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

Abstract

The present disclosure provides Inhibin subunit beta E (INHBE)-related double stranded oligonucleotides, compositions, and methods of using such double stranded oligonucleotides and compositions for preventing and / or treating various conditions, disorders, or diseases associated with expression of INHBE. In some embodiments, the provided double stranded oligonucleotides and compositions comprise nucleobase modifications, sugar modifications, internucleotidic linkage modifications and / or patterns thereof, and have improved properties, activities and / or selectivities. In some embodiments, the provided double stranded oligonucleotides and compositions target INHBE.

Description

[0001] INHIBIN SUBUNIT BETA E-RELATED DOUBLE STRANDED OLIGONUCLEOTIDE COMPOSITIONS AND METHODS RELATING THERETO

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] 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 herein by reference herein in their entirety.

[0004] TECHNICAL FIELD

[0005] Among other things, the present disclosure provides double stranded (ds) oligonucleotides, compositions and methods (e.g., of preparation, use, etc.) thereof. In some embodiments, provided technologies are useful for preventing and / or treating various conditions, disorders, or diseases associated with inhibin subunit beta E (INHBE) expression.

[0006] BACKGROUND

[0007] Metabolic disorders, e.g., metabolic syndrome, and related diseases, e.g., obesity, cardiovascular disease, diabetes, and hypertension are an increasing medical concern. Typical treatment for such disorders and diseases involves targeted approaches and / or changes in diet and lifestyle, e.g., increased physical activity. Particularly, treatment for obesity, which is a disorder involving excess body fat, entails exercise and a healthy, balanced diet leading to weight loss. There exists, however, a need for alternate treatment methods for metabolic diseases, particularly weight loss.

[0008] Double stranded (ds) oligonucleotides are useful in various applications, e.g., therapeutic, diagnostic, and / or research applications. Inhibin subunit beta E (INHBE), which is primarily expressed in the liver, has been linked with both body mass index and insulin resistance. As such, ds oligonucleotides targeting disorders or diseases associated with expression of INHBE can be useful treatment of such conditions including metabolic disorders, e.g., metabolic syndrome, and related diseases, e.g., obesity, cardiovascular disease, diabetes, and hypertension.

[0009] SUMMARY

[0010] In some embodiments, the present disclosure provides ds oligonucleotides targeting INHBE and compositions thereof that have significantly improved properties and / or high activities. Among other things, the present disclosure provides technologies for designing, manufacturing and utilizing such ds oligonucleotides and compositions. Particularly, in some embodiments, the present disclosure provides ds oligonucleotides comprising useful patterns of intemucleotidic linkages and / or patterns of sugar modifications, which, when combined with one or more other structural elements, e.g., base sequence (or portion thereof), nucleobase modifications (and patterns thereof), additional chemical moieties, etc., can provide ds oligonucleotides targeting INHBE and compositions thereof with high activities and / or desired properties, including but not limited to effective and efficient reduction of expression, levels and / or activities of INHBE transcripts and products encoded thereby. In some embodiments, ds oligonucleotides targeting INHBE and compositions reduce levels of a INHBE transcript, and are useful for treating and / or preventing INHBE-associated condition, disorder, or disease, including, but not limited to metabolic disorders, e.g., metabolic syndrome, and related diseases, e.g., obesity, cardiovascular disease, diabetes, and hypertension.

[0011] In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating knockdown of INHBE, wherein the level, expression and / or activity of INHBE or a product thereof are decreased. In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating pan-specific knockdown of INHBE, wherein the level, expression and / or activity of multiple or all INHBE alleles are decreased. In some embodiments, a ds oligonucleotide targeting INHBE has a base sequence that is complementary to a sequence which is common in multiple or all INHBE alleles.

[0012] In certain embodiments, such structural elements include one or more of: (1) chemical modifications (e.g., modifications of a sugar, base and / or intemucleotidic linkage) and patterns thereof; and (2) alterations in stereochemistry (e.g., stereochemistry of a backbone chiral intemucleotidic linkage) and patterns thereof. One or more of such structural elements can, in certain embodiments, be independently present in one or both oligonucleotides of a ds oligonucleotide. In certain embodiments, the properties and / or activities impacted by such structural elements include, but are not limited to, participation in, direction of a decrease in expression, activity or level of a gene or a gene product thereof, mediated, for example, by RNA interference (RNAi interference).

[0013] In certain embodiments, the present disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) with controlled structural elements provide unexpected properties and / or activities.

[0014] In certain embodiments, a dsRNAi agent capable of directing INHBE (Inhibin subunit PE)-specific RNA interference to induce lipolysis while preserving muscle mass, the dsRNAi agent comprises a guide strand and a passenger strand, wherein: a) the guide strand is complementary or substantially complementary to an INHBE target RNA sequence; b) the guide strand comprises: i. a non-negatively charged intemucleotidic linkage in the A'p configuration between the +3 nucleotide relative to the 5’ terminal nucleotide and the immediately downstream (+4) nucleotide; ii. a non-negatively charged intemucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide; iii. phosphorothioate intemucleotidic linkages in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide; and / or iv. phosphorothioate intemucleotidic linkages in Rp, A'p, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide; c) the guide strand further comprises a 5’ phosphate modification; d) the passenger strand comprises one or more chiral intemucleotidic linkages in Rp or Sp configuration; and e) the guide strand and the passenger strand each independently has a length of 15-49 nucleotides.

[0015] In alternative embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry of backbone chiral centers, can unexpectedly maintain or improve properties of ds oligonucleotides. For example, but not by way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising one or more of

[0016] (1) a guide strand comprising backbone non-negatively charged intemucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0017] (2) a guide strand comprising backbone non-negatively charged intemucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction; (3) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0018] (4) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0019] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0020] (6) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction;

[0021] (7) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification;

[0022] (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more backbone chiral centers in Rp or Sp configuration; and

[0023] (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide; wherein the ds oligonucleotide further comprises one or more of

[0024] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by a Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0025] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0026] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0027] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0028] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’-F modification, of the 3’ nucleotide of a nucleotide pair linked by a Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0029] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry of chiral centers at a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification of guide strands, can unexpectedly maintain or improve properties of the ds oligonucleotides described herein. For example, but not by way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide stranding comprising: (1) a phosphorothioate chiral center in Rp or Sp configuration; (2) an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage where the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage comprises a 2’ modification, e.g., a 2’ F; and (3) a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification selected from: a

[0030] (a) 5’ PO modifications, such as, but not limited to:

[0031] (b) 5’ VP modifications, such as, but not limited to: (c) 5’ MeP modifications, such as, but not limited to:

[0032] (d) 5’ PN and 5’ triazole-P modifications, such as, but not limited to: wherein Base is selected from A, C, G, T, U, abasic and modified nucleobases; R2is selected from H, OH, O-alkyl, F, MOE, locked nucleic acid (LNA) bridges and bridged nucleic acid (BNA) bridges to the 4’ C, such as, but not limited to:

[0033] In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non-negatively charged intemucleotidic linkage. In certain embodiments, the guide strand comprises, a backbone phosphorothioate chiral center in Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide, and a backbone phosphorothioate chiral center in the Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0034] In certain embodiments, the guide strand comprises a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification selected from, but not limited to, 5’ MeP modifications and 5’ triazole-P modifications. In certain embodiments, the 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification is . In some embodiments, the 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification i certain embodiments, the 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification is In certain embodiments, the guide strand comprises the

[0035] 5’ MeP modification , a backbone phosphorothioate chiral center in Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide, and a backbone phosphorothioate chiral center in the Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0036] In certain other embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry of chiral centers at the 5’ terminal nucleotide of guide strands, can unexpectedly maintain or improve properties of ds oligonucleotides wherein the guide strand of the ds oligonucleotide also comprises a phosphorothioate chiral center in Rp or Sp configuration. For example, but not by way of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide stranding comprising: (1) a phosphorothioate chiral center in Rp or Sp configuration; (2) an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage where the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage comprises a 2’ modification, e.g., a 2’ F; and (3) a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification selected from:

[0037] (a) 5’ PO nucleotides, such as, but not limited to:

[0038] (b) 5’ VP nucleotides, such as, but not limited to:

[0039] (c) 5’ MeP nucleotides, such as, but not limited to:

[0040] (d) 5’ PN and 5’ triazole-P nucleotides, such as, but not limited to:

[0041] (e) 5’ abasic VP and 5’ abasic MeP nucleotides, such as, but not limited to:

[0042] In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non-negatively charged intemucleotidic linkage. In certain embodiments, the present disclosure encompasses the recognition that non- naturally occurring internucleotidic linkages, e.g., neutral internucleotidic linkages, can, in certain embodiments, be used to link one or more molecules to the double-stranded oligonucleotides described herein. In certain embodiments, such linked molecules can facilitate targeting and / or delivery of the double-stranded oligonucleotide. For example, but not limitation, such linked molecules an include lipophilic molecules. In certain embodiments, the linked molecule is a molecule comprising one or more GalNAc moieties. In certain embodiments, the linked molecule is a receptor. In certain embodiments, the linked molecule is a receptor ligand.

[0043] In certain embodiments, the present disclosure provides technologies for incorporating various additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides, for example, reagents and methods for introducing additional chemical moieties through nucleobases (e.g., by covalent linkage, optionally via a linker, to a site on a nucleobase).

[0044] In certain embodiments, the present disclosure provides technologies, e.g., ds oligonucleotide compositions and methods thereof, that achieve allele-specific suppression, wherein transcripts from one allele of a particular target gene is selectively knocked down relative to at least one other allele of the same gene.

[0045] Among other things, the present disclosure provides structural elements, technologies and / or features that can be incorporated into ds oligonucleotides and can impart or tune one or more properties thereof (e.g., relative to an otherwise identical ds oligonucleotide lacking the relevant technology or feature). In certain embodiments, the present disclosure documents that one or more provided technologies and / or features can usefully be incorporated into ds oligonucleotides of various sequences.

[0046] In certain embodiments, the present disclosure demonstrates that certain provided structural elements, technologies and / or features are particularly useful for ds oligonucleotides that participate in and / or direct RNAi mechanisms (e.g., RNAi agents). Regardless, however, the teachings of the present disclosure are not limited to ds oligonucleotides that participate in or operate via any particular mechanism. In certain embodiments, the present disclosure pertains to any ds oligonucleotide, useful for any purpose, which operates through any mechanism, and which comprises any sequence, structure or format (or portion thereof) described herein. In certain embodiments, the present disclosure provides a ds oligonucleotide, useful for any purpose, which operates through any mechanism, and which comprises any sequence, structure or format (or portion thereof) described herein, comprising one or more of: (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0047] (2) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0048] (3) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0049] (4) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0050] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0051] (6) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction;

[0052] (7) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification; wherein the ds oligonucleotide further comprises one or more of

[0053] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0054] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0055] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0056] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0057] (5) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0058] In certain embodiments, the provided ds oligonucleotides may participate in (e.g., direct) RNAi mechanisms.

[0059] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N- 2) nucleotide, and one or more of: (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0060] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0061] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0062] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0063] (5) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkages, where n is about 1 to 49,

[0064] In certain embodiments, the present disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) with controlled structural elements provide unexpected properties and / or activities.

[0065] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of: (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0066] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0067] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0068] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0069] (5) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non-negatively charged intemucleotidic linkage. In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone phosphorothioate chiral centers in Sp configuration between the 3 ’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of

[0070] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0071] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0072] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0073] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0074] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0075] In certain embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the second (+2) and third (+3) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and the internucleotidic linkage to the penultimate 3’ (N-l) nucleotide, and one or more of:

[0076] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0077] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0078] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0079] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0080] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0081] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N- 2) nucleotide, and one or more of:

[0082] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0083] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0084] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0085] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0086] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0087] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0088] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0089] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0090] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0091] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of:

[0092] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0093] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide; (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0094] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0095] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0096] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0097] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0098] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0099] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0100] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0101] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0102] In certain embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non- negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non- negatively charged intemucleotidic linkage.

[0103] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N- 2) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non- negatively charged intemucleotidic linkage incorporated into the guide strand is an Rp non- negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non- negatively charged intemucleotidic linkage.

[0104] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged intemucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non-negatively charged intemucleotidic linkage.

[0105] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone phosphorothioate chiral centers in Sp configuration between the 3 ’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0106] In certain embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non- negatively charged internucleotidic linkage.

[0107] In certain embodiments, provided ds oligonucleotides may participate in exon skipping mechanisms. In certain embodiments, provided ds oligonucleotides may be aptamers. In certain embodiments, provided ds oligonucleotides may bind to and inhibit the function of a protein, small molecule, nucleic acid or cell. In certain embodiments, provided ds oligonucleotides may participate in forming a triplex helix with a double-stranded nucleic acid in the cell. In certain embodiments, provided ds oligonucleotides may bind to genomic (e.g., chromosomal) nucleic acid. In certain embodiments, provided ds oligonucleotides may bind to genomic (e.g, chromosomal) nucleic acid, thus preventing or decreasing expression of the nucleic acid (e.g., by preventing or decreasing transcription, transcriptional enhancement, modification, etc.). In certain embodiments, provided ds oligonucleotides may bind to DNA quadruplexes. In certain embodiments, provided ds oligonucleotides may be immunomodulatory. In certain embodiments, provided ds oligonucleotides may be immunostimulatory. In certain embodiments, provided oligonucleotides may be immunostimulatory and may comprise a CpG sequence. In certain embodiments, provided ds oligonucleotides may be immunostimulatory and may comprise a CpG sequence and may be useful as an adjuvant. In certain embodiments, provided ds oligonucleotides may be immunostimulatory and may comprise a CpG sequence and may be useful as an adjuvant in treating a disease (e.g., an infectious disease or cancer). In certain embodiments, provided ds oligonucleotides may be therapeutic. In certain embodiments, provided ds oligonucleotides may be non-therapeutic. In certain embodiments, provided ds oligonucleotides may be therapeutic or non-therapeutic. In certain embodiments, provided ds oligonucleotides are useful in therapeutic, diagnostic, research and / or nanomaterials applications. In certain embodiments, provided ds oligonucleotides may be useful for experimental purposes. In certain embodiments, provided ds oligonucleotides may be useful for experimental purposes, e.g., as a probe, in a microarray, etc. In certain embodiments, provided ds oligonucleotides may participate in more than one biological mechanism; in certain such embodiments, for example, provided ds oligonucleotides may participate in both RNAi and RNase H mechanisms.

[0108] In certain embodiments, provided ds oligonucleotides are directed to an INHBE target (e.g., an INHBE target sequence, an INHBE target RNA, an INHBE target mRNA, an INHBE target pre-mRNA, an INHBE target gene, etc.). An INHBE target gene is a gene with respect to which expression and / or activity of one or more INHBE gene products (e.g., INHBE RNA and / or protein products) are intended to be altered. In certain embodiments, an INHBE target gene is intended to be inhibited. Thus, when a ds oligonucleotide as described herein acts on an INHBE target gene, presence and / or activity of one or more INHBE gene products are altered when the ds oligonucleotide is present as compared with when it is absent.

[0109] In certain embodiments, an INHBE target is a specific INHBE allele with respect to which expression and / or activity of one or more products (e.g., INHBE RNA and / or protein products) are intended to be altered. In certain embodiments, an INHBE target allele is one whose presence and / or expression is associated (e.g., correlated) with presence, incidence, and / or severity, of one or more INHBE associated diseases and / or conditions. Alternatively or additionally, in certain embodiments, an INHBE target allele is one for which alteration of level and / or activity of one or more INHBE gene products correlates with improvement (e.g., delay of onset, reduction of severity, responsiveness to other therapy, etc.) in one or more aspects of an INHBE associated disease and / or condition.

[0110] In certain embodiments, e.g., where presence and / or activity of a particular INHBE allele (an INHBE disease-associated allele) is associated (e.g., correlated) with presence, incidence and / or severity of one or more disorders, diseases and / or conditions, a different INHBE allele exists and is not so associated, or is associated to a lesser extent (e.g., shows less significant, or statistically insignificant correlation), ds oligonucleotides and methods thereof as described herein may preferentially or specifically target the associated allele relative to the one or more less-associated / unassociated allele(s), thus mediating allele-specific suppression.

[0111] In certain embodiments, an INHBE target sequence is an INHBE sequence to which an oligonucleotide as described herein binds. In certain embodiments, an INHBE target sequence is identical to, or is an exact complement of, an INHBE sequence of a provided oligonucleotide, or of consecutive residues therein (e.g., a provided oligonucleotide includes an INHBE targetbinding sequence that is identical to, or an exact complement of, an INHBE target sequence). In certain embodiments, an INHBE target-binding sequence is an exact complement of an INHBE target sequence of an INHBE transcript (e.g., pre-mRNA, mRNA, etc.). An INHBE target-binding sequence / target sequence can be of various lengths to provided oligonucleotides with desired activities and / or properties. In certain embodiments, an INHBE target binding sequence / target sequence comprises 5-50 (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) bases. In certain embodiments, a small number of differences / mismatches is tolerated between (a relevant portion of) an oligonucleotide and its target sequence, including but not limited to the 5’ and / or 3’-end regions of the INHBE target and / or oligonucleotide sequence. In certain embodiments, an INHBE target sequence is present within an INHBE target gene. In certain embodiments, an INHBE target sequence is present within an INHBE transcript (e.g., an mRNA and / or a pre-mRNA) produced from an INHBE target gene.

[0112] In certain embodiments, an INHBE target sequence includes one or more allelic sites (i.e., positions within an INHBE target gene at which allelic variation occurs). In certain embodiments, an allelic site is a mutation. In certain embodiments, an allelic site is a SNP. In some such embodiments, a provided oligonucleotide binds to one allele preferentially or specifically relative to one or more other alleles. In certain embodiments, a provided oligonucleotide binds preferentially to a disease-associated allele. For example, in certain embodiments, an oligonucleotide (or a target-binding sequence portion thereof) provided herein has a sequence that is, fully or at least in part, identical to, or an exact complement of a particular allelic version of an INHBE target sequence.

