotide linkages include nucleotide linkages between PNA and PMO as well as Tmg nucleotide linkages.

[0721] In some embodiments, the uncharged internucleotide linkages have structures of formulas I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or their salts. In some embodiments, the uncharged internucleotide linkages comprise optionally substituted 3-20-membered heterocyclic or heteroaryl groups having 1-10 heteroatoms. In some embodiments, the uncharged internucleotide linkages comprise optionally substituted 3-20-membered heterocyclic or heteroaryl groups having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such heterocyclic or heteroaryl groups have a 5-membered ring. In some embodiments, such heterocyclic or heteroaryl groups have a 6-membered ring.

[0722] In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-20-membered heteroaryl group having 1-10 heteroatoms. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-20-membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-6-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the heteroaryl group is directly bonded to the linking phosphorus.

[0723] In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-20-membered heterocyclic group having 1-10 heteroatoms. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-20-membered heterocyclic group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-6-membered heterocyclic group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted 5-membered heterocyclic group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, the uncharged internucleotide linking comprises an optionally substituted triazole group. In some embodiments, the uncharged internucleotide linking comprises an unsubstituted triazole group, for example, In some embodiments, the non-negatively charged internucleotide linking comprises a substituted triazole group, for example, .

[0724] In some embodiments, the heterocyclic group is directly bonded to the linking phosphorus. In some embodiments, when the heterocyclic group is part of a guanidine moiety that is directly bonded to the linking phosphorus via its =N- group, the heterocyclic group is bonded to the linking phosphorus via a linker (e.g., =N-). In some embodiments, the non-negatively charged internucleotide linking comprises optionally substituted... Groups. In some embodiments, the internucleotide linkages without negative charge contain substituted groups. Groups. In some embodiments, the internucleotide linkages without negative charge include... Groups, wherein each R 1 Independently, it is -LR. In some embodiments, each R 1 Independently, C is optionally substituted 1-6 Alkyl group. In some embodiments, each R 1 It is methyl on its own.

[0725] In some embodiments, the modified internucleotide link (e.g., an uncharged internucleotide link) comprises a triazole or alkyne moiety, each optionally substituted. In some embodiments, the modified internucleotide link comprises a triazole moiety. In some embodiments, the modified internucleotide link comprises an unsubstituted triazole moiety. In some embodiments, the modified internucleotide link comprises a substituted triazole moiety. In some embodiments, the modified internucleotide link comprises an alkyl moiety. In some embodiments, the modified internucleotide link comprises an optionally substituted alkyne group. In some embodiments, the modified internucleotide link comprises an unsubstituted alkyne group. In some embodiments, the modified internucleotide link comprises a substituted alkyne group. In some embodiments, the alkyne group is directly bonded to the linked phosphorus.

[0726] In some embodiments, the ds oligonucleotide comprises different types of internucleotide phosphate linkages. In some embodiments, the chiral-controlled oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotide linkage. In some embodiments, the ds oligonucleotide comprises at least one natural phosphate linkage and at least one thiophosphate linkage. In some embodiments, the ds oligonucleotide comprises at least one uncharged internucleotide linkage. In some embodiments, the ds oligonucleotide comprises at least one natural phosphate linkage and at least one uncharged internucleotide linkage. In some embodiments, the ds oligonucleotide comprises at least one thiophosphate internucleotide linkage and at least one uncharged internucleotide linkage. In some embodiments, the ds oligonucleotide comprises at least one thiophosphate internucleotide linkage, at least one natural phosphate linkage, and at least one uncharged internucleotide linkage. In some embodiments, the ds oligonucleotide comprises one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) uncharged internucleotide bonds. In some embodiments, the uncharged internucleotide bonds are neutral because less than 50%, 40%, 40%, 30%, 20%, 10%, 5% or 1% of the internucleotide bonds are present in aqueous solution at a given pH as negatively charged salts. In some embodiments, the pH is about pH 7.4. In some embodiments, the pH is about 4-9. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, the internucleotide linkage is an uncharged internucleotide linkage because the neutral form of the internucleotide linkage in water does not have a pKa of more than about 1, 2, 3, 4, 5, 6, or 7. In some embodiments, no pKa is 7 or less. In some embodiments, no pKa is 6 or less. In some embodiments, no pKa is 5 or less. In some embodiments, no pKa is 4 or less. In some embodiments, no pKa is 3 or less. In some embodiments, no pKa is 2 or less. In some embodiments, no pKa is 1 or less. In some embodiments, the pKa of the neutral form of the internucleotide linkage may be represented as the pKa of the neutral form of a compound having the structure CH3−nucleotide linkage−CH3. For example, the pKa of the neutral form of the internucleotide linkage having the structure of Formula I may be represented by a compound having the structure CH3−nucleotide linkage−CH3. The pKa of the neutral form of the compound is represented by the structure (where X, Y, Z are independently -O-, -S-, -N(R')-; L is L). B And R 1 It is −L−R'). pKa can be determined by pKa This indicates that, in some embodiments, the uncharged nucleotide linkages are neutral nucleotide linkages. In some embodiments, the uncharged nucleotide linkages are positively charged nucleotide linkages. In some embodiments, the uncharged nucleotide linkages include a guanidine moiety. In some embodiments, the uncharged nucleotide linkages include a heteroaryl base moiety. In some embodiments, the uncharged nucleotide linkages include a triazole moiety. In some embodiments, the uncharged nucleotide linkages include an alkynyl moiety.

[0727] In some embodiments, the neutral or uncharged nucleotide linking has a structure of any of the following neutral or uncharged nucleotide linking: US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073、WO 2018 / 223081、WO 2018 / 237194、WO 2019 / 032607、WO2019 / 032612、WO 2019 / 055951、WO 2019 / 075357、WO 2019 / 200185、WO 2019 / 217784 and / or WO 2019 / 032612,2607, WO2019032612, WO 2019 / 055951, WO2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612, in which each of the neutral or uncharged nucleotides is linked by reference hereto.

[0728] In some embodiments, each R' is independently a optionally substituted C 1-6 Aliphatic. In some embodiments, each R' is independently an optionally substituted C. 1-6 Alkyl group. In some embodiments, each R' is independently −CH3. In some embodiments, each R s It is −H.

[0729] In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, the internucleotide linkages without negative charge have The structure. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the neutral nucleotide linking is the above-described uncharged nucleotide linking.

[0730] In some embodiments, the provided ds oligonucleotide comprises one or more nucleotide links of formula I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, as described in: US 9394333, US 9744183, US 9605019, US 9982257, US20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679、WO 2017 / 210647、WO 2018 / 098264、WO 2018 / 022473、WO 2018 / 223056、WO 2018 / 223073、WO 2018 / 223081、WO 2018 / 237194、WO 2019 / 032607, WO2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612,2607, WO2019032612, WO 2019 / 055951、WO WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO 2019 / 032612, formulas I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1 or II-d-2 or their salt forms, each of which is independently incorporated herein by reference.

[0731] In some embodiments, the ds oligonucleotide comprises neutral nucleotide links and chiral-controlled nucleotide links. In some embodiments, the ds oligonucleotide comprises neutral nucleotide links and chiral-controlled nucleotide links that are not neutral nucleotide links. In some embodiments, the ds oligonucleotide comprises neutral nucleotide links and chiral-controlled phosphate-thionucleotide links. In some embodiments, this disclosure provides a ds oligonucleotide comprising one or more negatively charged nucleotide links and one or more phosphate-thionucleotide links, wherein each phosphate-thionucleotide link in the oligonucleotide is independently a chiral-controlled nucleotide link. In some embodiments, this disclosure provides a ds oligonucleotide comprising one or more neutral nucleotide links and one or more phosphate-thionucleotide links, wherein each phosphate-thionucleotide link in the ds oligonucleotide is independently a chiral-controlled nucleotide link. In some embodiments, the ds oligonucleotide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chiral-controlled inter-nucleotide thiophosphate linkages. In some embodiments, the inter-nucleotide linkages of uncharged nucleotides are chiral-controlled. In some embodiments, the inter-nucleotide linkages of uncharged nucleotides are not chiral-controlled. In some embodiments, the inter-nucleotide linkages of neutral nucleotides are chiral-controlled. In some embodiments, the inter-nucleotide linkages of neutral nucleotides are not chiral-controlled.