[0113] In certain embodiments, an oligonucleotide (or a target-binding sequence portion thereof) provided herein has a sequence that is identical to, or an exact complement of an INHBE target sequence comprising an allelic site, or an allelic site, of a disease-associated allele. In certain embodiments, an oligonucleotide provided herein has an INHBE target binding sequence that is an exact complement of an INHBE target sequence comprising an allelic site of an INHBE transcript of an allele (in certain embodiments, a disease-associated allele), wherein the allelic site is a mutation. In certain embodiments, an oligonucleotide provided herein has an INHBE target binding sequence that is an exact complement of an INHBE target sequence comprising an allelic site of an INHBE transcript of an allele (in certain embodiments, a disease-associated allele), wherein the allelic site is a SNP. In certain embodiments, a sequence is any sequence disclosed herein.

[0114] Unless otherwise noted, all sequences (including, but not limited to base sequences and patterns of chemistry, modification, and / or stereochemistry) are presented in 5’ to 3’ order, with the 5’ terminal nucleotide identified as the “+1” position and the 3’ terminal nucleotide identified either by the number of nucleotides of the full sequence or by “N”, with the penultimate nucleotide identified, e.g., as “N-l”, and so on.

[0115] In certain embodiments, the present disclosure provides compositions and methods related to an oligonucleotide which is specific to an INHBE target and which has any format, structural element or base sequence of any oligonucleotide disclosed herein.

[0116] In certain embodiments, the present disclosure provides compositions and methods related to an oligonucleotide which is specific to an INHBE target and which has or comprises the base sequence of any oligonucleotide disclosed herein, or a region of at least 15 contiguous 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 contiguous nucleotides can be optionally replaced by T or DNA T.

[0117] In certain embodiments, the present disclosure provides compositions and methods for RNA interference directed by a RNAi agent (also referred to as a RNAi oligonucleotides). In certain embodiments, oligonucleotides of such compositions can have a format, structural element or base sequence of an oligonucleotide disclosed herein. In certain embodiments, the present disclosure provides compositions and methods for RNase H-mediated knockdown of an INHBE target gene RNA directed by an oligonucleotide (e.g., an antisense oligonucleotide).

[0118] Provided oligonucleotides and oligonucleotide compositions can have any format, structural element or base sequence of any oligonucleotide disclosed herein. In certain embodiments, a structural element is a 5 ’-end structure, 5 ’-end region, 5 ’-nucleotide, seed region, post-seed region, 3 ’-end region, 3 ’-terminal dinucleotide, 3 ’-end cap, or any portion of any of these structures, GC content, long GC stretch, and / or any modification, chemistry, stereochemistry, pattern of modification, chemistry or stereochemistry, or a chemical moiety (e.g., including but not limited to, a targeting moiety, a lipid moiety, a GalNAc moiety, a carbohydrate moiety, etc.), any component, or any combination of any of the above.

[0119] In certain embodiments, the present disclosure provides compositions and methods of use of an oligonucleotide.

[0120] In certain embodiments, the present disclosure provides compositions and methods of use of an oligonucleotide which can direct both RNA interference and RNase H-mediated knockdown of an INHBE target gene RNA. In certain embodiments, oligonucleotides of such compositions can have a format, structural element or base sequence of an oligonucleotide disclosed herein.

[0121] In certain embodiments, an oligonucleotide directing a particular event or activity participates in the particular event or activity, e.g., a decrease in the expression, level or activity of a target gene or a gene product thereof. In certain embodiments, an oligonucleotide is deemed to “direct” a particular event or activity when presence of the oligonucleotide in a system in which the event or activity can occur correlates with increased detectable incidence, frequency, intensity and / or level of the event or activity.

[0122] In certain embodiments, a provided oligonucleotide comprises any one or more structural elements of an oligonucleotide as described herein, e.g. , a base sequence (or a portion thereof of at least 15 contiguous bases); a pattern of internucleotidic linkages (or a portion thereof of at least 5 contiguous internucleotidic linkage); a pattern of stereochemistry of internucleotidic linkages (or a portion thereof of at least 5 contiguous internucleotidic linkages); a 5 ’-end structure; a 5 ’-end region; a first region; a second region; and a 3 ’-end region (which can be a 3’-terminal dinucleotide and / or a 3’-end cap); and an optional additional chemical moiety; and, in certain embodiments, at least one structural element comprises a chirally controlled chiral center. In certain embodiments, a 3 ’-terminal dinucleotide can comprise two total nucleotides. In certain embodiments, an oligonucleotide further comprises a chemical moiety selected from, as non-limiting examples, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, a lipid moiety, and any other chemical moiety described herein or known in the art. In certain embodiments, a moiety that binds APGR is a moiety of GalNAc, or a variant, derivative or modified version thereof, as described herein and / or known in the art. In certain embodiments, an oligonucleotide is a RNAi agent. In certain embodiments, a first region is a seed region. In certain embodiments, a second region is a post-seed region.

[0123] In certain embodiments, a provided oligonucleotide comprises any one or more structural elements of a RNAi agent as described herein, e.g., 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 can be a 3’-terminal dinucleotide and / or a 3’-end cap); and an optional additional chemical moiety; and, in certain embodiments, at least one structural element comprises a chirally controlled chiral center. In certain embodiments, a 3 ’-terminal dinucleotide can comprise two total nucleotides. In certain embodiments, an oligonucleotide further comprises a chemical moiety selected from, as non-limiting examples, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety. In certain embodiments, a moiety that binds APGR is any GalNAc, or variant, derivative or modification thereof, as described herein or known in the art.

[0124] In certain embodiments, a provided oligonucleotide comprises any one or more structural elements of an oligonucleotide as described herein, e.g., a 5 ’-end structure, a 5 ’-end region, a first region, a second region, a 3 ’-end region, and an optional additional chemical moiety, wherein at least one structural element comprises a chirally controlled chiral center. In certain embodiments, the oligonucleotide comprises a span of at least 5 total nucleotides without 2’ -modifications. In certain embodiments, the oligonucleotide further comprises an additional chemical moiety selected from, as non-limiting examples, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety. In certain embodiments, a provided oligonucleotide is capable of directing RNA interference. In certain embodiments, a provided oligonucleotide is capable of directing RNase H-mediated knockdown. In certain embodiments, a provided oligonucleotide is capable of directing both RNA interference and RNase H-mediated knockdown. In certain embodiments, a first region is a seed region. In certain embodiments, a second region is a post-seed region.

[0125] In certain embodiments, a provided oligonucleotide comprises any one or more structural elements of a RNAi agent, e.g., a 5 ’-end structure, a 5 ’-end region, a seed region, a post-seed region, and a 3 ’-end region and an optional additional chemical moiety, wherein at least one structural element comprises a chirally controlled chiral center; and, in certain embodiments, the oligonucleotide is also capable of directing RNase H-mediated knockdown of a target gene RNA. In certain embodiments, the oligonucleotide comprises a span of at least 5 total 2’-deoxy nucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from, as non-limiting examples, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety, and any other additional chemical moiety described herein.

[0126] In certain embodiments, the present disclosure demonstrates that oligonucleotide properties can be modulated through chemical modifications. In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides which have a common base sequence and comprise one or more internucleotidic linkage, sugar, and / or base modifications. In certain embodiments, the present disclosure provides an oligonucleotide composition capable of directing RNA interference and comprising a first plurality of oligonucleotides which have a common base sequence and comprise one or more internucleotidic linkage, and / or one or more sugar, and / or one or more base modifications. In certain embodiments, an oligonucleotide or oligonucleotide composition is also capable of directing RNase H-mediated knockdown of an INHBE target gene RNA. In certain embodiments, the present disclosure demonstrates that oligonucleotide properties, e.g., activities, toxi cities, etc., can be modulated through chemical modifications of sugars, nucleobases, and / or internucleotidic linkages. In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which have a common base sequence, and comprise one or more modified internucleotidic linkages (or “non-natural internucleotidic linkages”, linkages that can be utilized in place of a natural phosphate internucleotidic linkage (-OP(O)(OH)O-, which may exist as a salt form (-OP(O)(O")O-) at a physiological pH) found in natural DNA and RNA), one or more modified sugar moieties, and / or one or more natural phosphate linkages. In certain embodiments, provided oligonucleotides may comprise two or more types of modified internucleotidic linkages. In certain embodiments, a provided oligonucleotide comprises a non-negatively charged internucleotidic linkage. In certain embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In certain embodiments, a neutral internucleotidic linkage comprises a cyclic guanidine moiety. Such moieties an optionally substituted. In certain embodiments, a provided oligonucleotide comprises a neutral internucleotidic linkage and another internucleotidic linkage which is not a neutral backbone. In certain embodiments, a provided oligonucleotide comprises a neutral internucleotidic linkage and a phosphorothioate internucleotidic linkage. In certain embodiments, provided oligonucleotide compositions comprising a plurality of oligonucleotides are chirally controlled and level of the plurality of oligonucleotides in the composition is controlled or pre-determined, and oligonucleotides of the plurality share a common stereochemistry configuration at one or more chiral intemucleotidic linkages. For example, in certain embodiments, oligonucleotides of a plurality share a common stereochemistry 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 intemucleotidic linkages, each of which is independently Rp or Sp; in certain embodiments, oligonucleotides of a plurality share a common stereochemistry configuration at each chiral intemucleotidic linkages. In certain embodiments, a chiral intemucleotidic linkage where a controlled level of oligonucleotides of a composition share a common stereochemistry configuration (independently in the Rp or A'p configuration) is referred to as a chirally controlled intemucleotidic linkage. In certain embodiments, a modified intemucleotidic linkage is a non-negatively charged (neutral or cationic) intemucleotidic linkage in that at a pH, (e.g., human physiological pH (~ 7.4), pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.), etc.), it largely (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc.; in certain embodiments, at least 30%; in certain embodiments, at least 40%; in certain embodiments, at least 50%; in certain embodiments, at least 60%; in certain embodiments, at least 70%; in certain embodiments, at least 80%; in certain embodiments, at least 90%; in certain embodiments, at least 99%; etc.;) exists as a neutral or cationic form (as compared to an anionic form (e.g., -Q-P(O)(O )-O- (the anionic form of natural phosphate linkage), -O-P(O)(S")-O- (the anionic form of phosphorothioate linkage), etc.)), respectively. In certain embodiments, a modified intemucleotidic linkage is a neutral intemucleotidic linkage in that at a pH, it largely exists as a neutral form. In certain embodiments, a modified intemucleotidic linkage is a cationic intemucleotidic linkage in that at a pH, it largely exists as a cationic form. In certain embodiments, a pH is human physiological pH (~ 7.4). In certain embodiments, a modified intemucleotidic linkage is a neutral intemucleotidic linkage in that at pH 7.4 in a water solution, at least 90% of the intemucleotidic linkage exists as its neutral form. In certain embodiments, a modified intemucleotidic linkage is a neutral intemucleotidic linkage in that in a water solution of the oligonucleotide, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the intemucleotidic linkage exists in its neutral form. In certain embodiments, the percentage is at least 90%. In certain embodiments, the percentage is at least 95%. In certain embodiments, the percentage is at least 99%. In certain embodiments, a non-negatively charged intemucleotidic linkage, e.g., a neutral intemucleotidic linkage, when in its neutral form has no moiety with a pKa that is less than 8, 9, 10, 11. 12, 13, or 14. In certain embodiments, pKa of an intemucleotidic linkage in the present disclosure can be represented by pKa of CHs-the intemucleotidic linkage-QU (i.e., replacing the two nucleoside units connected by the intemucleotidic linkage with two -CH3 groups). Without wishing to be bound by any particular theory, in at least some cases, a neutral intemucleotidic linkage in an oligonucleotide can provide improved properties and / or activities, e.g., improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape and / or improved nuclear uptake, etc., compared to a comparable nucleic acid which does not comprises a neutral intemucleotidic linkage.

[0127] In certain embodiments, a non-negatively charged intemucleotidic linkage has the structure of e g., of formula l-n-1. 1-n-2, l-n-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 US 9394333, 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 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607,

[0128] WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO

[0129] 2019 / 032612 etc. In certain embodiments, a non-negatively charged intemucleotidic linkage comprises a cyclic guanidine moiety. In certain embodiments, a modified intemucleotidic linkage comprising a cyclic guanidine moiety has the structure of: embodiments, a neutral intemucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In certain embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral intemucleotidic linkage and at least one phosphorothioate intemucleotidic linkage.

[0130] In certain embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral intemucleotidic linkage and at least one phosphorothioate intemucleotidic linkage, wherein the phosphorothioate intemucleotidic linkage is a chirally controlled intemucleotidic linkage in the Sp configuration.

[0131] In certain embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral intemucleotidic linkage and at least one phosphorothioate intemucleotidic linkage, wherein the phosphorothioate is a chirally controlled intemucleotidic linkage in the Rp configuration.

[0132] In certain embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral intemucleotidic linkage of a neutral intemucleotidic linkage comprising a Tmg group and at least one phosphorothi oate .

[0133] In certain embodiments, each intemucleotidic linkage in an oligonucleotide is independently selected from a natural phosphate linkage, a phosphorothi oate linkage, and a non-negatively charged intemucleotidic linkage (e.g., nOOl, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n055). In some embodiments, each intemucleotidic linkage in an oligonucleotide is independently selected from a natural phosphate linkage, a phosphorothioate linkage, and a neutral intemucleotidic linkage (e.g., nOOl, n003, n004, n006, n008, n009, n013 n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n055).

[0134] In certain embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral intemucleotidic linkage of a neutral intemucleotidic linkage comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is a chirally controlled intemucleotidic linkage in the Sp configuration.

[0135] In certain embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral intemucleotidic linkage selected from a neutral intemucleotidic linkage of a neutral intemucleotidic linkage comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is a chirally controlled intemucleotidic linkage in the Rp configuration.

[0136] Various types of intemucleotidic linkages differ in properties. Without wishing to be bound by any theory, the present disclosure notes that a natural phosphate linkage (phosphodiester intemucleotidic linkage) is anionic and may be unstable when used by itself without other chemical modifications in vivo; a phosphorothioate intemucleotidic linkage is anionic, generally more stable in vivo than a natural phosphate linkage, and generally more hydrophobic; a neutral intemucleotidic linkage such as one exemplified in the present disclosure comprising a cyclic guanidine moiety is neutral at physiological pH, can be more stable in vivo than a natural phosphate linkage, and more hydrophobic.

[0137] In certain embodiments, a chirally controlled neutral intemucleotidic linkage sis neutral at physiological pH, chirally controlled, stable in vivo, hydrophobic, and may increase endosomal escape.

[0138] In certain embodiments, provided oligonucleotides comprise one or more regions, e.g., a block, wing, core, 5 ’-end, 3 ’-end, middle, seed, post-seed region, etc. In certain embodiments, a region (e.g., a block, wing, core, 5’-end, 3’-end, middle region, etc.) comprises a non-negatively charged intemucleotidic linkage, e.g., of formula I-n-1, 1-n-2, 1-n-3, II, Il-a-

[0139] 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

[0140] 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

[0141] 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081,

[0142] WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO

[0143] 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612. In certain embodiments, a region comprises a neutral intemucleotidic linkage. In certain embodiments, a region comprises an intemucleotidic linkage which comprises a cyclic guanidine. In certain embodiments, a region comprises an intemucleotidic linkage which comprises a cyclic guanidine moiety. In certain embodiments, a region comprises an intemucleotidic linkage having the structure of In certain embodiments, such intemucleotidic linkages are chirally controlled.

[0144] In certain embodiments, a nucleotide is a natural nucleotide. In certain embodiments, a nucleotide is a modified nucleotide. In certain embodiments, a nucleotide is a nucleotide analog. In certain embodiments, a base is a modified base. In certain embodiments, a base is protected nucleobase, such as a protected nucleobase used in oligonucleotide synthesis. In certain embodiments, a base is a base analog. In certain embodiments, a sugar is a modified sugar. In certain embodiments, a sugar is a sugar analog. In certain embodiments, an intemucleotidic linkage is a modified intemucleotidic linkage. In certain embodiments, a nucleotide comprises a base, a sugar, and an intemucleotidic linkage, wherein each of the base, the sugar, and the intemucleotidic linkage is independently and optionally naturally-occurring or non-naturally occurring. In certain embodiments, a nucleoside comprises a base and a sugar, wherein each of the base and the 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 which comprise one or more modifications at the base, sugar and / or intemucleotidic linkage. 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’-M0E modifications. In certain embodiments, an intemucleotidic linkage is a moiety which does not a comprise a phosphorus but serves to link two natural or non-natural sugars.

[0145] In certain embodiments, a composition comprises a multimer of two or more of any: oligonucleotides of a first plurality and / or oligonucleotides of a second plurality, wherein the oligonucleotides of the first and second plurality can independently direct knockdown of the same or different targets independently via RNA interference and / or RNase H-mediated knockdown.

[0146] In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides which share:

[0147] 1) a common base sequence;

[0148] 2) a common pattern of backbone linkages;

[0149] 3) common stereochemistry independently 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 internucleotidic linkages (“chirally controlled internucleotidic linkages”); which composition is chirally controlled in that level of the first plurality of oligonucleotides in the composition is predetermined.

[0150] In certain embodiments, an oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a first plurality of oligonucleotides) is chirally controlled in that oligonucleotides of the plurality share a common stereochemistry independently at one or more chiral internucleotidic linkages. In certain embodiments, oligonucleotides of the plurality share a common stereochemistry 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 internucleotidic linkages, each of which is independently Rp or Sp In certain embodiments, oligonucleotides of the plurality share a common stereochemistry configuration at each chiral internucleotidic linkages. In certain embodiments, a chiral internucleotidic linkage where a predetermined level of oligonucleotides of a composition share a common stereochemistry configuration (independently Rp or Sp) is referred to as a chirally controlled internucleotidic linkage.

[0151] In certain embodiments, a predetermined level of oligonucleotides of a provided composition, e.g., a first plurality of oligonucleotides of certain example compositions, comprise 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 chirally controlled internucleotidic linkages.