[0732] Without being bound by any particular theory, this disclosure states that neutral nucleotide inter-linking can be more hydrophobic than phosphate thioester (PS) inter-linking, which in turn can be more hydrophobic than native phosphate ester (PO) inter-linking. Typically, unlike PS or PO, neutral nucleotide inter-linking has less charge. Without being bound by any particular theory, this disclosure states that incorporating one or more neutral nucleotide inter-linkings into ds oligonucleotides can increase the ability of ds oligonucleotides to be taken up by cells and / or escaped from endosomes. Without being bound by any particular theory, this disclosure states that incorporating one or more neutral nucleotide inter-linkings can be used to modulate the melting temperature of the double strand formed between the ds oligonucleotide and its target nucleic acid.

[0733] Without being bound by any particular theory, this disclosure states that incorporating one or more uncharged nucleotide inter-linkings (e.g., neutral nucleotide inter-linkings) into ds oligonucleotides may enhance the ability of ds oligonucleotides to mediate functions such as target adenosine editing.

[0734] As will be understood by those skilled in the art, nucleotide linkages, such as natural phosphate ester linkages and those of formulas I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or their salts, typically link two nucleosides (which may be natural or modified), as in US9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741、WO 2017 / 192679、WO 2017 / 210647、WO 2018 / 098264、WO 2018 / 022473、WO 2018 / 223056、WO 2018 / 223073、WO 2018 / 223081、WO 2018 / 237194, WO 2019 / 032607, WO2019032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784 and / or WO As described in 2019 / 032612, formulas I, Ia, Ib, Ic, In-1, In-2, In-3, In-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or their salts, each of which is independently incorporated herein by reference. Typical linkages in natural DNA and RNA are nucleotide-to-nucleotide bonds formed by two sugars (which may be unmodified or modified as described herein). In many embodiments, as illustrated herein, the nucleotide-to-nucleotide bond is formed by its oxygen atom or heteroatom (e.g., Y and Z in the various formulas) with one optionally modified ribose or deoxyribose at its 5' carbon and another optionally modified ribose or deoxyribose at its 3' carbon. In some embodiments, each nucleoside unit linked by internucleotide bonds independently comprises a nucleobase that is independently an optionally substituted A, T, C, G or U or a substituted tautomer of A, T, C, G or U, or a nucleobase comprising an optionally substituted heterocyclic group and / or a heteroaryl ring having at least one nitrogen atom.

[0735] In some embodiments, the bonding has or includes −Y−P L (−X−R LThe structure of )−Z− or its salt form, wherein:

[0736] P L It is P, P(=W), P−>B(–L) L –R L )3 or P N ;

[0737] W is O, N(–L) L –R L ), S or Se;

[0738] P N It is P=N−C(–L) L –R')(=L N -R') or P=N–L L –R L ;

[0739] L N =N−L L1 -、=CH−L L1 - (where CH is optionally substituted) or = N + (R')(Q − )−L L1 -;

[0740] Q − It is an anion;

[0741] Each of X, Y, and Z is independently –O–, –S–, −L. L −N(–L L –R L )−L L −、−L L −N=C(–L L –R L )−L L -or L L ;

[0742] Each R L Independently is −L L –N(R')2、–L L –R'、−N=C(–L L –R')2、−L L –N(R')C(NR')N(R')2、−L L –N(R')C(O)N(R')2, carbohydrate or one or more other chemical components optionally connected by a linker;

[0743] L L1 and L L Each of them is L independently;

[0744] -CyIL -is-Cy-;

[0745] Each L is independently a covalent bond or selected from C. 1-30 Aliphatic groups and C with 1-10 heteroatoms 1-30 A divalent, optionally substituted, straight-chain or branched group of a heteroaliphatic group, wherein one or more methylene units are optionally and independently substituted by a group selected from the following optionally substituted groups: C 1-6 Alkylene, C 1-6 alkenyl, Divalent C1–C6 heteroaliphatic groups with 1–5 heteroatoms, −C(R')2−, −Cy−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(NR')N(R')−, −N(R')C(NR')N(R')−, −C(O)N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, −C(O)O−, −P(O)(OR')−, −P(O)(SR')−, −P(O)(R')−, −P(O)(NR')−, −P(S)(OR ')−、−P(S)(SR')−、−P(S)(R')−、−P(S)(NR')−、−P(R')−、−P(OR')−、−P(SR')−、−P(NR')−、−P(OR')[B(R')3]−、−OP(O)(OR')O−、−OP(O)(SR')O−、−OP(O)(R')O−、−OP(O)(NR')O−、−OP(OR')O−、−OP(SR')O−、−OP(NR')O−、−OP(R')O−、−OP(OR')[B(R')3]O− and −[C(R')2C(R')2O]n−, where n is 1-50, and one or more nitrogen or carbon atoms are optionally and independently controlled by Cy L Substitute;

[0746] Each -Cy- is independently a divalent 3- to 30-membered monocyclic, bicyclic, or polycyclic ring with 0 to 10 heteroatoms, which is optionally substituted.

[0747] Each Cy L Independently, it is a trivalent or tetravalent 3-30 member monocyclic, bicyclic or polycyclic, optionally substituted, having 0-10 heteroatoms;

[0748] Each R' is independently −R, −C(O)R, −C(O)N(R)2, −C(O)OR, or −S(O)2R;

[0749] Each R is independently -H, or a group selected from the following optionally substituted groups: C 1-30 Aliphatic groups, C with 1-10 heteroatoms 1-30 heteroaliphatic groups, C 6-30 Aryl, C 6-30 aryl aliphatic group, C with 1-10 heteroatoms 6-30 Aryl heteroaliphatic groups, 5-30 membered heteroaryl groups having 1-10 heteroatoms, and 3-30 membered heterocyclic groups having 1-10 heteroatoms, or

[0750] The two R groups optionally and independently form a covalent bond together, or:

[0751] Two or more R groups on the same atom optionally and independently form, together with the atom, a substituted 3-30 member monocyclic, bicyclic, or polycyclic atom having 0-10 heteroatoms in addition to the atom; or

[0752] Two or more R groups on two or more atoms optionally and independently combine with the atoms between them to form an optionally substituted 3-30 member monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms between them.

[0753] In some embodiments, the internucleotide bond has −O−P L (−X−R L The structure is −O−, where each variable is independent as described herein. In some embodiments, the internucleotide linkage has −O−P(=W)(−X−R). L The structure is −O−, where each variable is independent as described herein. In some embodiments, the internucleotide linking has −O−P(=W)[−N(–L L –R L )−R L The structure is −O−, where each variable is independent as described herein. In some embodiments, the internucleotide linkage has −O−P(=W)(−NH–L L –R L The structure is −O−P(=W)[−N(R')2]−O−, where each variable is independent as described herein. In some embodiments, the internucleotide linking has the structure −O−P(=W)(−NHR')−O−, where each variable is independent as described herein. In some embodiments, the internucleotide linking has the structure −O−P(=W)(−NHSO2R)−O−, where each variable is independent as described herein. In some embodiments, the internucleotide linking has the structure −O−P(=W)[−N=C(–L L–R')2]−O− structure, wherein each variable is independent as described herein. In some embodiments, the internucleotide link has the structure −O−P(=W)[−N=C[N(R')2]2]−O−, wherein each variable is independent as described herein. In some embodiments, the internucleotide link has the structure −OP(=W)(−N=C(R")2)−O−, wherein each variable is independent as described herein. In some embodiments, the internucleotide link has the structure −OP(=W)(−N(R")2)−O−, wherein each variable is independent as described herein. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, such an internucleotide link is an uncharged internucleotide link. In some embodiments, such an internucleotide link is a neutral internucleotide link.

[0754] In some embodiments, the internucleotide bond has −P L (−X−R L The structure is )−Z−, where eac...

Claims

1. A composition comprising a double-stranded RNAi (dsRNAi) agent capable of directing INHBE (inhibitor subunit βE)-specific RNA interference to induce lipolysis while preserving muscle mass, said dsRNAi agent comprising a guide strand and a transit strand, wherein: e) The guide strand is complementary to or substantially complementary to the INHBE target RNA sequence; f) The bootstrap chain includes: i. Sp-configured uncharged nucleotide linkage between the +3 nucleotide relative to the 5' terminal nucleotide and the immediately adjacent downstream (+4) nucleotide; ii. An Rp-configured, uncharged nucleotide bond between a +10 nucleotide and its immediate downstream (+11) nucleotide; iii. Sp-configured phosphate ester nucleotide linkages between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide; and / or iv. Linkage between phosphate thioester nucleotides in Rp, Sp, or alternating configurations between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide, and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide; c) The guiding chain further comprises 5' phosphate modification; d) The transient chain comprises one or more chiral nucleotides in the Rp or Sp configuration linked together; and e) The guide strand and the transit strand each have an independent length of 15-49 nucleotides.