[0152] In certain embodiments, at least 5 internucleotidic linkages are chirally controlled; in certain embodiments, at least 10 internucleotidic linkages are chirally controlled; in certain embodiments, at least 15 intemucleotidic linkages are chirally controlled; in certain embodiments, each chiral intemucleotidic linkage is chirally controlled.

[0153] In certain embodiments, l%-100% of chiral intemucleotidic linkages are chirally controlled. In certain 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 chiral intemucleotidic linkages are chirally controlled.

[0154] In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides which share:

[0155] 1) a common base sequence;

[0156] 2) a common pattern of backbone linkages; and

[0157] 3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of oligonucleotide in that a predetermined level of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers. In certain embodiments, the common pattern of backbone chiral centers comprises at least one intemucleotidic linkage comprising a chirally controlled chiral center. In certain embodiments, a 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 a provided composition. In certain embodiments, a 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 a provided composition that are of or comprise a common base sequence. In certain embodiments, all oligonucleotides in a provided composition that are of or comprise a common base sequence 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 certain embodiments, a 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 a provided composition that are of or comprise a common base sequence, base modification, sugar modification and / or modified intemucleotidic linkage. In certain embodiments, all oligonucleotides in a provided composition that are of or comprise a common base sequence, base modification, sugar modification and / or modified intemucleotidic linkage 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 certain embodiments, a 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 a provided composition that are of or comprise a common base sequence, pattern of base modification, pattern of sugar modification, and / or pattern of modified internucleotidic linkage. In certain embodiments, all oligonucleotides in a provided composition that are of or comprise a common base sequence, pattern of base modification, pattern of sugar modification, and / or pattern of modified internucleotidic linkage 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 certain embodiments, a 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 a provided composition that share a common base sequence, a common pattern of base modification, a common pattern of sugar modification, and / or a common pattern of modified internucleotidic linkages. In certain embodiments, all oligonucleotides in a provided composition that share a common base sequence, a common pattern of base modification, a common pattern of sugar modification, and / or a common pattern of modified internucleotidic linkages 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 certain embodiments, a predetermined level is 1-100%. In certain embodiments, a predetermined level is at least 1%. In certain embodiments, a predetermined level is at least 5%. In certain embodiments, a predetermined level is at least 10%. In certain embodiments, a predetermined level is at least 20%. In certain embodiments, a predetermined level is at least 30%. In certain embodiments, a predetermined level is at least 40%. In certain embodiments, a predetermined level is at least 50%. In certain embodiments, a predetermined level is at least 60%. In certain embodiments, a predetermined level is at least 10%. In certain embodiments, a predetermined level is at least 70%. In certain embodiments, a predetermined level is at least 80%. In certain embodiments, a predetermined level is at least 90%. In certain embodiments, a predetermined level is at least 5*(l / 2g), wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least 10*(l / 2g), wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least 100*(l / 2g), wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.80)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.80)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.80)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.85)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.90)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.95)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.96)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.97)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.98)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, a predetermined level is at least (0.99)g, wherein g is the number of chirally controlled internucleotidic linkages. In certain embodiments, to determine level of oligonucleotides having g chirally controlled internucleotidic linkages in a composition, product of diastereopurity of each of the g chirally controlled internucleotidic linkages: (diastereopurity of chirally controlled internucleotidic linkage 1) * (diastereopurity of chirally controlled internucleotidic linkage 2) * ... * (diastereopurity of chirally controlled internucleotidic linkage g) is utilized as the level, wherein diastereopurity of each chirally controlled internucleotidic linkage is independently represented by diastereopurity of a dimer comprising the same internucleotidic linkage and nucleosides flanking the internucleotidic linkage and prepared under comparable methods as the oligonucleotides (e.g., comparable or preferably identical oligonucleotide preparation cycles, including comparable or preferably identical reagents and reaction conditions). In certain embodiments, levels of oligonucleotides and / or diastereopurity can be determined by analytical methods, e.g., chromatographic, spectrometric, spectroscopic methods or any combinations thereof. Among other things, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that differ from one another, e.g., in the stereochemical structure (or stereochemistry) of individual backbone chiral centers within the oligonucleotide chain. Without control of stereochemistry of backbone chiral centers, stereorandom oligonucleotide preparations provide uncontrolled compositions comprising undetermined levels of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and / or chemical modifications, they are different chemical entities at least due to their different backbone stereochemistry, and they can have, as demonstrated herein, different properties, e.g., sensitivity to nucleases, activities, distribution, etc. In certain embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its length, its pattern of backbone linkages, and its pattern of backbone chiral centers. In certain embodiments, the present disclosure demonstrates that improvements in properties and activities achieved through control of stereochemistry within an oligonucleotide can be comparable to, or even better than those achieved through use of chemical modification.

[0158] Among other things, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that differ from one another, e.g., in the stereochemical structure (or stereochemistry) of individual backbone chiral centers within the oligonucleotide chain. Without control of stereochemistry of backbone chiral centers, stereorandom oligonucleotide preparations provide uncontrolled compositions comprising undetermined levels of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and / or chemical modifications, they are different chemical entities at least due to their different backbone stereochemistry, and they can have, as demonstrated herein, different properties, e.g., sensitivity to nucleases, activities, distribution, etc. In certain embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its length, its pattern of backbone linkages, and its pattern of backbone chiral centers. In certain embodiments, the present disclosure demonstrates that improvements in properties and activities achieved through control of stereochemistry within an oligonucleotide can be comparable to, or even better than those achieved through use of chemical modification

[0159] In some embodiments, a ds oligonucleotide targeting INHBE or ds oligonucleotide targeting INHBE composition is useful for prevention or treatment of a INHBE-associated condition, disorder, or disease, in a subject in need thereof. In some embodiments, the present disclosure provides a method for preventing or treating a INHBE-associated condition, disorder, or disease, comprising administering to a subject suffering therefrom or subject thereto a therapeutically effective amount of a provided ds oligonucleotide or a pharmaceutical composition that can deliver or comprise a therapeutically effective amount of a provided ds oligonucleotide. In some embodiments, the present disclosure provides pharmaceutical compositions which comprise a provided ds oligonucleotide targeting INHBE and a pharmaceutically acceptable carrier. In some embodiments, oligonucleotides in a pharmaceutical composition are in one or more pharmaceutically acceptable salt forms, e.g., a sodium salt form, an ammonium salt form, etc.

[0160] In some embodiments, an oligonucleotide or oligonucleotide composition is useful for the manufacture of a medicament for prevention or treatment of a INHBE-associated condition, disorder, or disease, such as metabolic disorders, e.g., metabolic syndrome, and related diseases, e.g., obesity, cardiovascular disease, diabetes, and hypertension, in a subject in need thereof.

[0161] Various INHBE-associated conditions, disorders, or diseases may be prevented and / or treated utilizing provided technologies (e.g., oligonucleotide, compositions, methods, etc.). In some embodiments, a condition, disorder, or disease is a metabolic disorder, e.g., metabolic syndrome, or related disease, e.g., obesity, cardiovascular disease, diabetes, or hypertension.

[0162] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0163] Technologies of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments.

[0164] Definitions

[0165] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0166] As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional / second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.

[0167] Unless otherwise specified, description of oligonucleotides and elements thereof (e.g., base sequence, sugar modifications, intemucleotidic linkages, linkage phosphorus stereochemistry, patterns thereof, etc.) is from 5’ to 3’. As those skilled in the art will appreciate, in some embodiments, oligonucleotides may be provided and / or utilized as salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts. Unless otherwise indicated, oligonucleotides include various forms of the oligonucleotides. As those skilled in the art will also appreciate, in some embodiments, individual oligonucleotides within a composition may be considered to be of the same constitution and / or structure even though, within such composition (e.g., a liquid composition), particular such oligonucleotides might be in different salt form(s) (and may be dissolved and the oligonucleotide chain may exist as an anion form when, e.g., in a liquid composition) at a particular moment in time. For example, those skilled in the art will appreciate that, at a given pH, individual internucleotidic linkages along an oligonucleotide chain may be in an acid (H) form, or in one of a plurality of possible salt forms (e.g., a sodium salt, or a salt of a different cation, depending on which ions might be present in the preparation or composition), and will understand that, so long as their acid forms (e.g., replacing all cations, if any, with H+) are of the same constitution and / or structure, such individual oligonucleotides may properly be considered to be of the same constitution and / or structure.

[0168] Analog: The term “analog” includes any chemical moiety which differs structurally from a reference chemical moiety or class of moieties, but which is capable of performing at least one function of such a reference chemical moiety or class of moieties. As non-limiting examples, a nucleotide analog differs structurally from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog differs structurally from a nucleobase but performs at least one function of a nucleobase; etc.

[0169] Antisense'. The term “antisense", as used herein, refers to a characteristic of an oligonucleotide or other nucleic acid having a base sequence complementary or substantially complementary to a target nucleic acid to which it is capable of hybridizing. In some embodiments, a target nucleic acid is a target gene mRNA. In some embodiments, hybridization is required for or results in at one activity, e.g., a decrease in the level, expression or activity of the target nucleic acid or a gene product thereof. The term “antisense oligonucleotide”, as used herein, refers to an oligonucleotide complementary to a target nucleic acid. In some embodiments, an antisense oligonucleotide is capable of directing a decrease in the level, expression or activity of a target nucleic acid or a product thereof. In some embodiments, an antisense oligonucleotide is capable of directing a decrease in the level, expression or activity of the target nucleic acid or a product thereof, via a mechanism that involves RNA interference. Chiral control: As used herein, “chiral control” refers to control of the stereochemical designation of the chiral linkage phosphorus in a chiral intemucleotidic linkage within an oligonucleotide. As used herein, a chiral intemucleotidic linkage is an intemucleotidic linkage whose linkage phosphorus is chiral. In some embodiments, a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in some embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation as described in the present disclosure, which chiral auxiliaries often are part of chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, a person having ordinary skill in the art appreciates that conventional oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry at a chiral intemucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral intemucleotidic linkage. In some embodiments, the stereochemical designation of each chiral linkage phosphorus in each chiral intemucleotidic linkage within an oligonucleotide is controlled.

[0170] Chirally controlled oligonucleotide composition: The terms “chirally controlled oligonucleotide composition”, “chirally controlled nucleic acid composition”, and the like, as used herein, refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids) which share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral intemucleotidic linkages (chirally controlled or stereodefined intemucleotidic linkages, whose chiral linkage phosphorus is Ap or Sp in the composition (“stereodefined”), not a random Ap and A'p mixture as non-chirally controlled intemucleotidic linkages). Level of the plurality of oligonucleotides (or nucleic acids) in a chirally controlled oligonucleotide composition is pre-determined / controlled (e.g., through chirally controlled oligonucleotide preparation to stereoselectively form one or more chiral intemucleotidic linkages). In some embodiments, about l%-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%, 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%) of all oligonucleotides in a chirally controlled oligonucleotide composition are oligonucleotides of the plurality. In some embodiments, about l%-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%, 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%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality. In some embodiments, a level is about l%-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%, 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%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, or of all oligonucleotides in a composition that share a common base sequence, a common patter of base modifications, a common pattern of sugar modifications, a common pattern of intemucleotidic linkage types, and / or a common pattern of intemucleotidic linkage modifications. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 chiral intemucleotidic linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about l%-100% of chiral intemucleotidic linkages. In some embodiments, oligonucleotides (or nucleic acids) of a plurality are of the same constitution (as appreciated by those skilled in the art, in some embodiments may exist in one or more forms, e.g., acid forms, salt forms, etc.). In some embodiments, level of the oligonucleotides (or nucleic acids) of the plurality is about 1%- 100% of all oligonucleotides (or nucleic acids) in a composition that share the same constitution as the oligonucleotides (or nucleic acids) of the plurality. In some embodiments, each chiral intemucleotidic linkage is a chiral controlled intemucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition. In some embodiments, oligonucleotides (or nucleic acids) of a plurality are structurally identical. In some embodiments, a chirally controlled intemucleotidic linkage has a diastereopurity of 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%. In some embodiments, a chirally controlled intemucleotidic linkage has a diastereopurity of at least 95%. In some embodiments, a chirally controlled intemucleotidic linkage has a diastereopurity of at least 96%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 97%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 98%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of at least 99%. In some embodiments, a percentage (e.g., a level as described herein) is or is at least (DS)nc, wherein DS is a diastereopurity as described in the present disclosure (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) and nc is the number of chirally controlled internucleotidic linkages as described in the present 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, a percentage (e.g., a level as described herein) is or is at least (DS)nc, wherein DS is 95%-100%. For example, when DS is 99% and nc is 10, the percentage is or is at least 90% ((99%)10~ 0.90 = 90%). In some embodiments, level of a plurality of oligonucleotides in a composition is represented as the product of the diastereopurity of each chirally controlled internucleotidic linkage in the oligonucleotides. In some embodiments, diastereopurity of an internucleotidic linkage connecting two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereopurity of an internucleotidic linkage of a dimer connecting the same two nucleosides, wherein the dimer is prepared using comparable conditions, in some instances, identical synthetic cycle conditions (e.g., for the linkage between Nx and Ny in an oligonucleotide ....NxNy , the dimer is NxNy). In some embodiments, not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, a non-chirally controlled internucleotidic linkage has a diastereopurity of less than about 80%, 75%, 70%, 65%, 60%, 55%, or of about 50%, as typically observed in stereorandom oligonucleotide compositions (e.g., as appreciated by those skilled in the art, from traditional oligonucleotide synthesis, e.g., the phosphoramidite method). In some embodiments, oligonucleotides (or nucleic acids) of a plurality are of the same type. In some embodiments, a chirally controlled oligonucleotide composition comprises nonrandom or controlled levels of individual oligonucleotide or nucleic acids types. For instance, in some embodiments a chirally controlled oligonucleotide composition comprises one and no more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a non-random or controlled level of a plurality of oligonucleotides of the oligonucleotide type.

[0171] Internucleotidic linkage: As used herein, the phrase “intemucleotidic linkage” refers generally to a linkage linking nucleoside units of an oligonucleotide or a nucleic acid. In some embodiments, an intemucleotidic linkage is a phosphodiester linkage, as extensively found in naturally occurring DNA and RNA molecules (natural phosphate linkage (-OP(=O)(OH)O-), which as appreciated by those skilled in the art may exist as a salt form). In some embodiments, an intemucleotidic linkage is a modified internucleotidic linkage (not a natural phosphate linkage). In some embodiments, an intemucleotidic linkage is a “modified intemucleotidic linkage” wherein at least one oxygen atom or -OH of a 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 the present disclosure. In some embodiments, an internucleotidic linkage is a phosphotriester linkage, phosphorothioate linkage (or phosphorothioate diester linkage, -OP(=O)(SH)O-, which as appreciated by those skilled in the art may exist as a salt form), or phosphorothioate triester linkage. In some embodiments, a modified intemucleotidic linkage is a phosphorothioate linkage. In some embodiments, an intemucleotidic linkage is one of, e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a neutral intemucleotidic linkage (e.g., nOOl in certain provided oligonucleotides). It is understood by a person of ordinary skill in the art that an internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage. In some embodiments, a modified internucleotidic linkages is a modified intemucleotidic linkages designated as s, si, s2, s3, s4, s5, s6, s7, s8, s9, slO, si 1, sl2, sl3, sl4, sl5, sl6, sl7 and sl8 as described in WO 2017 / 210647.

[0172] 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 cell culture, etc., rather than within an organism (e.g., animal, plant and / or microbe).

[0173] In vivo: As used herein, the term “in vivo" refers to events that occur within an organism (e.g., animal, plant and / or microbe).

[0174] Linkage phosphorus: as defined herein, the phrase “linkage phosphorus” is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in the intemucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a phosphodiester intemucleotidic linkage as occurs in naturally occurring DNA and RNA. In some embodiments, a linkage phosphorus atom is in a modified intemucleotidic linkage, wherein each oxygen atom of a phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, a linkage phosphorus atom is the P of Formula I as described herein. In some embodiments, a linkage phosphorus atom is chiral. In some embodiments, a linkage phosphorus atom is achiral (e.g., as in natural phosphate linkages).

[0175] Linker '. The terms “linker”, “linking moiety” and the like refer to any chemical moiety which connects one chemical moiety to another. As appreciated by those skilled in the art, a linker can be bivalent or trivalent or more, depending on the number of chemical moieties the linker connects. In some embodiments, a linker is a moiety which connects one oligonucleotide to another oligonucleotide in a multimer. In some embodiments, a linker is a moiety optionally positioned between the terminal nucleoside and the solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid. In some embodiments, in an oligonucleotide a linker connects a chemical moiety (e.g., a targeting moiety, a lipid moiety, a carbohydrate moiety, etc.) with an oligonucleotide chain (e.g., through its 5’-end, 3’-end, nucleobase, sugar, intemucleotidic linkage, etc.)

[0176] Modified nucleobase: The terms "modified nucleobase", "modified base" and the like refer to a chemical moiety which is chemically distinct from a nucleobase, but which is capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase which comprises a modification. In some embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases. In some embodiments, a modified nucleobase is substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobase in the context of oligonucleotides refer to a nucleobase that is not A, T, C, G or U.

[0177] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but which comprises a chemical modification which differentiates it from a natural nucleoside. Non-limiting examples of modified nucleosides include those which comprise a modification at the base and / or the sugar. Nonlimiting examples of modified nucleosides include those with a 2’ modification at a sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In some embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

[0178] Modified nucleotide: The term “modified nucleotide” includes any chemical moiety which differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, a modified nucleotide comprises a modification at a sugar, base and / or internucleotidic linkage. In some embodiments, a modified nucleotide comprises a modified sugar, modified nucleobase and / or modified internucleotidic linkage. In some embodiments, a modified nucleotide is capable of at least one function of a nucleotide, e.g., forming a subunit in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

[0179] Modified sugar : The term “modified sugar” refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. In some embodiments, as described in the present disclosure, a modified sugar is substituted ribose or deoxyribose. In some embodiments, a modified sugar comprises a 2’-modification. Examples of useful 2’-modification are widely utilized in the art and described herein. In some embodiments, a 2’-modification is 2’-OR, wherein R is optionally substituted Ci-io aliphatic. In some embodiments, a 2’ -modification is 2’-0Me. In some embodiments, a 2’ -modification is 2’-M0E. In some embodiments, a modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In some embodiments, in the context of oligonucleotides, a modified sugar is a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA.

[0180] Nucleic acid: The term “nucleic acid”, as used herein, includes any nucleotides and polymers thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and / or modified polynucleotides, such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0181] Nucleobase-. The term “nucleobase” refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a naturally- occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a naturally-occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase comprises a heteroaryl ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase comprises a heterocyclic ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase is a “modified nucleobase,” a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is substituted A, T, C, G or U. In some embodiments, a modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobases is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. In some embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. As used herein, the term “nucleobase” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, a “nucleobase” refers to a nucleobase unit in an oligonucleotide or a nucleic acid (e.g., A, T, C, G or U as in an oligonucleotide or a nucleic acid).

[0182] Nucleoside'. The term “nucleoside” refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar. In some embodiments, a nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxy cytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a “nucleoside” refers to a nucleoside unit in an oligonucleotide or a nucleic acid.

[0183] Nucleotide: The term “nucleotide” as used herein refers to a monomeric unit of a polynucleotide that consists of a nucleobase, a sugar, and one or more internucleotidic linkages (e.g., phosphate linkages in natural DNA and RNA). The naturally occurring bases [guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)] are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotidic linkages to form nucleic acids, or polynucleotides. Many internucleotidic linkages are known in the art (such as, though not limited to, phosphate, phosphorothioates, boranophosphates and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids, such as those described herein. In some embodiments, a natural nucleotide comprises a naturally occurring base, sugar and internucleotidic linkage. As used herein, the term “nucleotide” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, a “nucleotide” refers to a nucleotide unit in an oligonucleotide or a nucleic acid.

[0184] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, and may contain any combination of natural and non-natural nucleobases, sugars, and internucleotidic linkages.

[0185] Oligonucleotides can be single-stranded or double-stranded. A single-stranded oligonucleotide can have double-stranded regions (formed by two portions of the singlestranded oligonucleotide) and a double-stranded oligonucleotide, which comprises two oligonucleotide chains, can have single-stranded regions for example, at regions where the two oligonucleotide chains are not complementary to each other. Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, U1 adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

[0186] Oligonucleotides of the present disclosure can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, or triplestranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In some embodiments, the oligonucleotide is from about 9 to about 39 nucleosides in length. In some embodiments, 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 nucleosides in length. In some embodiments, the oligonucleotide is at least 19 nucleosides in length. In some embodiments, the oligonucleotide is at least 20 nucleosides in length. In some embodiments, the oligonucleotide is at least 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 30 nucleosides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 18 nucleosides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 21 nucleosides in length. In some embodiments, each nucleoside counted in an oligonucleotide length independently comprises A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G or U.

[0187] Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define an oligonucleotide that has a particular base sequence, pattern of backbone linkages (i.e., pattern of intemucleotidic linkage types, for example, phosphate, phosphorothioate, phosphorothioate triester, etc.), pattern of backbone chiral centers [i.e., pattern of linkage phosphorus stereochemistry (RpASp)], and pattern of backbone phosphorus modifications (e.g., pattern of “-XLR1” groups in Formula I as described herein). In some embodiments, oligonucleotides of a common designated “type” are structurally identical to one another.

[0188] One of skill in the art will appreciate that synthetic methods of the present disclosure provide for a degree of control during the synthesis of an oligonucleotide strand such that each nucleotide unit of the oligonucleotide strand can be designed and / or selected in advance to have a particular stereochemistry at the linkage phosphorus and / or a particular modification at the linkage phosphorus, and / or a particular base, and / or a particular sugar. In some embodiments, an oligonucleotide strand is designed and / or selected in advance to have a particular combination of stereocenters at the linkage phosphorus. In some embodiments, an oligonucleotide strand is designed and / or determined to have a particular combination of modifications at the linkage phosphorus. In some embodiments, an oligonucleotide strand is designed and / or selected to have a particular combination of bases. In some embodiments, an oligonucleotide strand is designed and / or selected to have a particular combination of one or more of the above structural characteristics. In some embodiments, the present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirally controlled oligonucleotide compositions). In some embodiments, all such molecules are of the same type (i.e., are structurally identical to one another). In some embodiments, however, provided compositions comprise a plurality of oligonucleotides of different types, typically in pre-determined relative amounts.

[0189] Optionally Substituted. As described herein, compounds, e.g., oligonucleotides, of the disclosure may contain optionally substituted and / or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Certain substituents are described below. Suitable monovalent substituents on a substitutable atom, e.g., a suitable carbon atom, are independently halogen; -(CH2)O 4R0; -(CH2)o4OR°; -0(CH2)o-4R°, -0-(CH2)o- 4C(O)OR°; -(CH2)O4CH(ORO)2; -(CH2)O4Ph, which may be substituted with R°; -(CH2)o- 40(CH2)o-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o-40(CH2)o-i-pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)o-4N(R°)2; -(CH2)O4N(RO)C(O)R°; -N(R°)C(S)R°; -(CH2)O4N(RO)C(O)NR°2;

[0190] -N(RO)C(S)NR°2; -(CH2)O4N(RO)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(RO)C(O)NRO2; -N(R°)N(R°)C(O)OR°; -(CH2)o4C(O)R°; -C(S)R°; -(CH2)o4C(O)OR°; -(CH2)o-4C(O)SR°; -(CH2)O4C(O)OSiR°3; -(CH2)o4OC(O)R°; -OC(0)(CH2)o4SR°, -SC(S)SR°; -(CH2)O4SC(O)RO; -(CH2)O4C(O)NRO2; -C(S)NRO2; -C(S)SR°; -(CH2)O-

[0191] 4OC(O)NRO2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)O4SSRO; -(CH2)O4S(O)2RO; -(CH2)O4S(O)2ORO; -(CH2)O4OS(O)2RO; -S(O)2NRO2; -(CH2)O4S(O)RO; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -Si(R°)3; - OSi(R°)3; -B(R°)2; -OB(RO)2; -OB(ORO)2; -P(RO)2; -P(ORO)2; -P(R°)(OR°); -OP(R°)2; -OP(OR°)2; -OP(R°)(OR°); -P(O)(RO)2; -P(O)(ORO)2; -OP(O)(RO)2; -OP(O)(ORO)2; -OP(O)(OR°)(SR°); -SP(O)(R°)2; -SP(O)(ORO)2; -N(RO)P(O)(R°)2; -N(RO)P(O)(OR°)2; -P(RO)2[B(R°)3]; -P(ORO)2[B(R°)3]; -OP(RO)2[B(R°)3]; -OP(ORO)2[B(R°)3]; -(CI^ straight or branched alkylene)O-N(R°)2; or -(Ci^t straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined herein and is independently hydrogen, C1-20 aliphatic, C1-20 heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, -CH2-(C6-i4 aryl), -0(CH2)o-i(C6-i4 aryl), -CH2-(5- 14 membered heteroaryl ring), 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, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), 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.

[0192] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), -(CH2)O 2OH, -(CH2)o-2OR*, -(CH2)o-2CH(OR*)2; -O(haloR’), -CN, -N3, - (CH2)O2C(O)R*, -(CH2)O2C(O)OH, -(CH2)O2C(O)OR*, -(CH2)O2SR*, -(CH2)O2SH, - (CH2)O 2NH2, -(CH2)O2NHR*, -(CH2)O2NR*2, -N02, -SiR%, -OSiR%, -C(O)SR* -(Ci^ straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, -CH2Ph, -0(CH2)o iPh, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0193] Suitable divalent substituents, e.g., on a suitable carbon atom, are independently the following: =0, =S, =NNR*2, =NNHC(0)R*, =NNHC(0)0R*, =NNHS(0)2R*, =NR*, =N0R*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: - O(CR*2)2 -3O- , wherein each independent occurrence of R* is selected from hydrogen, Ci- 6 aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, and aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0194] Suitable substituents on the aliphatic group of R* are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci 4 aliphatic, -CH2PI1, -0(CH2)o iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0195] In some embodiments, suitable substituents on a substitutable nitrogen are independently -R\ -NR\ -C(O)Rf, -C(O)ORf, -C(O)C(O)Rt, -C(O)CH2C(O)Rt, -S(O)2Rf, -S(O)2NRt2, -C(S)NRt2, -C(NH)NR^2, or -N(Rt)S(O)2Rt; wherein each R:is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R1', taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0196] Suitable substituents on the aliphatic group of R? are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0197] Oral: The phrases “oral administration” and “administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition.

[0198] Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.

[0199] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0200] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0201] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0202] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve 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; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0203] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, z.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salt include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with 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, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)s, wherein each R is independently defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises more than one acid groups, for example, an oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotidic linkages). In some embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), all ionizable hydrogen (e.g., in an aqueous solution with a pKa no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no more than about 7; in some embodiments, no more than about 6; in some embodiments, no more than about 5; in some embodiments, no more than about 4; in some embodiments, no more than about 3) in the acidic groups are replaced with cations. In some embodiments, each phosphorothioate and phosphate group independently exists in its salt form (e.g., if sodium salt, -O-P(O)(SNa)-O- and -O-P(O)(ONa)-O-, respectively). In some embodiments, each phosphorothioate and phosphate internucleotidic linkage independently exists in its salt form (e.g., if sodium salt, -O-P(O)(SNa)-O- and -O-P(O)(ONa)-O-, respectively). In some embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In some embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, wherein each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, exists as a salt form (all sodium salt).

[0204] Protecting group: The term “protecting group,” as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Also included are those protecting groups specially adapted for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage etal. 06 / 2012, the entirety of Chapter 2 is incorporated herein by reference. Suitable amino-protecting groups include but are not limited to described herein and / or in: WO 2018 / 022473, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, and / or WO 2019 / 075357, or US Provisional patent applications 62 / 825766 and 62 / 911339, the description of the protecting groups of each of which is independently incorporated herein by reference.

[0205] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and / or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and / or purification of certain components, etc.

[0206] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a provided compound (e.g., a provided oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic 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, a subject is a human. In some embodiments, a subject may be suffering from and / or susceptible to a disease, disorder and / or condition.

[0207] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. A base sequence which is substantially complementary to a second sequence is not identical to the second sequence, but is mostly or nearly identical to the second sequence. In addition, one of ordinary skill in the biological and / or chemical arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0208] Sugar: The term “sugar” refers to a monosaccharide or polysaccharide in closed and / or open form. In some embodiments, sugars are monosaccharides. In some embodiments, sugars are polysaccharides. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term “sugar” also encompasses structural analogs used in lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of the nucleic acid analog, glycol nucleic acid (“GNA”), etc. As used herein, the term “sugar” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is a RNA or DNA sugar (ribose or deoxyribose). In some embodiments, a sugar is a modified ribose or deoxyribose sugar, e.g., 2’ -modified, 5 ’-modified, etc. As described herein, in some embodiments, when used in oligonucleotides and / or nucleic acids, modified sugars may provide one or more desired properties, activities, etc. In some embodiments, a sugar is optionally substituted ribose or deoxyribose. In some embodiments, a “sugar” refers to a sugar unit in an oligonucleotide or a nucleic acid.

[0209] Susceptible to\ An individual who is “susceptible to” a disease, disorder and / or condition is one who has a higher risk of developing the disease, disorder and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition is predisposed to have that disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may exhibit symptoms of the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not exhibit symptoms of the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0210] Therapeutic agent: As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a population of subjects suffering from and / or 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 criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In some embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms or features of a disease, disorder, and / or condition in a subject when administered to the subject in an effective amount. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. In some embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

[0211] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0212] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0213] Unsaturated: The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation. Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0214] As those skilled in the art will appreciate, methods and compositions described herein relating to provided compounds (e.g., ds oligonucleotides) generally also apply to pharmaceutically acceptable salts of such compounds.

[0215] Description of Certain Embodiments

[0216] Double stranded oligonucleotides are useful tools for a wide variety of applications. For example, ds oligonucleotides targeting INHBE (e.g., NCBI Gene ID: 345275 for human INHBE and related sequences from other organisms) from are useful in therapeutic, diagnostic, and research applications, including the treatment of a variety of INHBE-associated 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 endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings and / or to further improve various properties and activities. These include synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications, etc., which, among other things, render these molecules less susceptible to degradation and improve other properties and / or activities. From a structural point of view, modifications to intemucleotidic linkages can introduce chirality and / or alter charge, and certain properties may be affected by configurations of linkage phosphorus atoms of oligonucleotides. For example, binding affinity, sequence specific binding to complementary RNA, stability against nucleases, cleavage of target nucleic acids, delivery, pharmacokinetics, etc., can be affected by, inter alia, chirality and / or charge of backbone linkage atoms.

[0217] In certain embodiments, a dsRNAi agent capable of directing INHBE (Inhibin subunit PE)-specific RNA interference to induce lipolysis while preserving muscle mass, the dsRNAi agent comprises a guide strand and a passenger strand, wherein: c) the guide strand is complementary or substantially complementary to an INHBE target RNA sequence; d) the guide strand comprises: i. a non-negatively charged intemucleotidic linkage in the A'p configuration between the +3 nucleotide relative to the 5’ terminal nucleotide and the immediately downstream (+4) nucleotide; ii. a non-negatively charged intemucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide; iii. phosphorothioate intemucleotidic linkages in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide; and / or iv. phosphorothioate intemucleotidic linkages in Rp, A'p, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide; c) the guide strand further comprises a 5’ phosphate modification; d) the passenger strand comprises one or more chiral intemucleotidic linkages in Rp or Sp configuration; and e) the guide strand and the passenger strand each independently has a length of 15-49 nucleotides.

[0218] In alternative embodiments, a ds oligonucleotide, e.g.a dsRNAi agent, targeting INHBE comprises one or more of

[0219] (1) a guide strand comprising backbone non-negatively charged intemucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0220] (2) a guide strand comprising backbone non-negatively charged intemucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0221] (3) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide; (4) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0222] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0223] (6) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction;

[0224] (7) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification; wherein the ds oligonucleotide further comprises one or more of

[0225] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0226] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0227] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide; (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0228] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0229] In some embodiments, a ds oligonucleotide targeting INHBE comprises: (1) a phosphorothioate chiral center in Rp or Sp configuration; (2) an Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage where the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage comprises a 2’ modification, e.g., a 2’ F; and (3) a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification selected from:

[0230] (a) 5’ PO modifications, such as, but not limited to:

[0231] (b) 5’ VP modifications, such as, but not limited to:

[0232] (c) 5’ MeP modifications, such as, but not limited to:

[0233] (d) 5’ PN and 5’ triazole-P modifications, such as, but not limited to: wherein Base is selected from A, C, G, T, U, abasic and modified nucleobases;

[0234] R2is selected from H, OH, O-alkyl, F, MOE, locked nucleic acid (LNA) bridges and bridged nucleic acid (BNA) bridges to the 4’ C, such as, but not limited to: In certain embodiments, the one or more Rp,

[0235] Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged intemucleotidic linkage. In certain embodiments, the guide strand comprises, a backbone phosphorothioate chiral center in Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide, and a backbone phosphorothioate chiral center in the Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0236] In certain embodiments, the guide strand comprises a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification selected from, but not limited to, 5’ MeP modifications and 5’ triazole-P modifications. In certain embodiments, the guide strand comprises the 5’ MeP modification , a backbone phosphorothioate chiral center in Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide, and a backbone phosphorothioate chiral center in the Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0237] In some embodiments, a ds oligonucleotide targeting INHBE comprises: (1) a phosphorothioate chiral center in Rp or Sp configuration; (2) an Rp, Sp, or stereorandom non- negatively charged intemucleotidic linkage where the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage comprises a 2’ modification, e.g., a 2’ F; and (3) a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification selected from:

[0238] (a) 5’ PO nucleotides, such as, but not limited to:

[0239] (b) 5’ VP nucleotides, such as, but not limited to:

[0240] (c) 5’ MeP nucleotides, such as, but not limited to:

[0241] (d) 5’ PN and 5’ triazole-P nucleotides, such as, but not limited to:

[0242] (e) 5’ abasic VP and 5’ abasic MeP nucleotides, such as, but not limited to: . In certain embodiments, the one or more

[0243] Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0244] In some embodiments, a ds oligonucleotide targeting INHBE comprises one or more of: (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0245] (2) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0246] (3) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0247] (4) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0248] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0249] (6) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction;

[0250] (7) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification; wherein the ds oligonucleotide further comprises one or more of

[0251] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0252] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0253] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0254] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand;

[0255] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0256] In some embodiments, a ds oligonucleotide targeting INHBE comprises non-naturally occurring internucleotidic linkages, e.g., neutral internucleotidic linkages, which can, in certain embodiments, be used to link one or more molecules to the double-stranded oligonucleotides described herein. In certain embodiments, such linked molecules can facilitate targeting and / or delivery of the double-stranded oligonucleotide. For example, but not limitation, such linked molecules an include lipophilic molecules. In certain embodiments, the linked molecule is a molecule comprising one or more GalNac moieties. In certain embodiments, the linked molecule is a receptor. In certain embodiments, the linked molecule is a receptor ligand.

[0257] In certain embodiments, the present disclosure provides technologies (e.g., compounds, methods, etc.) for improving oligonucleotide stability while maintaining or increasing activity, including compositions of improved-stability oligonucleotides.

[0258] In certain embodiments, the present disclosure provides technologies for incorporating various additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides, for example, reagents and methods for introducing additional chemical moieties through nucleobases (e.g., by covalent linkage, optionally via a linker, to a site on a nucleobase).

[0259] In certain embodiments, the present disclosure provides technologies, e.g., ds oligonucleotide compositions and methods thereof, that achieve allele-specific suppression, wherein transcripts from one allele of a particular target gene is selectively knocked down relative to at least one other allele of the same gene.

[0260] Among other things, the present disclosure provides structural elements, technologies and / or features that can be incorporated into ds oligonucleotides and can impart or tune one or more properties thereof (e.g., relative to an otherwise identical ds oligonucleotide lacking the relevant technology or feature). In certain embodiments, the present disclosure documents that one or more provided technologies and / or features can usefully be incorporated into ds oligonucleotides of various sequences.