2. The composition of claim 1, wherein the leading chain comprises a 5' phosphate modification selected from: Bases: A, C, G, T, U, no bases, and modified nucleobases; R: H, OH, O-alkyl, F, MOE, LNA bridge to 4' position, BNA bridge to 4' position.

3. The composition according to claim 2, wherein the leading chain comprises a 5' phosphate modification selected from 5' MeP modification and 5' triazole-P modification.

4. The double-stranded oligonucleotide or composition according to claim 14, wherein the 5' MeP modification is... .

5. The composition of claim 1, wherein the leader chain comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotide bonds, the internucleotide bonds occurring between any two adjacent nucleotides between the second (+2) nucleotide of the 5' terminal nucleotide of the leader chain and the penultimate 3' (N-1) nucleotide of the leader chain, wherein N is the 3' terminal nucleotide, and the guest chain comprises one or more skeletal chiral centers in the form of Rp or Sp.

6. The composition of claim 1, wherein the leading chain comprises a skeletal phosphate chiral center in the Sp configuration, the skeletal phosphate chiral center being located between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately preceding (N-2) upstream nucleotide, and the guest chain comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide bonds, wherein n is about 1 to 49, and one or more skeletal chiral centers in the Rp or Sp configuration.

7. The composition of claim 1, wherein the leading chain comprises a skeletal phosphate chiral center in an Rp, Sp, or alternating configuration, the skeletal phosphate chiral center being located between the 5' terminal (+1) nucleotide and the immediately adjacent downstream (+2) nucleotide and between the +2 nucleotide and the immediately adjacent downstream (+3) nucleotide, and the guest chain comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide bonds, wherein n is about 1 to 49, and one or more skeletal chiral centers in an Rp or Sp configuration.

8. The composition of claim 1, wherein the leading chain comprises one or more skeletal phosphate thioester chiral centers of Rp or Sp configuration, the skeletal phosphate thioester chiral centers being upstream of the Sp configuration skeletal phosphate thioester chiral centers between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately adjacent upstream (N-2) nucleotide, and the guest chain comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide bonds, wherein n is about 1 to 49, and one or more skeletal chiral centers of Rp or Sp configuration.

9. The composition of claim 1, wherein the leader chain comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotide links, the internucleotide links occurring between any two adjacent nucleotides between the second (+2) nucleotide of the 5' terminal nucleotide of the leader chain and the penultimate 3' (N-1) nucleotide of the leader chain, wherein N is the 3' terminal nucleotide, and the guest chain comprises 0 to n non-negatively charged internucleotide links, wherein n is about 1 to 49, and one or more skeletal chiral centers in the Rp or Sp configuration.

10. The composition according to any one of the preceding claims, wherein the inter-linking of Rp, Sp or stereorandom non-negatively charged backbone nucleotides has a neutral charge.

11. The composition according to claim 10, wherein the neutral backbone nucleotides are linked as follows: .

12. The composition of claim 11, wherein the guide chain comprises having the following structure The bonding, wherein the bonding is between the third (+3) and fourth (+4) nucleotides of the leader chain, between the tenth (+10) and eleventh (+11) nucleotides of the leader chain, or both.

13. The composition of claim 12, wherein the transit chain comprises having the following structure The bond is at the 5' of the central nucleotide of the transit chain, at the 3' of the central nucleotide of the transit chain, or both.

14. The composition according to claim 1, wherein the leader strand and guest strand that independently share a common base sequence, a common base modification pattern, a common sugar modification pattern and / or a common nucleotide linking pattern in the composition are at least 90% of all leader strands and guest strands in the composition.

15. The composition according to any one of the preceding claims, wherein the double-stranded oligonucleotide comprises a carbohydrate moiety optionally linked by a linker at the nucleoside / nucleotide link.

16. The composition according to any one of the preceding claims, wherein the double-stranded oligonucleotide comprises a lipid moiety optionally linked to the double-stranded oligonucleotide via a linker at a nucleoside-nucleotide inter-linkage site.

17. The composition according to any one of the preceding claims, wherein one or both strands of the double-stranded oligonucleotide contain a target portion optionally linked by a linker at the nucleoside / nucleotide link.

18. The composition according to any one of the preceding claims, wherein at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the internucleotide linkages of the double-stranded oligonucleotide are independently chiral internucleotide linkages.