[0261] In certain embodiments, the present disclosure demonstrates that certain provided structural elements, technologies and / or features are particularly useful for ds oligonucleotides that participate in and / or direct RNAi mechanisms (e.g., RNAi agents). Regardless, however, the teachings of the present disclosure are not limited to ds oligonucleotides that participate in or operate via any particular mechanism.

[0262] In certain embodiments, the present disclosure pertains to any ds oligonucleotide, useful for any purpose, which operates through any mechanism, and which comprises any sequence, structure or format (or portion thereof) described herein.

[0263] In some embodiments, a ds oligonucleotide targeting INHBE comprises a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction; and one or more of: (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0264] (2) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0265] (3) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0266] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0267] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e. in the 5’ direction; (1) a guide strand comprising backbone non- negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0268] (6) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification.

[0269] In some embodiments, a ds oligonucleotide targeting INHBE comprises a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction; and one or more of

[0270] (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0271] (3) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0272] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0273] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction;

[0274] (6) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification.

[0275] In some embodiments, a ds oligonucleotide targeting INHBE comprises backbone phosphorothioate chiral centers in Sp configuration between the 3 ’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of

[0276] (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0277] (2) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0278] (3) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0279] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0280] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction;

[0281] (6) a guide strand comprising a 5’ terminal modification, e.g. 5’ phosphate modification, e.g. 5’ triazole phosphate modification; wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0282] In some embodiments, a ds oligonucleotide targeting INHBE comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of

[0283] (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction; (2) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0284] (3) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0285] (4) a guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5’ direction, relative to backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, where the upstream backbone phosphorothioate chiral centers are in Rp or Sp configuration;

[0286] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0287] In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of:

[0288] (1) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction;

[0289] (2) a guide strand comprising backbone non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction;

[0290] (3) a guide strand comprising backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream, i.e., in the 5’ direction, (N-2) nucleotide;

[0291] (4) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

[0292] (5) a guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the N-2 nucleotide and the immediately upstream (N- 3) nucleotide, i.e., in the 5’ direction; and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In some embodiments, the guide strand comprises one or more Sp non-negatively charged intemucleotidic linkage occurs between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3’ direction, and one or more of:

[0293] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0294] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0295] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0296] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0297] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0298] In some embodiments, the guide strand comprises one or more Rp non-negatively charged intemucleotidic linkage occurs between the +10 nucleotide and the immediately downstream (+11) nucleotide, i.e., in the 3’ direction, and one or more of:

[0299] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0300] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0301] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0302] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0303] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.

[0304] In some embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the second (+2) and third (+3) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and the internucleotidic linkage to the penultimate 3’ (N-l) nucleotide, and one or more of:

[0305] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0306] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide; (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0307] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0308] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0309] In some embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N- 2) nucleotide, and one or more of:

[0310] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide; (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0311] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0312] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0313] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0314] In some embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of:

[0315] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0316] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0317] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0318] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0319] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage. In certain of the embodiments disclosed herein, the backbone phosphorothioate chiral centers are in Rp configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide. In certain embodiments described herein, the backbone phosphorothioate chiral centers are in Sp configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide. In certain embodiments, backbone phosphorothioate chiral centers in Rp and Sp configurations, respectively, between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide. In certain embodiments described herein, the backbone phosphorothioate chiral centers are in Sp, Rp configurations, respectively, between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0320] In some embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of

[0321] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged intemucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged intemucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0322] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0323] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0324] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0325] In some embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide, as well as between one or both of: (a) the (+3) nucleotide and the (+4) nucleotide; and (b) the (+5) nucleotide and the (+6) nucleotide, and one or more of:

[0326] (1) a guide strand where one or both of the 5’ and 3’ terminal dinucleotides are not linked by non-negatively charged internucleotidic linkages, i.e., the guide strand comprises one more non-negatively charged internucleotidic linkages downstream, i.e., in the 3’ direction, relative to the linkage between the 5’ terminal dinucleotide and / or upstream, i.e., in the 5’ direction, relative to the linkage between the 3’ terminal dinucleotide;

[0327] (2) a guide strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide;

[0328] (3) a guide strand where an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between the third (+3) and fourth (+4) nucleotides, relative to the 5’ terminal nucleotide, of the guide strand and / or between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5’ terminal nucleotide;

[0329] (4) a passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand; and

[0330] (5) Passenger strand where one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand, and wherein the ds oligonucleotide further comprises a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0331] In some embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration.

[0332] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N- 2) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

[0333] In certain embodiments, the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide or passenger strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0334] In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone phosphorothioate chiral centers in Sp configuration between the 3 ’ terminal nucleotide and the penultimate (N-l) nucleotide and as between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is an Sp non-negatively charged internucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage is a stereorandom non-negatively charged internucleotidic linkage.

[0335] In certain embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-l) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, a 2’ modification, e.g., a 2’ F modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage incorporated into the guide strand is an Rp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is an Sp non-negatively charged intemucleotidic linkage. In certain embodiments, the one or more Rp, Sp, or stereorandom non-negatively charged intemucleotidic linkage is a stereorandom non- negatively charged intemucleotidic linkage.

[0336] In certain embodiments, intemucleotidic linkages of an 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 chirally controlled intemucleotidic linkages. In certain embodiments, the present disclosure provides a dsRNAi oligonucleotide composition wherein the dsRNAi oligonucleotides comprise at least one chirally controlled intemucleotidic linkage. In certain embodiments, the present disclosure provides a dsRNAi oligonucleotide composition wherein the dsRNAi oligonucleotides are stereorandom or not chirally controlled. In certain embodiments, in a dsRNAi oligonucleotide, at least one intemucleotidic linkage is stereorandom and at least one intemucleotidic linkage is chirally controlled.

[0337] In certain embodiments, intemucleotidic linkages of an oligonucleotide comprise or consist of one or more neutrally charged intemucleotidic linkages.

[0338] INHBE

[0339] In some embodiments, INHBE refers to a gene or a gene product thereof (including but not limited to, a nucleic acid, including but not limited to a DNA or RNA, a transcript, a protein encoded thereby; can be from any form of INHBE, e.g., wide-type or mutant alleles) from any species. In some embodiments, it refers to the gene and product thereof in human. In some embodiments, it refers to the gene and product thereof in a non-human primate. Various INHBE sequences, including variants thereof, from human, mouse, rat, monkey, etc., are readily available to those of skill in the art. In some embodiments, INHBE is a human or mouse INHBE, which is wild-type or mutant. It has been reported that INHBE can have a number of functions. Various technologies, e.g., assays, cells, animal models, etc., have also been reported and can be utilized for characterization and / or assessment of provided technologies (e.g., oligonucleotides, compositions, methods, etc.) in accordance with the present disclosure.

[0340] In some embodiments, a INHBE gene, transcript (e.g., mRNA before or after splicing), or protein variant or isoform comprises a mutation. In some embodiments, a INHBE gene, transcript or protein is or a transcription or translation product of an alternatively spliced variant or isoform.

[0341] INHBE-Associated Conditions, disorders, or diseases

[0342] Various conditions, disorders, or diseases are reported to be associated with INHBE. Generally, a disease, disorder, or condition is associated with INHBE if the presence, level, activity, and / or form of INHBE and / or products (e.g., transcripts, encoded proteins, etc.) thereof correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, a condition, disorder, or disease associated with INHBE may be treated and / or prevented by reducing expression, level and / or activity of INHBE transcripts and / or proteins.

[0343] Various INHBE-associated conditions, disorders, or diseases are reported. In some embodiments, an INHBE-associated condition, disorder, or disease is metabolic syndromes such as heart diseases, type 2 and type 1 diabetes, kidney diseases, and obesity. In some embodiments, an INHBE-associated condition, disorder, or disease is type 2 diabetes. In some embodiments, an INHBE-associated condition, disorder, or disease is obesity.

[0344] Among other things, provided technologies are useful for treating or preventing a condition, disorder, or disease 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, the present disclosure pertains to the use of a ds oligonucleotide targeting INHBE or a composition thereof in the treatment of a INHBE- associated disorder, disease or condition, including, but not limited to, metabolic disorders, e.g., metabolic syndrome, and related diseases, e.g., obesity, cardiovascular disease, diabetes, and hypertension.

[0345] In some embodiments, treatment or prevention with provided technologies reduces rate of INHBE production and reduces or halts or reverses accumulation of INHBE. In some embodiments, treatment or prevention with provided technologies increases the rate of weight loss or otherwise allows for control of body weight.

[0346] As appreciated by those skilled in the art, mechanisms, genotypes, symptoms, biomarkers, etc. of such conditions, disorders, or diseases may be utilized in accordance with the present disclosure to characterize / assess provided technologies.

[0347] Double Stranded Oligonucleotides

[0348] Among other things, the present disclosure provides ds oligonucleotides of various designs, which may comprise various nucleobases and patterns thereof, sugars and patterns thereof, intemucleotidic linkages and patterns thereof, and / or additional chemical moieties and patterns thereof as described in the present disclosure. In some embodiments, provided ds oligonucleotides targeting INHBE can direct a decrease in the expression, level and / or activity of a INHBE gene and / or one or more of its products (e.g., transcripts, mRNA, proteins, etc.). In some embodiments, provided ds oligonucleotides targeting INHBE can direct a decrease in the expression, level and / or activity of a INHBE gene and / or one or more of its products in a cell of a subject or patient. In some embodiments, a cell normally expresses INHBE or produces INHBE protein. In some embodiments, provided ds oligonucleotides targeting INHBE can direct a decrease in the expression, level and / or activity of a INHBE target gene or a gene product and has a base sequence which consists of, comprises, or comprises a portion (e.g., a span of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous bases) of the base sequence of a ds oligonucleotide targeting INHBE disclosed herein, wherein each T can be independently substituted with U and vice versa, and the ds oligonucleotide comprises at least one non-naturally-occurring modification of a base, sugar and / or intemucleotidic linkage.

[0349] In some embodiments, ds oligonucleotides targeting INHBE can direct a decrease in the expression, level and / or activity of a target gene, e.g., a INHBE target gene, or a product thereof. In some embodiments, ds oligonucleotides targeting INHBE can direct a decrease in the expression, level and / or activity of a INHBE target gene or a product thereof via RNase H- mediated knockdown. In some embodiments, ds oligonucleotides targeting INHBE can direct a decrease in the expression, level and / or activity of a INHBE target gene or a product thereof by sterically blocking translation after binding to a INHBE target gene mRNA, and / or by altering or interfering with mRNA splicing. Regardless, however, the present disclosure is not limited to any particular mechanism. In some embodiments, the present disclosure provides oligonucleotides, compositions, methods, etc., capable of operating via double-stranded RNA interference.

[0350] In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating a decrease in the expression, level and / or activity of INHBE. In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating a decrease in the level of INHBE proteins. In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating a decrease in the level of INHBE proteins.

[0351] In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating a decrease in the expression, level and / or activity of INHBE via a mechanism involving mRNA degradation.

[0352] In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating a decrease in the expression, level and / or activity of more than one INHBE allele.

[0353] In some embodiments, the present disclosure pertains to a method of treatment of a INHBE-associated disease, disorder or condition, wherein INHBE is expressed, comprising the step of administering a therapeutically effective amount of a ds oligonucleotide targeting INHBE capable of mediating a decrease in the expression, level and / or activity of INHBE. In some embodiments, multiple forms, e.g., alleles, of INHBE may exist, and provided technologies can reduce expression, level and / or activity of two or more or all of the forms and products thereof.

[0354] In some embodiments, the present disclosure pertains to a method of treatment of a INHBE-associated disease, disorder or condition, comprising the step of administering a therapeutic amount of a ds oligonucleotide targeting INHBE capable of mediating a decrease in the expression, level and / or activity of INHBE.

[0355] In some embodiments, a ds oligonucleotide targeting INHBE is capable of mediating a decrease in the expression, level and / or activity of INHBE via a mechanism involving splicing modulation, e.g., exon skipping.

[0356] In some embodiments, a ds oligonucleotide targeting INHBE comprises a structural element or a portion thereof described herein, e.g., in Table 1. In some embodiments, a ds oligonucleotide targeting INHBE comprises a base sequence (or a portion thereof) described herein, wherein each T can be independently substituted with U and vice versa, a chemical modification or a pattern of chemical modifications (or a portion thereof), and / or a format or a portion thereof described herein. In some embodiments, a ds oligonucleotide targeting INHBE has a base sequence which comprises the base sequence (or a portion thereof) wherein each T can be independently substituted with U, pattern of chemical modifications (or a portion thereof), and / or a format of an oligonucleotide disclosed herein, e.g., in Table 1, or otherwise disclosed herein. In some embodiments, such oligonucleotides, e.g., ds oligonucleotides targeting INHBE, reduce expression, level and / or activity of a gene, e.g., a INHBE gene, or a gene product thereof.

[0357] Among other things, ds oligonucleotides targeting INHBE can hybridize to their target nucleic acids (e.g., pre-mRNA, mature mRNA, etc.). For example, in some embodiments, a ds oligonucleotide targeting INHBE can hybridize to a INHBE nucleic acid derived from a DNA strand (either strand of the INHBE gene). In some embodiments, a ds oligonucleotide targeting INHBE can hybridize to a INHBE transcript. In some embodiments, a ds oligonucleotide targeting INHBE can hybridize to a INHBE nucleic acid in any stage of RNA processing, including but not limited to a pre-mRNA or a mature mRNA. In some embodiments, a ds oligonucleotide targeting INHBE can hybridize to any element of a INHBE nucleic acid or its complement, including but not limited to: a promoter region, an enhancer region, a transcriptional stop region, a translational start signal, a translation stop signal, a coding region, a non-coding region, an exon, an intron, an intron / exon or exon / intron junction, the 5' UTR, or the 3' UTR. In some embodiments, ds oligonucleotides targeting INHBE can hybridize to their targets with no more than 2 mismatches. In some embodiments, ds oligonucleotides targeting INHBE can hybridize to their targets with no more than one mismatch. In some embodiments, ds oligonucleotides targeting INHBE can hybridize to their targets with no mismatches (e.g., when all C-G and / or A-T / U base paring).

[0358] In some embodiments, an oligonucleotide can hybridize to two or more variants of transcripts. In some embodiments, a ds oligonucleotide targeting INHBE can hybridize to two or more or all variants of INHBE transcripts. In some embodiments, a ds oligonucleotide targeting INHBE can hybridize to two or more or all variants of INHBE transcripts derived from the sense strand.

[0359] In some embodiments, a INHBE target of a ds oligonucleotide targeting INHBE is a INHBE RNA which is not a mRNA.

[0360] In some embodiments, oligonucleotides, e.g., ds oligonucleotides targeting INHBE, contain increased levels of one or more isotopes. In some embodiments, oligonucleotides, e.g., ds oligonucleotides targeting INHBE, are labeled, e.g., by one or more isotopes of one or more elements, e.g., hydrogen, carbon, nitrogen, etc. In some embodiments, oligonucleotides, e.g., ds oligonucleotides targeting INHBE, in provided compositions, e.g., oligonucleotides of a plurality of a composition, comprise base modifications, sugar modifications, and / or internucleotidic linkage modifications, wherein the oligonucleotides contain an enriched level of deuterium. In some embodiments, oligonucleotides, e.g., ds oligonucleotides targeting INHBE, are labeled with deuterium (replacing -XH with -2H) at one or more positions. In some embodiments, one or more1H of an oligonucleotide chain or any moiety conjugated to the oligonucleotide chain (e.g., a targeting moiety, etc.) is substituted with2H. Such oligonucleotides can be used in compositions and methods described herein. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which:

[0361] 1) have a common base sequence complementary to a target sequence (e.g., a INHBE target sequence) in a transcript; and

[0362] 2) comprise one or more modified sugar moieties and / or modified internucleotidic linkages.

[0363] In some embodiments, ds oligonucleotides targeting INHBE having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, etc. In some embodiments, a pattern of nucleoside modifications may be represented by a combination of locations and modifications. In some embodiments, a pattern of backbone linkages comprises locations and types (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.) of each internucleotidic linkage.

[0364] In some embodiments, oligonucleotides of a plurality, e.g., in provided compositions, are of the same oligonucleotide type. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of sugar modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of base modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of an oligonucleotide type have the same constitution. In some embodiments, oligonucleotides of an oligonucleotide type are identical. In some embodiments, oligonucleotides of a plurality are identical. In some embodiments, oligonucleotides of a plurality share the same constitution.

[0365] In some embodiments, as exemplified herein, ds oligonucleotides targeting INHBE are chiral controlled, comprising one or more chirally controlled internucleotidic linkages. In some embodiments, ds oligonucleotides targeting INHBE are stereochemically pure. In some embodiments, ds oligonucleotides targeting INHBE are substantially separated from other stereoisomers.

[0366] In some embodiments, ds oligonucleotides targeting INHBE comprise one or more modified nucleobases, one or more modified sugars, and / or one or more modified internucleotidic linkages.

[0367] In some embodiments, ds oligonucleotides targeting INHBE comprise one or more modified sugars. In some embodiments, oligonucleotides of the present disclosure comprise one or more modified nucleobases. Various modifications can be introduced to a sugar and / or nucleobase in accordance with the present disclosure. For example, in some embodiments, a modification is a modification described in US 9006198. In some embodiments, a modification is a modification described in US 9394333, 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 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, WO 2019 / 032612, and / or WO 2020 / 191252, the sugar, base, and intemucleotidic linkage modifications of each of which are independently incorporated herein by reference.

[0368] As used in the present 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” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten.

[0369] As used in the present 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.

[0370] In some embodiments, a ds oligonucleotide targeting INHBE is or comprises a ds oligonucleotide targeting INHBE described in Table 1.

[0371] As demonstrated in the present disclosure, in some embodiments, a provided oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) is characterized in that, when it is contacted with the transcript in a knockdown system, knockdown of its target (e.g., a INHBE transcript for a ds oligonucleotide targeting INHBE) is achieved.

[0372] In some embodiments, ds oligonucleotides are provided as salt forms. In some embodiments, ds oligonucleotides are provided as salts comprising negatively-charged internucleotidic linkages (e.g., phosphorothioate internucleotidic linkages, natural phosphate linkages, etc.) existing as their salt forms. In some embodiments, ds oligonucleotides are provided as pharmaceutically acceptable salts. In some embodiments, ds oligonucleotides are provided as metal salts. In some embodiments, oligonucleotides are provided as sodium salts. In some embodiments, ds oligonucleotides are provided as metal salts, e.g., sodium salts, wherein each negatively-charged internucleotidic linkage is independently in a salt form (e.g., for sodium salts, -O-P(O)(SNa)-O- for a phosphorothioate internucleotidic linkage, -O-P(O)(ONa)-O- for a natural phosphate linkage, etc.).

[0373] Double Stranded Oligonucleotide Base Sequences

[0374] In some embodiments, a ds oligonucleotide targeting INHBE comprises a base sequence described herein or a portion (e.g., a span of 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 at least 10, at least 15, contiguous nucleobases) thereof with 0-5 (e.g., 0, 1, 2, 3, 4 or 5) mismatches, wherein each T can be independently substituted with U and vice versa. In some embodiments, a ds oligonucleotide targeting INHBE comprises a base sequence described herein, or a portion thereof, wherein a portion is a span of at least 10 contiguous nucleobases, or a span of at least 15 contiguous nucleobases with 1-5 mismatches. In some embodiments, ds oligonucleotides targeting INHBE comprise a base sequence described herein, or a portion thereof, wherein a portion is a span of at least 10 contiguous nucleobases, or a span of at least 10 contiguous nucleobases with 1-5 mismatches, wherein each T can be independently substituted with U and vice versa. In some embodiments, base sequences of oligonucleotides comprise 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) contiguous bases of a base sequence that is identical to or complementary to a base sequence of a INHBE gene or a transcript (e.g., mRNA) thereof (e.g., in an intron).

[0375] Base sequences of ds oligonucleotides targeting INHBE, as appreciated by those skilled in the art, typically have sufficient length and complementarity to their targets, e.g., RNA transcripts (e.g., pre-mRNA, mature mRNA, etc.) to mediate target-specific knockdown. In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE has a sufficient length and identity to a INHBE transcript target to mediate target-specific knockdown. In some embodiments, the ds oligonucleotide targeting INHBE is complementary to a portion of a INHBE transcript (a INHBE transcript target sequence). In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE has 90% or more identity with the base sequence of an oligonucleotide disclosed in Table 1, wherein each T can be independently substituted with U and vice versa. In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE has 95% or more identity with the base sequence of an oligonucleotide disclosed in Table 1, wherein each T can be independently substituted with U and vice versa. In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE comprises a continuous span of 15 or more bases of an oligonucleotide disclosed in Table 1, wherein each T can be independently substituted with U and vice versa, except that one or more bases within the span are abasic (e.g., a nucleobase is absent from a nucleotide). In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE comprises a continuous span of 19 or more bases of a ds oligonucleotide targeting INHBE disclosed herein, except that one or more bases within the span are abasic (e.g., a nucleobase is absent from a nucleotide). In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE comprises a continuous span of 19 or more bases of an oligonucleotide disclosed herein, wherein each T can be independently substituted with U and vice versa, except for a difference in the 1 or 2 bases at the 5’ end and / or 3’ end of the base sequences.

[0376] In some embodiments, the present disclosure pertains to an oligonucleotide having a base sequence which comprises the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with U and vice versa.

[0377] In some embodiments, the present disclosure pertains to an oligonucleotide having a base sequence which comprises at least 15 contiguous bases of the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with U and vice versa.

[0378] In some embodiments, the present disclosure pertains to an oligonucleotide having a base sequence which is at least 90% identical to the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with U and vice versa.

[0379] In some embodiments, the present disclosure pertains to an oligonucleotide having a base sequence which is at least 95% identical to the base sequence of any oligonucleotide disclosed herein, wherein each T may be independently replaced with U and vice versa.

[0380] In some embodiments, a ds oligonucleotide targeting INHBE is selected from Table 1.

[0381] In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE is complementary to that of a INHBE transcript or a portion thereof.

[0382] In some embodiments, the base sequence of a ds oligonucleotide targeting INHBE is complementary to a portion of a INHBE nucleic acid sequence, e.g., a INHBE gene sequence, a INHBE transcript, a INHBE mRNA sequence, etc. In some embodiments, a ds oligonucleotide targeting INHBE is identical to a portion of a INHBE nucleic acid sequence, e.g., a INHBE gene sequence, a INHBE transcript, a INHBE mRNA sequence, etc. In some embodiments, the base sequence of such a portion is characteristic of INHBE in that no other genomic or transcript sequences in a system contain the same sequence as the portion. In some embodiments, no other genomic or transcript sequences in a system contain a sequence that differs from such a portion at no more than 1 nucleobase. In some embodiments, no other genomic or transcript sequences in a system contain a sequence that differs from such a portion at no more than 2 nucleobases. In some embodiments, a portion of a gene that is complementary to an oligonucleotide is referred to as a target sequence of the oligonucleotide. In some embodiments, a system is or comprises a cell, sample, tissue, organ, or a species. For example, for oligonucleotides targeting human INHBE, a relevant species in many embodiments is human. In some embodiments, a system can be or comprises multiple species, e.g., when cross-species activities and / or properties are characterized and / or assessed. 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 an intron and an exon. In some embodiments, such a portion spans two exons. In some embodiments, such a portion is in a 5’-UTR region. In some embodiments, such a portion is in a 3’-UTR region.

[0383] In some embodiments, a ds oligonucleotide targeting INHBE targets two or more or all alleles (if multiple alleles exist in a relevant system) of INHBE. In some embodiments, an oligonucleotide reduces expressions, levels and / or activities of both wild-type INHBE and mutant INHBE, and / or transcripts and / or products thereof.

[0384] In some embodiments, base sequences of provided oligonucleotides are fully complementary to both human and a non-human primate (NHP) INHBE target sequences. In some embodiments, such sequences can be particularly useful as they can be readily assessed in both human and non-human primates.

[0385] In some embodiments, a ds oligonucleotide targeting INHBE comprises a base sequence or portion thereof described in the Tables, wherein each T may be independently replaced with U and vice versa, and / or a sugar, nucleobase, and / or internucleotidic linkage modification and / or a pattern thereof described in Table 1, and / or an additional chemical moiety (in addition to an oligonucleotide chain, e.g., a target moiety, a lipid moiety, a carbohydrate moiety, etc.) described in Table 1.

[0386] In some embodiments, the terms “complementary,” “fully complementary” and “substantially complementary” may be used with respect to the base matching between n oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) base sequence and a target sequence (e.g., a INHBE target sequence), as will be understood by those skilled in the art from the context of their use. It is noted that substitution of T for U, or vice versa, generally does not alter 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 sequence (e.g., a ds oligonucleotide targeting INHBE) which is substantially complementary has 1, 2, 3, 4 or 5 mismatches when aligned to its target sequence. In some embodiments, a ds oligonucleotide targeting INHBE has a base sequence which is substantially complementary to a INHBE target sequence. In some embodiments, a ds oligonucleotide targeting INHBE has a base sequence which is substantially complementary to the complement of the sequence of a ds oligonucleotide targeting INHBE disclosed herein. As appreciated by those skilled in the art, in some embodiments, sequences of oligonucleotides need not be 100% complementary to their targets for the oligonucleotides to perform their functions (e.g., knockdown of target nucleic acids. Typically, when determining complementarity, A and T (or U) are complementary nucleobases and C and G are complementary nucleobases.

[0387] In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE comprising a sequence found in an oligonucleotide described in a Table. In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE comprising a sequence found in an oligonucleotide described in Table 1, wherein one or more U is independently and optionally replaced with T or vice versa. In some embodiments, a ds oligonucleotide targeting INHBE can comprise at least one T and / or at least one U. In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE comprising a sequence found in an oligonucleotide described in a Table, wherein the said sequence has over 50% identity with the sequence of the oligonucleotide described in the Table. In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE comprising the sequence of an oligonucleotide disclosed in Table 1. In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE whose base sequence is the sequence of an oligonucleotide disclosed in Table 1, wherein each T may be independently replaced with U and vice versa. In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE comprising a sequence found in an oligonucleotide in Table 1, wherein the oligonucleotides have a pattern of backbone linkages, pattern of backbone chiral centers, and / or pattern of backbone phosphorus modifications of the same oligonucleotide or another oligonucleotide in Table 1.

[0388] Among other things, the present disclosure presents, in Table 1 and elsewhere, various ds oligonucleotides, each of which has a defined base sequence. In some embodiments, the present disclosure, the present disclosure provides an oligonucleotide whose base sequence which is, comprises, or comprises a portion of the base sequence of an oligonucleotide disclosed herein, e.g., in Table 1 herein, wherein each T may be independently replaced with U and vice versa. In some embodiments, the disclosure provides an oligonucleotide having a base sequence which is, comprises, or comprises a portion of the base sequence of an oligonucleotide disclosed herein, e.g., in Table 1, wherein each T may be independently replaced with U and vice versa, wherein the oligonucleotide further comprises a chemical modification, stereochemistry, format, an additional chemical moiety described herein (e.g., a targeting moiety, lipid moiety, carbohydrate moiety, etc.), and / or another structural feature.

[0389] In some embodiments, a “portion” (e.g., of a base sequence or a pattern of modifications) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomeric units long (e.g., for a base sequence, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases long). In some embodiments, a “portion” of a base sequence is at least 5 bases long. In some embodiments, a “portion” of a base sequence is at least 10 bases long. In some embodiments, a “portion” of a base sequence is at least 15 bases long. In some embodiments, a “portion” of a base sequence is at least 16, 17, 18, 19 or 20 bases long. In some embodiments, a “portion” of a base sequence is at least 20 bases long. In some embodiments, a portion of a base sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous (consecutive) bases. In some embodiments, a portion of a base sequence is 15 or more contiguous (consecutive) bases. In some embodiments, a portion of a base sequence is 16, 17, 18, 19 or 20 or more contiguous (consecutive) bases. In some embodiments, a portion of a base sequence is 20 or more contiguous (consecutive) bases.

[0390] In some embodiments, the present disclosure provides an oligonucleotide (e.g., a ds oligonucleotide targeting INHBE) whose base sequence is a base sequence of an oligonucleotide in Table 1 or a portion thereof, wherein each T may be independently replaced with U and vice versa. In some embodiments, the present disclosure provides a ds oligonucleotide targeting INHBE of a sequence of an oligonucleotide in Table 1, wherein the oligonucleotide is capable of directing a decrease in the expression, level and / or activity of a INHBE gene or a gene product thereof. As appreciated by those skilled in the art, in provided base sequence, each U may be optionally and independently replaced by T or vice versa, and a sequence can comprise a mixture of U and T. In some embodiments, C may be optionally and independently replaced with 5mC.

[0391] In some embodiments, a portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides. In some embodiments, a portion is a span 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 span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0-3 mismatches, wherein a span with 0 mismatches is complementary and a span with 1 or more mismatches is a non-limiting example of substantial complementarity. In some embodiments, a base comprises a portion characteristic of a nucleic acid (e.g., a gene) in that the portion is identical or complementary to a portion of the nucleic acid or a transcript thereof, and is not identical or complementary to a portion of any other nucleic acid (e.g., a gene) or a transcript thereof in the same genome. In some embodiments, a portion is characteristic of human INHBE.

[0392] In some embodiments, a provided oligonucleotide, e.g., a ds oligonucleotide targeting INHBE, has a length of no more than about 49, 45, 40, 30, 35, 25, or 23 total nucleotides as described herein. In some embodiments, wherein the sequence recited herein starts with a U or T at the 5 ’-end, the U can be deleted and / or replaced by another base. In some embodiments, an oligonucleotide has a base sequence which is or comprises or comprises a portion of the base sequence of an oligonucleotide in a Table, wherein each T may be independently replaced with U and vice versa, which has a format or a portion of a format disclosed herein.

[0393] In some embodiments, oligonucleotides, e.g., ds oligonucleotides targeting INHBE are stereorandom. In some embodiments, ds oligonucleotides targeting INHBE are chirally controlled. In some embodiments, a ds oligonucleotide targeting INHBE is chirally pure (or “stereopure”, “stereochemically pure”), wherein the oligonucleotide exists as a single stereoisomeric form (in many cases a single diastereoisomeric (or “diastereomeric”) form as multiple chiral centers may exist in an oligonucleotide, e.g., at linkage phosphorus, sugar carbon, etc.). As appreciated by those skilled in the art, a chirally pure oligonucleotide is separated from its other stereoisomeric forms (to the extent that some impurities may exist as chemical and biological processes, selectivities and / or purifications etc. rarely, if ever, go to absolute completeness). In a chirally pure oligonucleotide, each chiral center is independently defined with respect to its configuration (for a chirally pure oligonucleotide, each internucleotidic linkage is independently stereodefined or chirally controlled). In contrast to chirally controlled and chirally pure oligonucleotides which comprise stereodefined linkage phosphorus, racemic (or “stereorandom”, “non-chirally controlled”) oligonucleotides comprising chiral linkage phosphorus, e.g., from traditional phosphoramidite oligonucleotide synthesis without stereochemical control during coupling steps in combination with traditional sulfurization (creating stereorandom phosphorothioate internucleotidic linkages), refer to a random mixture of various stereoisomers (typically diastereoisomers (or “diastereomers”) as there are multiple chiral centers in an oligonucleotide; e.g., from traditional oligonucleotide preparation using reagents containing no chiral elements other than those in nucleosides and linkage phosphorus). For example, for A*A*A wherein * is a phosphorothioate internucleotidic linkage (which comprises a chiral linkage phosphorus), a racemic oligonucleotide preparation includes four diastereomers [22= 4, considering the two chiral linkage phosphorus, each of which can exist in either of two configurations (Sp or Ap)]: A *S A *S A, A *S A *R A, A *R A *S A, and A *R A *R A, wherein *S represents a Sp phosphorothioate internucleotidic linkage and *R represents a Ap phosphorothioate internucleotidic linkage. For a chirally pure oligonucleotide, e.g., A *S A *S A, it exists in a single stereoisomeric form and it is separated from the other stereoisomers (e.g., the diastereomers A *S A *R A, A *R A *S A, and A *R A *R A).

[0394] In some embodiments, ds oligonucleotides targeting INHBE comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom internucleotidic linkages (mixture of Rp and Sp linkage phosphorus at the internucleotidic linkage, e.g., from traditional non-chirally controlled oligonucleotide synthesis). In some embodiments, ds oligonucleotides targeting INHBE comprise 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) chirally controlled internucleotidic linkages (Rp or A'p linkage phosphorus at the internucleotidic linkage, e.g., from chirally controlled oligonucleotide synthesis). In some embodiments, an internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, an internucleotidic linkage is a stereorandom phosphorothioate internucleotidic linkage. In some embodiments, an internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage.

[0395] Among other things, the present disclosure provides technologies for preparing chirally controlled (in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, oligonucleotides are stereochemically pure. In some embodiments, oligonucleotides of the present disclosure 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, intemucleotidic linkages of 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 intemucleotidic linkages, each of which independently has a diastereopurity of 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%. In some embodiments, oligonucleotides of the present disclosure, e.g., Ds oligonucleotides targeting INHBE, have a diastereopurity of (DS)CIL, wherein DS is a diastereopurity as described in the present disclosure (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) and CIL is the number of chirally controlled intemucleotidic linkages (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 intemucleotidic linkage is independently chirally controlled, and CIL is the number of chirally controlled intemucleotidic linkages.

[0396] As examples, certain ds oligonucleotides targeting INHBE comprising certain example base sequences, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties are presented in Table 1, below. Among other things, oligonucleotides, e.g., those in Table 1, may be utilized to target a INHBE transcript, e.g., to reduce the level of a INHBE transcript and / or a product thereof.

[0397] In certain exemplary embodiments, the ds oligonucleotide targeting INHBE of the present disclosure comprises the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of: DSR-0104068, DSR-0104099, DSR-0104072, DSR-0104075, DSR-0104083, DSR- 0104091, and DSR-0104071. In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of: DSR-0104068, DSR-0104099, DSR-0104072, DSR-0104075, DSR-0104083, DSR-0104091, and DSR-0104071. In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprises a passenger strand comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104068. In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprises a guide strand comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties DSR-0104068, DSR-0104099, DSR-0104072, DSR- 0104075, DSR-0104083, DSR-0104091, and DSR-0104071 comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104068.

[0398] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104068 comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof.

[0399] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104099.

[0400] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104072.

[0401] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104075.

[0402] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104083.

[0403] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104091.

[0404] In certain exemplary embodiments, a ds oligonucleotide targeting INHBE of the present disclosure comprising the base sequence, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, intemucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties of DSR-0104071.

[0405] Table 1. Example Double Stranded Oligonucleotides Targeting INHBE.