19. The composition according to any one of the preceding claims, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the nucleotide units of the double-stranded oligonucleotide independently comprise 2'-substitution.

20. The composition according to any one of the preceding claims, wherein the 2'-substitution of the oligonucleotide is 2'-F.

21. The composition according to any one of the preceding claims, wherein the 2'-substitution of the oligonucleotide is 2'-OR1.

22. The composition according to any one of the preceding claims, wherein the 2'-substituted oligonucleotide is -L-, wherein L links C2 and C4 of the sugar unit.

23. The composition according to any one of the preceding claims, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the nucleotide units of the double-stranded oligonucleotide do not contain 2'-substitution.

24. The composition according to any one of the preceding claims, wherein the guide chain comprises an INHBE target-binding sequence that is completely complementary to the INHBE target sequence, wherein the INHBE target-binding sequence has a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases, wherein each base is optionally substituted adenine, cytosine, guanosine, thymine or uracil.

25. The composition according to any one of the preceding claims, wherein the INHBE target sequence comprises two SNPs.

26. The composition according to any one of the preceding claims, wherein the INHBE target sequence comprises an allele site, and the INHBE target binding sequence is completely complementary to the INHBE target sequence of a disease-associated allele, but not completely complementary to the target sequence of an allele with low association with the disease.

27. The composition according to any one of the preceding claims, wherein The double-stranded oligonucleotide includes a guide strand that binds to the transcript of the INHBE target nucleic acid sequence. For the INHBE target nucleic acid sequence, multiple alleles exist in the population, and each of the multiple alleles contains a specific nucleotide sequence element that defines the allele relative to other alleles of the same INHBE target nucleic acid sequence. The base sequence of the guide strand is or contains a sequence complementary to the characteristic sequence element defining the target allele, and The guide strand is characterized in that, when brought into contact with a cell containing a transcript of the INHBE target nucleic acid sequence, it exhibits inhibition of the transcript of the target allele or the protein encoded by the transcript at a level higher than that observed for another allele of the same nucleic acid sequence.

28. The composition according to claim 1, comprising DSR-0104068, DSR-0104099, DSR-0104072, DSR-0104075, DSR-0104083, DSR-0104091, DSR-0104071 or DSR-0104108.

29. A method for reducing the level and / or activity of an INHBE transcript or a protein encoded therefrom, the method comprising administering to cells expressing the INHBE transcript a composition according to any one of the preceding claims, wherein the guide strand of the double-stranded oligonucleotide or composition comprises an INHBE binding sequence that is completely complementary to the INHBE target sequence in the transcript.

30. The method of claim 29, wherein the cell is a hepatocyte.

31. A method for treating a metabolic disorder, the method comprising administering a composition according to any one of claims 1 to 28.

32. The method of claim 31, wherein the metabolic disorder is metabolic syndrome.

33. A method for treating obesity, the method comprising administering a composition according to any one of claims 1 to 28.

34. A method for treating cardiovascular disease, the method comprising administering a composition according to any one of claims 1 to 28.

35. A method for treating diabetes, the method comprising administering the composition according to any one of claims 1 to 28.

36. A method for treating hypertension, the method comprising administering a composition according to any one of claims 1 to 28.

37. The method according to any one of claims 31 to 36, wherein the application of a second therapeutic agent is included.

38. The method of claim 37, wherein the second therapeutic agent is selected from the group consisting of: insulin; sulfonylureas; megglitinide; biguanide; thiazolidinedione; α-glucosidase inhibitors; SGLT2 inhibitors; DPP4 inhibitors; glucagon-like peptide-1 receptor agonists (GLP-1RA); glucose-dependent insulinotropic peptide agonists (GIP RA); glucagon receptor agonists (Gcg RA); co-agonists of GLP-1R, GIP R and / or Gcg R; HMG-CoA reductase inhibitors; statins; PCSK9 inhibitors; ApoC3 inhibitors; ANGPTL3 inhibitors; ATP citrate lyase (bepaidic acid); ezetimibe; Lp(a) inhibitors; LPL activators; and combinations thereof.

39. The method of claim 38, wherein the composition of any one of claims 1 to 28 is administered simultaneously with the second therapeutic agent.

40. The method of claim 39, wherein the composition of any one of claims 1 to 28 is administered sequentially with the second therapeutic agent.

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