[0406] Table la. Examples of Guide Sequences Targeting INHBE

[0407] Table lb. Examples of Passenger Sequences Targeting INHBE

[0408] Notes:

[0409] Such notation 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. Description, Base Sequence and Stereochemistry / Linkage, due to their length, may be divided into multiple lines in Table 1. Unless otherwise specified, all oligonucleotides in Table 1 are single-stranded. As appreciated by those skilled in the art, nucleoside units are unmodified and contain unmodified nucleobases and 2’ -deoxy sugars unless otherwise indicated (e.g., with r, m, etc.); linkages, unless otherwise indicated, are natural phosphate linkages; and acidic / basic groups may independently exist in their salt forms. If a sugar is not specified, the sugar is a natural DNA sugar; and if an internucleotidic linkage is not specified, the internucleotidic linkage is a natural phosphate linkage. Moieties and modifications: m: 2’-0Me; f or fl2r: 2’-F;

[0410] O, PO, or p: phosphodiester (phosphate). It can a linkage or be an end group (or a component thereof), e.g., a linkage between a linker and an oligonucleotide chain, an internucleotidic linkage (a natural phosphate linkage), etc. Phosphodiesters are typically indicated with “O” in the Stereochemistry / Linkage column and are typically not marked in the Description column (if it is an end group, e.g., a 5’-end group, it is indicated in the Description and typically not in Stereochemistry / Linkage); if no linkage is indicated in the Description column, it is typically a phosphodiester unless otherwise indicated. Note that a phosphate linkage between a linker (e.g., L001) and an oligonucleotide chain may not be marked in the Description column, but may be indicated with “O” in the Stereochemistry / Linkage column; *, PS: Phosphorothioate. It can be an end group (if it is an end group, e.g., a 5 ’-end group, it is indicated in the Description and typically not in Stereochemistry / Linkage), or a linkage, e.g., a linkage between linker (e.g., L001) and an oligonucleotide chain, an internucleotidic linkage (a phosphorothioate internucleotidic linkage), etc.;

[0411] A, rip, or Rsp: Phosphorothioate in the rip configuration. Note that * R in Description indicates a single phosphorothioate linkage in the rip configuration;

[0412] 5, rip or Ssp: Phosphorothioate in the rip configuration. Note that * S in Description indicates a single phosphorothioate linkage in the rip configuration;

[0413] X: stereorandom phosphorothioate or phosphoryl guanidine; nX: stereorandom nOOl; nR or nOOIR or [nOOIR]: nOOl in Rp configuration; configuration; nX: stereorandom n002; nR or n002R: n002 in Rp configuration; nS or n002S: n002 in Sp configuration; nX: stereorandom n003; nR or n003R: n003 in Rp configuration; nS or n003S: n003 in Sp configuration; nX: stereorandom n004; nR or n004R: n004 in Rp configuration; nS or n004S: n004 in Sp configuration; nX: stereorandom n006; nR or n006R: n006 in Rp configuration; nS or n006S: n006 in Sp configuration; nX: stereorandom n008; nR or n008R: n008 in Rp configuration; nS or n008S: n008 in Sp configuration; nX: stereorandom n009; nR or n009R: n009 in Rp configuration; nS or n009S: n009 in Sp configuration; nX: stereorandom n012; nR or nO12R: n012 in Rp configuration; nS or nO12S: n012 in Sp configuration; nX: stereorandom n020; nR or n020R: n020 in Rp configuration; nS or n020S: n020 in Sp configuration; nX: stereorandom n021; nR or nO21R: n021 in Rp configuration; nS or nO21S: n021 in Sp configuration; nX: stereorandom n025; nR or nO25R: n025 in Rp configuration; nS or nO25S: n025 in Sp configuration; nX: stereorandom n026; nR or nO26R: n026 in Rp configuration; nS or nO26S: n026 in Sp configuration; nX: stereorandom n029; nR or nO29R: n029 in Rp configuration; nS or nO29S: n029 in Sp configuration; nX: stereorandom n030; nR or n030R: n030 in Rp configuration; nS or n030S: n030 in Sp configuration; n031: nX: stereorandom n031; nR or n031R: n031 in Rp configuration; nS or nO31S: n031 in Sp configuration; n033: nX: stereorandom n033; nR or nO33R: n033in Rp configuration; nS or nO33S: n033 in Sp configuration; n034 nX: stereorandom n034; nR or nO34R: n034in Rp configuration; nS or nO34S: n034 in Sp configuration; nO35: nX: stereorandom n035; nR or nO35R: n035in Rp configuration; nS or nO35S: n035 in Sp configuration; n036 nX: stereorandom n036; nR or nO36R: n036in Rp configuration; nS or nO36S: n036 in Sp configuration; nX: stereorandom n037; nR or nO37R: n037in Rp configuration; nS or nO37S: n037 in Sp configuration; nX: stereorandom n039; nR or nO39R: n039 in Rp configuration; nS or nO39S: n039 in Sp configuration; nX: stereorandom n040; nR or n040R: n040in Rp configuration; nS or n040S: n040 in Sp configuration; nX: stereorandom n041; nR or nO41R: n041in Rp configuration; nS or nO41S: n041 in Sp configuration; nX: stereorandom n043; nR or nO43R: n043 in Rp configuration; nS or nO43S: n043 in Sp configuration; nX: stereorandom n045; nR or nO45R: n045 in Rp configuration; nS or nO45S: n045 in Sp configuration; nX: stereorandom n046; nR or nO46R: n046in Rp configuration; nS or nO46S: n046 in Sp configuration; nX: stereorandom n047; nR or nO47R: n047in Rp configuration; nS or nO47S: n047 in Sp configuration; nX: stereorandom n051; nR or n051R: n051in Rp configuration; nS or n051S: n051 in Sp configuration; nX: stereorandom n052; nR or nO52R: n052in Rp configuration; configuration; nX: stereorandom n054; nR or nO54R: n054 in Rp configuration; nS or nO54S: n054 in Sp configuration; nX: stereorandom n055; nR or n055R: n055 in Rp configuration; nS or n055S: n055 in Sp configuration;

[0414] nX: stereorandom n057; nR or nO57R: n057 in Rp configuration; nS or nO57S: n057 in Sp configuration; nX: stereorandom n058; nR or nO58R: n058 in Rp configuration; nS or n058S: n058 in Sp configuration; nX: stereorandom n060; nR or n060R: n060 in Rp configuration; nS or n060S: n060 in Sp configuration; nX: stereorandom n061; nR or nO61R: n061 in Rp configuration; nS or nO61S: n061 in Sp configuration; nX: stereorandom n062; nR or nO62R: n062 in Rp configuration; nS or nO62S: n062 in Sp configuration; nX: stereorandom n065; nR or nO65R: n065 in Rp configuration; nS or nO65S: n065 in Sp configuration; nX: stereorandom n066; nR or nO66R: n066 in Rp configuration; nS or nO66S: n066 in Sp configuration; nX: stereorandom n068; nR or nO68R: n068 in Rp configuration; nS or nO68S: n068 in Sp configuration; nX: stereorandom n069; nR or nO69R: n069 in Rp configuration; nS or nO69S: n069 in Sp configuration; nX: stereorandom n070; nR or n070R: n070 in Rp configuration; nS or n070S: n070 in Sp configuration; nX: stereorandom n071; nR or nO71R: n071 in Rp configuration; nS or nO71S: n071 in Sp configuration; nX: stereorandom n072; nR or nO72R: n072 in Rp configuration; nS or nO72S: n072 in Sp configuration; nX: stereorandom n073; nR or nO73R: n073 in Rp configuration; nS or nO73S: n073 in Sp configuration; nX: stereorandom n076; nR or nO76R: n076in Rp configuration; nS or nO76S: n076 in Sp configuration; nX: stereorandom n077; nR or nO77R: n077in Rp configuration; nS or nO77S: n077 in Sp configuration;

[0415] X: stereorandom phosphorothioate or phosphoryl guanidine;

[0416]

[0417]

[0418] i.e. morpholine carbamate internucleotidic linkage

[0419]

[0420]

[0421] L001 : - NH- (CH2)6_linker (C6 linker, C6 amine linker or C6 amino linker), connected to Mod (e.g., ModOOl) through -NH- and, in the case of, for example, WV-38061, the 5’-end of the oligonucleotide chain through a phosphate linkage (O or PO). For example, in WV- 38061, L001 is connected to ModOOl through -NH- (forming an amide group -C(O)-NH-), and is connected to the oligonucleotide chain through a phosphate linkage (O). some embodiments, when LO 10 is present in the middle of an oligonucleotide, it is bonded to internucleotidic linkages as other sugars (e.g., DNA sugars), e.g., its 5’-carbon is connected to another unit (e.g., 3’ of a sugar) and its 3’-carbon is connected to another unit (e.g., a 5’-carbon of a carbon) independently, e.g., via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (can be either not chirally controlled or chirally controlled fS'p or Rp )));

[0422] L012:-CH2CH20CH2CH20CH2CH2- When L012 is present in the middle of an oligonucleotide, each of its two ends is independently bonded to an internucleotidic linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (can be either not chirally controlled or chirally controlled fS'p or Rp ))); wherein L022 is connected to the rest of a molecule through a phosphate unless indicated otherwise;

[0423] L023: HO-(CH2)6_, wherein CH2is connected to the rest of a molecule through a phosphate unless indicated otherwise. For example, in WV-42644 (wherein the O in OnRnRnRnRSSSSSSSSSSSSSSSSSSnRSSSSSnRSSnR indicates a phosphate linkage connecting L023 to the rest of the molecule); , wherein the -CH2- connection site is utilized as a C5 connection site of a sugar (e.g., a DNA sugar) and is connected to another unit (e.g., 3’ of a sugar), and the connection site on the ring is utilized as a C3 connection site and is connected to another unit (e.g., a 5’-carbon of a carbon), each of which is independently, e.g., via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (can be either not chirally controlled or chirally controlled (Sp or Rp ))). When L025 is at a5’-end without any modifications, its -CH2- connection site is bonded to -OH. For example, L025L025L025- in various oligonucleotides has the structure (may exist as various salt forms) and is connected to 5 ’-carbon of an oligonucleotide chain via a linkage as indicated (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (can be either not chirally controlled or chirally controlled fS'p or Rp )));

[0424] L016: . wherein L016 is connected to the rest of a molecule through a phosphate unless indicated otherwise; L016 is utilized with nOOl to form L016n001, which has the structure Double Stranded Oligonucleotide Lengths

[0425] As appreciated by those skilled in the art, ds oligonucleotides targeting INHBE can be of various lengths to provide desired properties and / or activities for various uses. Many technologies for assessing, selecting and / or optimizing oligonucleotide length are available in the art and can be utilized in accordance with the present disclosure. As demonstrated herein, in many embodiments, ds oligonucleotides targeting INHBE are of suitable lengths to hybridize with their targets and reduce levels of their targets and / or an encoded product thereof. In some embodiments, an oligonucleotide is long enough to recognize a target nucleic acid (e.g., a INHBE mRNA). In some embodiments, an oligonucleotide is sufficiently long to distinguish between a target nucleic acid and other nucleic acids (e.g., a nucleic acid having a base sequence which is not INHBE) to reduce off-target effects. In some embodiments, a ds oligonucleotide targeting INHBE is sufficiently short to reduce complexity of manufacture or production and to reduce cost of products.

[0426] In some embodiments, the base sequence of an oligonucleotide is about 10-500 nucleobases in length. In some embodiments, a base sequence is about 10-500 nucleobases in length. In some embodiments, a base sequence is about 10-50 nucleobases in length. In some embodiments, a base sequence is about 15-50 nucleobases in length. In some embodiments, a base sequence is from about 15 to about 30 nucleobases in length. In some embodiments, a base sequence is from about 10 to about 25 nucleobases in length. In some embodiments, a base sequence is from about 15 to about 22 nucleobases in length. In some embodiments, a base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases in length. In some embodiments, a base sequence is about 18 nucleobases in length. In some embodiments, a base sequence is about 19 nucleobases in length. In some embodiments, a base sequence is about 20 nucleobases in length. In some embodiments, a base sequence is about 21 nucleobases in length. In some embodiments, a base sequence is about 22 nucleobases in length. In some embodiments, a base sequence is about 23 nucleobases in length. In some embodiments, a base sequence is about 24 nucleobases in length. In some embodiments, a base sequence is about 25 nucleobases in length. In some embodiments, each nucleobase is optionally substituted A, T, C, G, U, or an optionally substituted tautomer of A, T, C, G, or U.

[0427] Double Stranded Oligonucleotide Internucleotidic Linkages

[0428] In some embodiments, ds oligonucleotides targeting INHBE comprise base modifications, sugar modifications, and / or intemucleotidic linkage modifications. Various internucleotidic linkages can be utilized in accordance with the present disclosure to link units comprising nucleobases, e.g., nucleosides. In some embodiments, ds oligonucleotides targeting INHBE comprise both one or more modified intemucleotidic linkages and one or more natural phosphate linkages. As widely known by those skilled in the art, natural phosphate linkages are widely found in natural DNA and RNA molecules; they have the structure of -OP(O)(OH)O-, connect sugars in the nucleosides in DNA and RNA, and may be in various salt forms, for example, at physiological pH (about 7.4), natural phosphate linkages are predominantly exist in salt forms with the anion being -OP(O)(O )O- A modified internucleotidic linkage, or a non-natural phosphate linkage, is an intemucleotidic linkage that is not natural phosphate linkage or a salt form thereof. Modified internucleotidic linkages, depending on their structures, may also be in their salt forms. For example, as appreciated by those skilled in the art, phosphorothioate internucleotidic linkages which have the structure of -OP(O)(SH)O- may be in various salt forms, e.g., at physiological pH (about 7.4) with the anion being -OP(O)(S )O~.

[0429] In some embodiments, an oligonucleotide comprises an intemucleotidic linkage which is a modified internucleotidic linkage, e.g., phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroamidate, thiophosphate, 3 ’-thiophosphate, or 5 ’-thiophosphate.

[0430] In some embodiments, a modified intemucleotidic linkage is a chiral internucleotidic linkage which comprises a chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is chirally controlled with respect to its chiral linkage phosphorus. In some embodiments, a chiral intemucleotidic linkage is stereochemically pure with respect to its chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is not chirally controlled. In some embodiments, a pattern of backbone chiral centers comprises or consists of positions and linkage phosphorus configurations of chirally controlled intemucleotidic linkages (Rp or A'p) and positions of achiral internucleotidic linkages (e.g., natural phosphate linkages).

[0431] In certain embodiments, an intemucleotidic linkage comprises a P-modification, wherein a P-modification is a modification at a linkage phosphorus. In certain embodiments, a modified internucleotidic linkage is a moiety which does not comprise a phosphorus but serves to link two sugars or two moieties that each independently comprises a nucleobase, e.g., as in peptide nucleic acid (PNA).

[0432] In certain embodiments, a ds oligonucleotide comprises a modified intemucleotidic linkage, e.g., those having the structure of Formula I, I-a, I-b, or I-c and described herein and / or in: WO 2018 / 022473, WO 2018 / 098264, 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, the intemucleotidic linkages (e.g., those of Formula I, I-a, I-b, I-c, etc.) of each of which are independently incorporated herein by reference. In certain embodiments, a modified intemucleotidic linkage is a chiral intemucleotidic linkage. In certain embodiments, a modified intemucleotidic linkage is a phosphorothioate intemucleotidic linkage.

[0433] In certain embodiments, a modified intemucleotidic linkage is a non-negatively charged intemucleotidic linkage. In certain embodiments, provided ds oligonucleotides comprise one or more non-negatively charged intemucleotidic linkages. In certain embodiments, a non- negatively charged intemucleotidic linkage is a positively charged intemucleotidic linkage. In certain embodiments, a non-negatively charged intemucleotidic linkage is a neutral intemucleotidic linkage. In certain embodiments, the present disclosure provides ds oligonucleotides comprising one or more neutral intemucleotidic linkages. In certain embodiments, a non-negatively charged intemucleotidic linkage has the structure of Formula I-n-1, I-n-2, I-n-3, 1-n-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 a salt form thereof, as described herein and / or in 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, the non- negatively charged intemucleotidic linkages (e.g., those of Formula I-n-1, I-n-2, I-n-3, 1-n-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 a suitable salt form thereof) of each of which are independently incorporated herein by reference.

[0434] In certain embodiments, a non-negatively charged intemucleotidic linkage can improve the delivery and / or activities (e.g., adenosine editing activity). In certain embodiments, a modified intemucleotidic linkage (e.g., a non-negatively charged intemucleotidic linkage) comprises optionally substituted triazolyl. In certain embodiments, a modified intemucleotidic linkage (e.g., a non-negatively charged intemucleotidic linkage) comprises optionally substituted alkynyl. In certain embodiments, a modified intemucleotidic linkage comprises a triazole or alkyne moiety. In certain embodiments, a triazole moiety, e.g., a triazolyl group, is optionally substituted. In certain embodiments, a triazole moiety, e.g., a triazolyl group) is substituted. In certain embodiments, a triazole moiety is unsubstituted. In certain embodiments, a modified intemucleotidic linkage comprises an optionally substituted cyclic guanidine moiety. In certain embodiments, a modified intemucleotidic linkage has the structure and is optionally chirally controlled, wherein R1is -L-R’, wherein L is LBas described herein, and R’ is as described herein. In certain embodiments, each R1is independently R’. In certain embodiments, each R’ is independently R. In certain embodiments, two R1are R and are taken together to form a ring as described herein. In certain embodiments, two R1on two different nitrogen atoms are R and are taken together to form a ring as described herein. In certain embodiments, R1is independently optionally substituted C1-6 aliphatic as described herein. In certain embodiments, R1is methyl. In certain embodiments, two R’ on the same nitrogen atom are R and are taken together to form a ring as described herein. In certain embodiments, a modified intemucleotidic linkage has the structure optionally chirally controlled. In certain embodiments, In certain embodiments, a modified intemucleotidic linkage comprises an optionally substituted cyclic guanidine moiety and has the structure of or , wherein W is O or S. In certain embodiments, W is O. In certain embodiments, W is S. In certain embodiments, a non-negatively charged internucleotidic linkage is stereochemically controlled.

[0435] In certain embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is an internucleotidic linkage comprising a triazole moiety. In some embodiments, an internucleotidic linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) has the structure In some embodiments, an internucleotidic linkage comprising a triazole moiety has the structure some embodiments, an internucleotidic linkage comprising a triazole moiety has the formula some embodiments, an internucleotidic linkage comprising an alkyne moiety (e.g., an optionally substituted alkynyl group) has the formula of , wherein W is O or S. In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, a neutral internucleotidic linkage, comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety has the structure some embodiments, a non- negatively charged internucleotidic linkage, or a neutral internucleotidic linkage, is or comprising a structure selected from , wherein W is O or S. In certain embodiments, an intemucleotidic linkage, e.g., a non-negatively charged intemucleotidic linkage, a neutral intemucleotidic linkage, comprises a cyclic guanidine moiety. In certain embodiments, an intemucleotidic linkage comprising a cyclic guanidine moiety has the structure In certain embodiments, a non-negatively charged intemucleotidic linkage, or a neutral intemucleotidic linkage, is or comprising a structure , wherein W is O or S.

[0436] In certain embodiments, an intemucleotidic linkage comprises a Tmg group ( certain embodiments, an intemucleotidic linkage comprises a Tmg group and has the structure (the “Tmg intemucleotidic linkage”). In certain embodiments, neutral intemucleotidic linkages include intemucleotidic linkages of PNA and PMO, and a Tmg intemucleotidic linkage.

[0437] In certain embodiments, a non-negatively charged intemucleotidic linkage has the structure of Formula I, I-a, I-b, I-c, l-n-1. I-n-2, l-n-3. 1-n-4, II, II-a-1, II-a-2, II-b-1, I l-b- 2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In certain embodiments, a non- negatively charged intemucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In certain embodiments, a non-negatively charged intemucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, such a heterocyclyl or heteroaryl group is of a 5-membered ring. In certain embodiments, such a heterocyclyl or heteroaryl group is of a 6- membered ring. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, a non- negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, a heteroaryl group is directly bonded to a linkage phosphorus.

[0438] In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5- membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, at least two heteroatoms are nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an unsubstituted triazolyl group, e.g., In some embodiments, a non-negatively

[0439] N=N charged internucleotidic linkage comprises a substituted triazolyl group, e.g.,

[0440] In certain embodiments, a heterocyclyl group is directly bonded to a linkage phosphorus. In certain embodiments, a heterocyclyl group is bonded to a linkage phosphorus through a linker, e.g., =N- when the heterocyclyl group is part of a guanidine moiety who directed bonded to a linkage phosphorus through its =N- In certain embodiments, a non-

[0441] H yrN negatively charged internucleotidic linkage comprises an optionally substituted HN^y group. In certain embodiments, a non-negatively charged intemucleotidic linkage comprises an substituted group. In certain embodiments, a non-negatively charged intemucleotidic linkage comprises a R group, wherein each R is independently -L-R. In certain embodiments, each R1is independently optionally substituted C1-6 alkyl. In certain embodiments, each R1is independently methyl.

[0442] In certain embodiments, a modified intemucleotidic linkage, e.g., a non-negatively charged intemucleotidic linkage, comprises a triazole or alkyne moiety, each of which is optionally substituted. In certain embodiments, a modified intemucleotidic linkage comprises a triazole moiety. In certain embodiments, a modified intemucleotidic linkage comprises a unsubstituted triazole moiety. In certain embodiments, a modified intemucleotidic linkage comprises a substituted triazole moiety. In certain embodiments, a modified intemucleotidic linkage comprises an alkyl moiety. In certain embodiments, a modified intemucleotidic linkage comprises an optionally substituted alkynyl group. In certain embodiments, a modified intemucleotidic linkage comprises an unsubstituted alkynyl group. In certain embodiments, a modified intemucleotidic linkage comprises a substituted alkynyl group. In certain embodiments, an alkynyl group is directly bonded to a linkage phosphorus.

[0443] In certain embodiments, a ds oligonucleotide comprises different types of intemucleotidic phosphorus linkages. In certain embodiments, a chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) intemucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In certain embodiments, a ds oligonucleotide comprises at least one non-negatively charged intemucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises at least one natural phosphate linkage and at least one non-negatively charged intemucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises at least one phosphorothioate intemucleotidic linkage and at least one non-negatively charged intemucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises at least one phosphorothioate intemucleotidic linkage, at least one natural phosphate linkage, and at least one non-negatively charged intemucleotidic linkage. In certain embodiments, ds oligonucleotides comprise 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 non-negatively charged internucleotidic linkages. In certain embodiments, a non-negatively charged internucleotidic linkage is not negatively charged in that at a given pH in an aqueous solution less than 50%, 40%, 40%, 30%, 20%, 10%, 5%, or 1% of the internucleotidic linkage exists in a negatively charged salt form. In certain embodiments, a pH is about pH 7.4. In certain embodiments, a pH is about 4-9. In certain embodiments, the percentage is less than 10%. In certain embodiments, the percentage is less than 5%. In certain embodiments, the percentage is less than 1%. In certain embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage in that the neutral form of the internucleotidic linkage has no pKa that is no more than about 1, 2, 3, 4, 5, 6, or 7 in water. In certain embodiments, no pKa is 7 or less. In certain embodiments, no pKa is 6 or less. In certain embodiments, no pKa is 5 or less. In certain embodiments, no pKa is 4 or less. In certain embodiments, no pKa is 3 or less. In certain embodiments, no pKa is 2 or less. In certain embodiments, no pKa is 1 or less. In certain embodiments, pKa of the neutral form of an internucleotidic linkage can be represented by pKa of the neutral form of a compound having the structure of CHs-the internucleotidic linkage-CHv For example, pKa of the neutral form of an internucleotidic linkage having the structure of Formula I may be represented by the pKa of the neutral form of a compound having the structure of (wherein each of X, Y, Z is independently -Q-, -S-, -N(R’)-; L is LB, and R1is -L-R’), pKa of can be represented by pKa jn certajnembodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In certain embodiments, a non-negatively charged internucleotidic linkage is a positively-charged internucleotidic linkage. In certain embodiments, a non-negatively charged internucleotidic linkage comprises a guanidine moiety. In certain embodiments, a non-negatively charged internucleotidic linkage comprises a heteroaryl base moiety. In certain embodiments, a non- negatively charged internucleotidic linkage comprises a triazole moiety. In certain embodiments, a non-negatively charged internucleotidic linkage comprises an alkynyl moiety.

[0444] In certain embodiments, a neutral or non-negatively charged internucleotidic linkage has the structure of any neutral or non-negatively charged internucleotidic linkage described in any of: 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, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612, each neutral or non-negatively charged intemucleotidic linkage of each of which is hereby incorporated by reference.

[0445] In certain embodiments, each R’ is independently optionally substituted C1-6 aliphatic. In certain embodiments, each R’ is independently optionally substituted C1-6 alkyl. In certain embodiments, each R’ is independently -CH3. In certain embodiments, each Rsis -H.

[0446] In certain embodiments, a non-negatively charged intemucleotidic linkage has the structure of . In certain embodiments, a non-negatively charged intemucleotidic linkage has the structure certain embodiments, a non- negatively charged intemucleotidic linkage has the structure some embodiments, a non-negatively charged intemucleotidic linkage has the structure of . In some embodiments, a non-negatively charged intemucleotidic linkage has the structure In some embodiments, a non-negatively charged internucleotidic linkage has the structure In some embodiments, a non negatively charged internucleotidic linkage has the structure . in some embodiments, a non-negatively charged internucleotidic linkage has the structure of some embodiments, a non-negatively charged internucleotidic linkage has the structure In some embodiments, a non-negatively charged internucleotidic linkage has the structure In some embodiments, a non- negatively charged internucleotidic linkage has the structure In some embodiments, a non-negatively charged internucleotidic linkage has the structure of . In some embodiments, W is O. In some embodiments, W is S. In some embodiments, a neutral internucleotidic linkage is a non-negatively charged internucleotidic linkage described above.

[0447] In certain embodiments, provided ds oligonucleotides comprise 1 or more internucleotidic linkages of Formula I, I-a, I-b, I-c, I-n-1, 1-n-2, 1-n-3, 1-n-4, II, II-a-1, Il-a- 2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, which are described in 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, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612, the Formula I, I-a, I-b, I-c, I-n-1, 1-n-2, 1-n-3, 1-n-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 salt forms thereof, each of which are independently incorporated herein by reference.

[0448] In certain embodiments, a ds oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage which is not the neutral internucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled phosphorothioate internucleotidic linkage. In certain embodiments, the present disclosure provides a ds oligonucleotide comprising one or more non-negatively charged internucleotidic linkages and one or more phosphorothioate internucleotidic linkages, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In certain embodiments, the present disclosure provides a ds oligonucleotide comprising one or more neutral internucleotidic linkages and one or more phosphorothioate internucleotidic linkage, wherein each phosphorothioate internucleotidic linkage in the ds oligonucleotide is independently a chirally controlled internucleotidic linkage. In certain embodiments, a ds oligonucleotide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotidic linkages. In certain embodiments, non-negatively charged internucleotidic linkage is chirally controlled. In certain embodiments, non-negatively charged internucleotidic linkage is not chirally controlled. In certain embodiments, a neutral internucleotidic linkage is chirally controlled. In certain embodiments, a neutral internucleotidic linkage is not chirally controlled.

[0449] Without wishing to be bound by any particular theory, the present disclosure notes that a neutral internucleotidic linkage can be more hydrophobic than a phosphorothioate internucleotidic linkage (PS), which can be more hydrophobic than a natural phosphate linkage (PO). Typically, unlike a PS or PO, a neutral internucleotidic linkage bears less charge. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages into a ds oligonucleotide may increase the ds oligonucleotides’ ability to be taken up by a cell and / or to escape from endosomes. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages can be utilized to modulate melting temperature of duplexes formed between a ds oligonucleotide and its target nucleic acid. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more non-negatively charged intemucleotidic linkages, e.g., neutral internucleotidic linkages, into a ds oligonucleotide may be able to increase the ds oligonucleotide’s ability to mediate a function such as target adenosine editing.

[0450] As appreciated by those skilled in the art, internucleotidic linkages such as natural phosphate linkages and those of Formula I, I-a, I-b, I-c, I-n-1, 1-n-2, I-n-3, I-n-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 salt forms thereof typically connect two nucleosides (which can either be natural or modified) as described in 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, WO2019032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612, the Formula I, I-a, I-b, I-c, I-n-1, 1-n-2, I-n-3, I-n-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 salt forms thereof, each of which are independently incorporated herein by reference. A typical connection, as in natural DNA and RNA, is that an intemucleotidic linkage forms bonds with two sugars (which can be either unmodified or modified as described herein). In many embodiments, as exemplified herein an intemucleotidic linkage forms bonds through its oxygen atoms or heteroatoms (e.g., Y and Z in various formulae) with one optionally modified ribose or deoxyribose at its 5’ carbon, and the other optionally modified ribose or deoxyribose at its 3’ carbon. In certain embodiments, each nucleoside units connected by an intemucleotidic linkage independently comprises a nucleobase which 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 heterocyclyl and / or a heteroaryl ring having at least one nitrogen atom.

[0451] In some embodiments, a linkage has the structure of or comprises -Y-PL(-X-RL)-Z-, or a salt form thereof, wherein:

[0452] LNis =N- LL1— , =CH- LL1— wherein CH is optionally substituted, or =N+(R’)(Q )-LL1-;

[0453] Q is an anion; each of X, Y and Z is independently -O-, -S-, -LL-N(-LL-RL)-LL-, -LL-N=C(-LL-RL)-LL- or LL; each RLis independently -LL-N(R’)2, -LL-R’, -N=C(-LL-R’)2, -LL- N(R’)C(NR’)N(R’)2, -LL-N(R’)C(0)N(R’)2, a carbohydrate, or one or more additional chemical moieties optionally connected through a linker; each of LL1and LLis independently L;

[0454] -CyIL- is -Cy-; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene,c=c, a bivalent C1-C6 heteroaliphatic group having 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-, wherein n is 1-50, and one or more nitrogen or carbon atoms are optionally and independently replaced with CyL; each -Cy- is independently an optionally substituted bivalent 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each CyLis independently an optionally substituted trivalent or tetraval ent, 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R’ is independently -R, -C(O)R, -C(O)N(R)2, -C(O)OR, or -S(O)2R; each R is independently -H, or an optionally substituted group selected from Ci -3...

Claims

CLAIMS What is claimed is:

1. A composition comprising a double-stranded RNAi (dsRNAi) agent capable of directing INHBE (Inhibin subunit βE)-specific RNA interference to induce lipolysis while preserving muscle mass, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: e) the guide strand is complementary or substantially complementary to an INHBE target RNA sequence; f) the guide strand comprises: i. a non-negatively charged internucleotidic linkage in the Sp configuration between the +3 nucleotide relative to the 5’ terminal nucleotide and the immediately downstream (+4) nucleotide; ii. a non-negatively charged internucleotidic linkage in the Rp configuration between the +10 nucleotide and the immediately downstream (+11) nucleotide; iii. phosphorothioate internucleotidic linkages in Sp configuration 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; and / or iv. phosphorothioate internucleotidic linkages in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide; c) the guide strand further comprises a 5’ phosphate modification; d) the passenger strand comprises one or more chiral internucleotidic linkages in Rp or Sp configuration; and e) the guide strand and the passenger strand each independently has a length of 15- 49 nucleotides.

2. The composition of claim 1, wherein the guide strand comprises a 5’ phosphate modification selected from:Base: A, C, G, T, U, abasic, and modified nucleobases; R: H, OH, O-alkyl, F, MOE, LNA bridge to the 4’ position, BNA bridge to the 4’ position.

3. The composition of claim 2 wherein the guide strand comprises a 5’ phosphate modification selected from 5’ MeP modifications and 5’ triazole-P modifications.

4. The double stranded oligonucleotide or composition of claim 14 wherein the 5’ MePmodification is .

4. The composition of claim 4, further comprising a backbone phosphorothioate chiral center in Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream(+2) nucleotide, and a backbone phosphorothioate chiral center in the Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

5. The composition of claim 1, wherein the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-1) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration.

6. The composition of claim 1, wherein the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N- 1) nucleotide and as between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non- negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

7. The composition of claim 1, wherein the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

8. The composition of claim 1, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of backbone phosphorothioate chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-1) nucleotide and as between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

9. The composition of claim 1, wherein the guide strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5’ terminal nucleotide of the guide strand and the penultimate 3’ (N-1) nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the passenger strand comprises 0-n non-negatively charged internucleotidic linkages, where n is about 1 to 49 and one or more backbone chiral centers in Rp or Sp configuration.

10. The composition of any one of the preceding claims, wherein the Rp, Sp, or stereorandom non-negatively charged backbone internucleotidic linkages have neutral charge.

11. The composition of claim 10, wherein the neutral backbone internucleotidic linkages is.

12. The composition of claim 11, wherein the guide strand comprises a linkage having the following structurebetween the third (+3) and fourth (+4) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, or both.

13. The composition of claim 12, wherein the passenger strand comprises a linkage having the following structurethe central nucleotide of the passenger strand, 3’ to the central nucleotide of the passenger strand, or both.

14. The composition of claim 1, where the guide and passenger strands in the composition that independently share a common base sequence, a common pattern of base modification, acommon pattern of sugar modification, and / or a common pattern of internucleotidic linkages are at least 90% of all the guide and passenger strands in the composition.

15. The composition of any of the preceding claims, wherein the double stranded oligonucleotide comprises a carbohydrate moiety connected at a nucleoside, an internucleotidic linkage, optionally through a linker.

16. The composition of any of the preceding claims, wherein the double stranded oligonucleotide comprises a lipid moiety connected to the double stranded oligonucleotide at a nucleoside, an internucleotidic linkage, optionally through a linker.

17. The composition of any of the preceding claims, wherein one or both strands of the double stranded oligonucleotide comprises a target moiety connected at a nucleoside, an internucleotidic linkage, optionally through a linker.

18. The composition of 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 internucleotidic linkages of the double stranded oligonucleotide are independently chiral internucleotidic linkages.

19. The composition of 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 nucleotidic units of the double stranded oligonucleotide independently comprise a 2’-substitution.

20. The composition of any one of the preceding claims, wherein a 2’-substitution of the oligonucleotide is 2’-F.

21. The composition of any one of the preceding claims, wherein a 2’-substitution of the oligonucleotide is 2’-OR1.

22. The composition of any one of the preceding claims, wherein a 2’-substitution of the oligonucleotide is−L−, wherein L connects C2 and C4 of the sugar unit.

23. The composition of 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 nucleotidic units of the double stranded oligonucleotide comprise no 2’- substitution.

24. The composition of any one of the preceding claims, wherein the guide strand comprises an INHBE target-binding sequence that is completely complementary to an 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 of any one of the preceding claims, wherein the INHBE target sequence comprises two SNPs.

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

27. The composition of any one of the preceding claims, wherein the double stranded oligonucleotide comprises a guide strand that binds with a transcript of an INHBE target nucleic acid sequence for which a plurality of alleles exist within a population, each of which contains a specific nucleotide characteristic sequence element that defines the allele relative to other alleles of the same INHBE target nucleic acid sequence, wherein the base sequence of the guide strand is or comprises a sequence that is complementary to the characteristic sequence element that defines a target allele, andthe guide strand being characterized in that, when it is contacted with a cell comprising transcripts of INHBE target nucleic acid sequence, it shows suppression of transcripts of the target allele, or a protein encoded thereby, at a level that is greater than a level of suppression observed for another allele of the same nucleic acid sequence.

28. The composition of 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 level and / or activity of an INHBE transcript or a protein encoded thereby, comprising administering to a cell expressing the INHBE transcript a composition of any one of the preceding claims, wherein the guide strand of double stranded oligonucleotide or composition comprises a INHBE-binding sequence that is completely complementary to an INHBE target sequence in the transcript.

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

31. A method of treating a metabolic disorder, comprising administering a composition of any one of claims 1-28.

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

33. A method of treating obesity, comprising administering a composition of any one of claims 1-28.

34. A method of treating cardiovascular disease, comprising administering a composition of any one of claims 1-28.

35. A method of treating diabetes, comprising administering a composition of any one of claims 1-28.

36. A method of treating hypertension, comprising administering a composition of any one of claims 1-28.

37. The method of any one of claims 31-36, comprising administration of a second therapeutic.

38. The method of claim 37, wherein the second therapeutic is selected from the group consisting of: insulin; sulfonylurea; meglitinide; biguanide; thiazolidinedione; alphaglucosidase inhibitor; SGLT2 inhibitor; DPP4 inhibitor; glucagon like peptide 1 receptor agonist (GLP-1RA); glucose-dependent insulinotropic polypeptide agonist (GIP RA); glucagon receptor agonist (Gcg RA); co-agonist of GLP-1R, GIP R, and / or Gcg R; HMG-CoA reductase inhibitor, statins; PCSK9 inhibitor; ApoC3 inhibitor; ANGPTL3 inhibitor; ATP citrate lyase (bempedoic acid), ezetimide; Lp(a) inhibitor, LPL activator, and combinations thereof.

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

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

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